Sertoli Cell Biology

SERTOLI CELL BIOLOGY

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SERTOLI CELL BIOLOGY

Michael K. Skinner Michael D. Griswold Center for Reproductive Biology School of Molecular Biosciences Washington State University Pullman, WA 99164-4660

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Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald's Road, London WC1X 8RR, UK This book is printed on acid-free paper. ∞ Copyright © 2005, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Customer Support” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data: Application Submitted

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 0-12-647751-5 For all information on all Academic Press publications visit our Web site at www.academicpress.com Printed in the United States of America 04 05 06 07 08 9 8 7 6 5 4 3 2 1

Dedication

This book is dedicated to the memory of Professor Lonnie Russell of Southern Illinois University. Lonnie died in a drowning accident in Brazil in 2001 at the height of a successful research career in reproductive physiology. Lonnie used his expertise in anatomy to help define the intricacies of spermatogenesis with a focus on the function of the Sertoli cells. In 1993 Lonnie Russell and Michael Griswold edited a book titled The Sertoli Cell, which has served as a major resource and inspiration for investigators interested in male reproduction. It is appropriate that now—more than 10 years later—the advances in this field are summarized in this new text. It is also appropriate that this volume is a tribute to the many scientific contributions made by Lonnie Russell. It can only be hoped that this volume generates a small part of the energy and enthusiasm for science that Lonnie was able to stimulate with his wit and enquiring mind. Michael K. Skinner Michael D. Griswold

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Contents

Contributors Preface

xi xv P A R T

I INTRODUCTION CHAPTER 1

History of the Sertoli Cell Discovery

3

Rex A. Hess and Luiz R. França CHAPTER 2

Perspective on the Function of Sertoli Cells

15

Michael D. Griswold CHAPTER 3

Structure of the Sertoli Cell

19

Rex A. Hess and Luiz R. França P A R T

II SERTOLI CELL DEVELOPMENT CHAPTER 4

Embryonic Sertoli Cell Differentiation

43

Andrea S. Cupp and Michael K. Skinner CHAPTER 5

Sertoli Cell Biology in Fishes and Amphibians

71

Jerry Bouma and Joseph G. Cloud CHAPTER 6

Sertoli Cell Biology in Seasonal Breeders

81

Amiya P. Sinha Hikim and Andrzej Bartke

vii

viii

Contents P A R T

III SERTOLI CELL FUNCTION AND GENE EXPRESSION CHAPTER 7

Sertoli Cell Gene Expression and Protein Secretion

95

Michael D. Griswold and Derek McLean

CHAPTER 8

Sertoli Cell Secreted Regulatory Factors

107

Michael K. Skinner

CHAPTER 9

Proteases and Protease Inhibitors

121

Martin Charron and William W. Wright

P A R T

IV SERTOLI CELL ENDOCRINOLOGY AND SIGNAL TRANSDUCTION CHAPTER 10

FSH Regulation at the Molecular and Cellular Levels: Mechanisms of Action and Functional Effects

155

Ilpo Huhtaniemi and Jorma Toppari

CHAPTER 11

In Vivo FSH Actions

171

Charles M. Allan and David J. Handelsman

CHAPTER 12

Sertoli Cell Endocrinology and Signal Transduction: Androgen Regulation

199

Richard M. Sharpe

CHAPTER 13

Thyroid Hormone Regulation of Sertoli Cell Development

217

Paul S. Cooke, Denise R. Holsberger, and Luiz R. França

CHAPTER 14

The Transforming Growth Factor β Superfamily in Sertoli Cell Biology Kate L. Loveland and David M. Robertson

227

Contents

ix

P A R T

V SERTOLI CELL TRANSCRIPTIONAL REGULATION CHAPTER 15

Transcription Factors in Sertoli Cells

251

Jaideep Chaudhary and Michael K. Skinner

CHAPTER 16

Structure and Regulation of the FSH Receptor Gene

281

Leslie L. Heckert

P A R T

VI CELL–CELL INTERACTIONS INVOLVING SERTOLI CELLS CHAPTER 17

The Role of the Sertoli Cell in Spermatogonial Stem Cell Fate

303

Martin Dym and Lixin Feng

CHAPTER 18

Sertoli Cell–Somatic Cell Interactions

317

Michael K. Skinner

CHAPTER 19

Sertoli Cell Lines

329

Kenneth P. Roberts

P A R T

VII SERTOLI CELL PATHOPHYSIOLOGY CHAPTER 20

Sertoli Cell Toxicants

345

Kim Boekelheide, Kamin J. Johnson, and John H. Richburg

CHAPTER 21

Conditions Affecting Sertoli Cells Wael A. Salameh and Ronald S. Swerdloff

383

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Contents P A R T

VIII SPERMATOGONIAL STEM CELLS CHAPTER 22

Gonocyte Development and Differentiation

417

Peter J. Donovan and Maria P. de Miguel

CHAPTER 23

Hormones and Spermatogonial Development

437

Marvin L. Meistrich, Gunapala Shetty, Olga U. Bolden-Tiller, and Karen L. Porter

CHAPTER 24

Long-Term Cultures of Mammalian Spermatogonia

449

Marie-Claude C. Hofmann and Martin Dym

CHAPTER 25

Transplantation

471

Ina Dobrinski

Index

487

Contributors

Charles M. Allan Senior Scientist Andrology Laboratory ANZAC Research Institute Sydney, NSW 2139, Australia Phone: +61-2-9767 9100 Fax: +61-2-9767 9101 E-mail: [email protected] Andrzej Bartke Geriatrics Research Department of Medicine Southern Illinois University School of Medicine P.O. Box 19628 Springfield, IL 62794 Phone: 217-545-7962 E-mail: [email protected] Kim Boekelheide Professor of Medical Sciences Department of Pathology & Laboratory Medicine Division of Biology and Medicine Brown University, Box G-E Providence, RI 02912 Phone: 401-863-1783 E-mail: [email protected] Olga U. Bolden-Tiller Department of Experimental Radiation Oncology University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd. Houston, TX 77030 Phone: 713-792-4848 Fax: 713-794-5369 E-mail: [email protected] Jerry Bouma The Jackson Laboratory 600 Main Street Bar Harbor, ME 04609 Phone: 207-288-6344 E-mail: [email protected]

Joseph G. Cloud Professor of Zoology Department of Biological Sciences University of Idaho Moscow, ID 83844-3051 Phone: 208-885-6388 Fax: 208-885-7905 E-mail: [email protected] Martin Charron Division of Reproductive Biology Department of Biochemistry & Molecular Biology Bloomberg School of Public Health Johns Hopkins University Baltimore, MA 21205-2179 Phone: 410-955-7831 Fax: 410-955-2926 E-mail: [email protected] Jaideep Chaudhary Center for Reproductive Biology School of Molecular Biosciences Washington State University Pullman, WA 99164-4231 Phone: 509-335-1945 Fax: 509-335-2176 E-mail: [email protected] Paul S. Cooke Department of Veterinary Biosciences and Division of Nutritional Sciences University of Illinois Urbana, IL 61802 Phone: 217-333-6825 Fax: 217-244-1652 E-mail: [email protected] Andrea S. Cupp Department of Animal Science University of Nebraska Lincoln, NE 68583-0908 Phone: 402-472-6424 Fax: 402-472-6362 E-mail: [email protected]

xi

xii Ina Dobrinski Center for Animal Transgenesis and Germ Cell Research 145 Myrin Bldg., New Bolton Center School of Veterinary Medicine University of Pennsylvania 382 West Street Rd. Kennett Square, PA 19348 Phone: 610-925-6563 Fax: 610-925-8121 E-mail: [email protected] Peter J. Donovan Stem Cell Program Institute for Cell Engineering Johns Hopkins University School of Medicine Broadway Research Building 733 North Broadway Baltimore, MD 21205-2179 Phone: 443-287-5591 Fax: 443-287-5611 E-mail: [email protected] Martin Dym Department of Cell Biology Georgetown University School of Medicine Washington, DC 20007 Phone: 202-687-1157 Fax: 202-687-9864 E-mail: [email protected] Lixin Feng Department of Cell Biology Georgetown University School of Medicine Washington, DC 20057 Phone: 202-687-1194 E-mail: [email protected] Luiz R. França Laboratory of Cellular Biology Department of Morphology Institute of Biological Sciences Federal University of Minas Gerais Belo Horizonte, Brazil 31270-901 Fax: +55-31-34992780 E-mail: [email protected] Michael D. Griswold Dean of Science Professor of Molecular Biosciences Washington State University Pullman, WA 99164-4660 Phone: 509-335-6281 Fax: 509-335-9688 E-mail: [email protected]

Contributors

David J. Handelsman Professor of Reproductive Endocrinology and Andrology University of Sydney Director, ANZAC Research Institute Sydney, NSW 2139, Australia Phone: +61-2-9767 9100 Fax: +61-2-9767 9101 E-mail: [email protected] Leslie L. Heckert Department of Molecular and Integrative Physiology University of Kansas Medical Center 3901 Rainbow Blvd. Kansas City, KS 66160 Phone: 913-588-7488 Fax: 913-588-7430 E-mail: [email protected] Rex A. Hess Professor Reproductive Biology and Toxicology Department of Veterinary Biosciences University of Illinois 2001 S. Lincoln Urbana, IL 61802 Phone: 217-333-8933 Fax: 217-244-1652 E-mail: [email protected] Amiya P. Sinha Hikim Division of Endocrinology Department of Medicine Harbor–UCLA Medical Center David Geffen School of Medicine at UCLA 1000 West Carson Street Torrance, CA 90509 Phone: 310-222-1867 E-mail: [email protected] Marie-Claude C. Hofmann Department of Biology Sciences Center The University of Dayton 300 College Park Dayton, OH 45469 Office/Lab: SC-303C/347 Phone: 937-229-2894/2507 Fax: 937-229-2021 E-mail: [email protected] Denise R. Holsberger Department of Veterinary Biosciences (P.S.C.) Division of Nutritional Sciences University of Illinois Urbana, IL 61802 Phone: 217-244-5782 Fax: 217-244-1652 E-mail: [email protected]

Contributors

Ilpo Huhtaniemi Professor of Reproductive Biology Institute of Reproductive and Developmental Biology (IRDB) Imperial College London Du Cane Road London, W12 0NN, U.K. Phone: +44-20-75942104 Fax: +44-20-75942184 E-mail: [email protected] Kamin J. Johnson Division of Biological Sciences CIIT Centers for Health Research Research Triangle Park, NC 27709 Phone: 919-558-1439 E-mail: [email protected] Kate L. Loveland Monash Institute of Reproduction and Development ARC Centre of Excellence in Biotechnology and Development Monash University 27-31 Wright Street Clayton, Victoria 3168 Australia Phone: +61-3-9594 7125 Fax: +61-3-9594 7111 E-mail: [email protected] Derek McLean Center for Reproductive Biology Department of Animal Sciences Washington State University Pullman, WA 99164-6353 Phone: 509-335-8759 E-mail: [email protected] Marvin L. Meistrich Department of Experimental Radiation Oncology University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd. Houston, TX 77030 Phone: 713-792-4866 Fax: 713-794-5369 E-mail: [email protected] Maria P. de Miguel Cell Therapy Laboratory La Paz Hospital Paseo Castellanan 261 Madrid, 28045 Spain Phone: 34-91-7271940 E-mail: [email protected]

xiii

Karen L. Porter Department of Experimental Radiation Oncology University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd. Houston, TX 77030 Phone: 713-792-4848 Fax: 713-794-5369 E-mail: [email protected] John H. Richburg Division of Pharmacology and Toxicology College of Pharmacy The University of Texas at Austin 1 University Station, A1915 Austin, TX 78712 Phone: 512-471-4736 E-mail: [email protected] Kenneth P. Roberts Associate Professor Department of Urologic Surgery University of Minnesota 420 Delaware St. SE MMC 394 Minneapolis, MN 55455 Phone: 612-625-9977 E-mail: [email protected] David M. Robertson Prince Henry’s Institute of Medical Research P.O. Box 5152 Clayton, Victoria 3168 Australia Phone: +6-3-95944386 Fax: +6-3-95946125 E-mail: [email protected] Wael A. Salameh David Geffen School of Medicine at UCLA Harbor–UCLA Medical Center Division of Endocrinology Rb-1 1124 West Carson Street Torrance, CA 90502 Phone: 310-222-1867 Fax: 310-533-0627 E-mail: [email protected] Richard M. Sharpe MRC Human Reproductive Sciences Unit Centre for Reproductive Biology University of Edinburgh Chancellor’s Building 49 Little France Crescent Old Dalkeith Road Edinburgh, EH16 4SB Phone: +44-131-242-6387 Fax: +44-131-242-6231 E-mail: [email protected]

xiv

Contributors

Gunapala Shetty Department of Experimental Radiation Oncology University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd. Houston, TX 77030 Phone: 713-794-4858 Fax: 713-794-5369 E-mail: [email protected] Michael K. Skinner Director and Professor Center for Reproductive Biology School of Molecular Bioscience Washington State University Pullman, WA 99164-4231 Phone: 509-335-1524 Fax: 509-335-2176 E-mail: [email protected] Ronald S. Swerdloff David Geffen School of Medicine at UCLA Harbor–UCLA Medical Center Division of Endocrinology Rb-1 1124 West Carson Street Torrance, CA 90502 Phone: 310-222-1867 Fax: 310-533-0627 E-mail: [email protected]

Jorma Toppari Departments of Physiology and Pediatrics University of Turku Kiinamyllynkatu 10 20520 Turku, Finland Phone: +358-2-333-7297 Fax: +358-2-2502621 E-mail: [email protected] William W. Wright Department of Biochemistry Reproductive Biology Bloomberg School of Public Health Johns Hopkins University East Baltimore Campus W3508 Wolfe Street Building Baltimore, MD 21205-2179 Fax: 410-614-2356 E-mail: [email protected]

Preface

In 1993 Lonnie Russell and Michael Griswold edited a book titled The Sertoli Cell, which has served as a major resource for investigators interested in male reproduction. It is appropriate that now—more than 10 years later—the advances in this field are summarized in this new text. It is also appropriate that this volume is a tribute to the many scientific contributions made by Professor Lonnie Russell of Southern Illinois University. Lonnie died in a drowning accident in Brazil in 2001 at the height of a successful research career in reproductive physiology. Lonnie used his expertise in anatomy to help define the intricacies of spermatogenesis with a focus on the function of Sertoli cells. He was first an anatomist and physiologist, and he used those tools to produce an enormous amount of information on the testis. His laboratory published the first and only visualization of intact Sertoli cells reconstructed from serial sections in the electron microscope. These papers allowed those of us in the field a fundamental visualization of the beauty and complexity of the Sertoli cells. It is essential for the molecular biologists and biochemists to place their findings in the context of the complex biology of the testis. Part of the goal of this current text is to make that process easier. The editors of this text (the Mikes) have a combined nearly 50 years of experience focused on exposing the secrets of the Sertoli cells. Both of us have spent many years reporting on specific gene products of Sertoli cells and over the combined 50 years we have thoroughly investigated perhaps a dozen gene products. The biggest change in the field has been the use of expression arrays and gene knockout technologies that allow the monitoring of thousands of expressed sequences and the more laborious testing of their physiological functions. In The Sertoli Cell, Russell and Griswold reported that nearly 300 papers were published in 1990 that somehow involved these cells. Since the millennium there have been nearly twice that

number of papers per year, suggesting these new technologies and other factors have stimulated an increased interest in the Sertoli cell. The current text attempts to present a systems biology approach to an understanding of the Sertoli cell. A combination of molecular, cellular, and physiological aspects of Sertoli cells are presented. Due to the essential role Sertoli cells play in the process of spermatogenesis, topics such as spermatogonial stem cells and germ cell transplantation are also presented. An attempt was made to identify areas that have had significant advances over the past decade, as well as suggest important areas for the future analysis of Sertoli cell biology. Information is presented to provide the novice with basic information on the topics as well as to provide experts with new details that have advanced the field. We hope you find Sertoli Cell Biology a useful reference and that it provides insight for an understanding of the Sertoli cell and male reproduction.

ACKNOWLEDGMENTS The chapters provided and hard work of the contributing authors is what made this book possible. The editors thank Ms. Mica Haley, Ms. Jacqueline Garrett and the staff at Elsevier/Academic Press for their assistance and patience. We thank Dr. Rex Hess and acknowledge his interest in the book, particularly in the design of the cover. The cover includes an electron micrograph of a Sertoli cell from Dr. Lonnie Russell and his three-dimensional reconstruction of the cell, as well as a light micrograph of the Sertoli cell provided by Dr. Hess. This book would not have been possible without the editorial and administrative support of Ms. Jill Griffin. Jill’s dedication and efficiency were critical for the book and indispensable to the editors.

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P A R T

I INTRODUCTION

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C H A P T E R

1 History of the Sertoli Cell Discovery

I. II. III. IV. V. VI. VII. VIII. IX.

REX A. HESS

LUIZ R. FRANÇA

Reproductive Biology & Toxicology, Veterinary Biosciences, University of Illinois, Urbana, Illinois

Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil

Brian P. Setchell [10,11], whose foreword in the first book provided a sincere and deserved admirable look at Enrico Sertoli [12].

INTRODUCTION THE STUDENT THE MICROSCOPE THE LABORATORY THE DISCOVERY COMPLETING THE MANUSCRIPT AFTER GRADUATION TRANSITION TO THE MODERN SERTOLI CELL POSTSCRIPT References

II. THE STUDENT Enrico Sertoli was 18 years old when he began his studies in medicine and research at the University of Pavia in Northern Italy in 1860 [3]. He studied general medical subjects at first and then after 2 years began his research studies in the laboratory of the distinguished physiologist and histologist, Professor Eusebio Oehl (1827–1903). It is interesting that Camillo Golgi, for whom the Golgi apparatus is named, was a fellow student at the university and he and Sertoli both studied under Professor Oehl [13] and graduated in the same year, 1865. Sertoli was born on June 6, 1842, to a noble family in the small town of Sondrio, located North of Milano along the Italian–Swiss border [3]. His noble birth in all probability meant that he was expected to attend university and study medicine. Unfortunately, as Sertoli entered his teenage years, the countryside was not at peace and there was talk of war between Prussia and Austria during this period. Accordingly, Sertoli may have had the same urges of young people today to join the local forces and defend his country, but his father surely urged him to complete his medical training before entering the army, which Sertoli did when war broke out after he graduated in 1865 [12].

I. INTRODUCTION The first edition of The Sertoli Cell was an appropriate vision of the late Professor Lonnie D. Russell, because he studied the Sertoli cell in more depth than most other modern-day scientists. He published more than 200 papers, of which nearly half were focused on the Sertoli cell, including the first book devoted to the cell, which he coedited with Michael D. Griswold [1]. Therefore, this chapter is written in honor of Lonnie because he was a fun-loving friend and visionary scientist who always used the microscope and his imagination to find new insights into complex scientific problems of the testis, and in particular the Sertoli cell. Lonnie’s devotion to this cell was exemplified by the license plate that he attached to his automobile, which read “Sertol 1,” and by his cat whose name was also “Sertoli.” Factual events surrounding Sertoli’s life were gathered from reading numerous reviews [2–9], particularly those of the distinguished scholar SERTOLI CELL BIOLOGY Edited by M. K. Skinner and M. D. Griswold

3

Copyright 2005, Elsevier Science (USA). All rights reserved.

4

Rex A. Hess and Luiz R. França

The University of Pavia was an old, well-established center of higher education located just south of Milano, which according to historical records was established by edict of the Emperor Lotarius in the year 825. The culture surrounding this famous university was surely one that encouraged the highest standards of achievement and bred intellectual inquiry that was capable of producing Nobel Prize–winning scientists, such as Golgi, who also studied under Bizzozero and was later nominated for the first Nobel Prize in Physiology and Medicine in 1901 and then every year until 1906, when he shared the prize with Santiago Ramón y Cajal.

III. THE MICROSCOPE The cellule ramificate or branched cell was discovered using the personal microscope of Enrico Sertoli (Fig. 1.1). He had purchased the microscope in 1862, after he began his research studies under Professor Oehl. The Belthle microscope that he purchased from the Kellner Optical Institute in Wetzlar, Germany, was a state-of-the-art light microscope at that time. Instruments produced by the Kellner Optical Institute were compound microscopes with three or more lenses, and each came with 10× and 20 × magnifier eyepieces. They also had a screw system for lowering the compound lenses to the slide, so that there was less breakage of the glass coverslips.

FIGURE 1.1 The Belthle microscope that Sertoli personally purchased in 1862 and the same microscope that he used to make the famous discovery of the cell that now carries his name. (Photograph kindly provided by Michi Sertoli, the great nephew of Professor Enrico Sertoli, of Milan, Italy.)

In contrast, some of the more common microscopes found in laboratories in 1862, such as the van Deyl and Beck microscopes, were no more than high-powered magnifying lenses. Such microscopes would have posed difficulties for a serious student and may have been reason enough for Sertoli to purchase one of the highest quality microscopes available, knowing that the instrument chosen would become the limiting factor in his histological research accomplishments. We do not know the complete story behind the purchase of this microscope, but it was surely a momentous occasion. It is possible that microscope purchases were required for every medical student on entry into the histology course. Regardless, we can envision that some students were capable of purchasing a compound microscope, whereas others found it necessary to settle for the less expensive brands that provided only a magnifying lens. The quality of the Belthle microscope and its personal importance are evidenced by the care that Sertoli devoted to it, which has permitted its survival for more than 100 years, along with a wooden storage box and the original two-page letter of guarantee that was provided by Belthle from the Kellner Optical Institute (Fig. 1.2).

IV. THE LABORATORY To our knowledge, there are no photographs of Sertoli in his laboratory; therefore, we must assume that the room was similar to others that were photographed in the late 1800s. Professor Oehl’s laboratory was likely a typical large room with a high ceiling and large windows. Because the Edison lamp did not arrive until 1890, few professors, except in the department of physics, would have had electric lights in their offices and laboratories. The furniture would likely have been made from a hardwood and coated with dark stain. Tall wood encasings would have lined the outer walls. A bench would have run the full length of the room with hardwood shelving down the center. It would have encased chemical solutions, flasks, stopcock bottles, and other supplies common to the research lab. Natural gas was used quite extensively in Europe by the late 1860s; however, harnessing the gas was not easy, and Robert Bunsen had not as yet invented the famous “Bunsen burner.” This marvelous invention in 1885 permitted the controlled burning of natural gas through a metal tubule by regulating the amount of gas and air proportions individually, which essentially allowed for increases in the temperature and in the intensity of the flame. Until this device came along, everyone

Chapter 1 History of the Sertoli Cell Discovery

5

FIGURE 1.2 (A) This wooden box is the original storage box for the microscope, which Sertoli maintained so meticulously. (B) The original two-page document that was sent along with the microscope from the German company Belthle in Wetzlar. The paper is a guarantee from the Optics Institute of von C. Kellner. (The date 1862 has been magnified digitally for emphasis. Image kindly provided by Michi Sertoli, the great nephew of Professor Enrico Sertoli, of Milan, Italy.)

simply used a straight tubule with a round base and controlled the flow of gas by a single valve. This led to many accidents and explosions, but the flame was still superior to candle or alcohol lamps. It was common to have a large blackboard and chalk in the laboratory. We can imagine the blackboard being covered with drawings of Sertoli’s observations and maybe even outlines of experiments dealing with Professor Oehl’s own research. On the bench may have been another typical microscope that was often used in physiology, the Cuff simple microscope, which was made by Dollond of London. This monocular-type scope was used for dissections. It was capable of magnifying with fairly good resolution up to 10× to 20×. The instrument had a tube body mounted above a stage, similar to the compound microscope, but it was strictly a magnifying lens on a stand. Thus, this crude magnifier was the precursor of the dissecting microscope. Along that same bench would have been glass jars with specimens preserved in alcohol and acids and other types of solutions that were used in histology. On the opposite side could have been stacks of histology glass slides and possibly an open copy of the first textbook of histology, written by the Swiss scientist, Albert Kölliker [14]. Like many scientists during that period, Sertoli would have worn the typical white smock-like lab coat with five buttons up the front. The coat would protect his white shirt and dark tie, the formal dress of a university student.

V. THE DISCOVERY In anticipation of the microscope’s arrival, Sertoli likely collected several pieces of human testes preserved in a sublimate solution (a precipitating solution formed by adding ammonia to mercuric chloride) that he later reported as the incubation solution of choice at that time [15]. Without being aware, Sertoli was using a very nice model to investigate the seminiferous epithelium, because in humans, unlike in mice and rats, the Sertoli cell occupies about 37% of the epithelium. In contrast, the Sertoli cell occupies about 15–20% in the rodent species. The human testis has a higher ratio of Sertoli to germ cells, due to the reduced efficiency of spermatogenesis. It appears that Sertoli worked only with human testes throughout his career. Thus, working in a medical school was an advantage to Sertoli from the very beginning. Sertoli used several different types of preparations of testes, including microdissections of individual seminiferous tubules, thin sections of the testis after sublimate incubation, pieces of fresh tissue, and frayed sections of tubules. Like all young students in the laboratory, he probably worried that the tissue may have remained in the solution too long. With such concerns, perhaps he had numerous conversations with Golgi and other students regarding the latest methodologies that were being tested in histology to better preserve structures and improve the visualization of cells.

6

Rex A. Hess and Luiz R. França

After the new microscope arrived, Sertoli probably spent the first few weeks working obsessively with the shiny new instrument. He no doubt spent endless hours getting the angle of the sunlight just right so that the cells would appear more clearly. To a scientist today, the methods used by Sertoli were crude and harsh. It is astounding that such important discoveries were made during this early period of development of what we now accept as routine histological procedures. These early observations were made without the benefit of fixatives that could crosslink proteins and bind lipids in the tissue. Alcohols and acids were the primary methods for treatment of fresh samples. Although formaldehyde was discovered in 1859, it was not until after the commercial synthesis of formalin from methanol by Hoffman in 1868 that sufficient quantities became available for testing in various medical and chemical procedures. The actual use of formalin for hardening of tissue did not come about until 1892 when a chemist, Blum, was asked to test formalin as a potential antiseptic agent. Formalin hardened the skin on his fingertips, and the rest is now history for this widely used fixative. At first, Sertoli may have called the cell a tree-like cell or stringy cell or some other description that suggested that this cell had long extensions. On the first page of his publication [15], the term mother cells is used, which suggests that his observations were quite perceptive and even intuitive of the true Sertoli cell function. In 1863, it was necessary to draw observations made through a microscope. Although a photo was actually produced in 1827 on materials hardened after exposure to light for up to 8 hours [16], the word photography was not invented until 1839, by Sir John Herschel. This early type of photography was time consuming and quite expensive; therefore, it was not routine in the scientific laboratory, and a box for holding the film that could be used on a microscope was not invented until 1884, when George Eastman introduced flexible film and the boxed camera [16]. Therefore, Sertoli would have spent many hours drawing what he observed. Sertoli’s first drawings must have been simple (probably similar to those in Figure I of the original plate, reproduced here as Fig. 1.3). Looking through a microscope and drawing what you see is difficult even when the tissue is well preserved and well stained. Unfortunately, the corrosive solutions that Sertoli used extracted the cells and left them rather transparent. Nevertheless, he would have been very excited, as every student would be upon using for the first time a new instrument or observing for the first time a new organelle or cell. In truth, the discoveries by Sertoli were things that had never before been described by others.

FIGURE 1.3 Drawings taken from the original paper (Fig. I a–d) of Sertoli [15].

Reproductive biology literature was scarce in the 1800s. However, Sertoli was reading the work of Kölliker [17] and referred to his work as being the “most authoritative one.” But Kölliker claimed that these cells of interest to Sertoli were polygonal in shape, instead of conical or cylindrical in shape as described by Sertoli, and such statements in the early scientific literature tended to become dogma. To discover something that contradicts the published literature surely provides the ultimate excitement for a scientist, and such exhilaration may have been even greater during that period in time if we take into consideration the culture of science in Europe in 1863. Such discoveries can also be intimidating, especially when one considers that their observation may contradict a famous scientist, such as Kölliker. One cannot help but wonder what Sertoli wrote in his notebook. Today we publish only small portions of the total data that are collected during an experiment. However, in the 1800s, every observation was novel and worthy of discussion. He probably had numerous drawings lying on the bench, and his notebooks were likely filled with drawings and intricate descriptions. We know he described the cell as having branches and blobs at the ends. Maybe the branches surrounded germ cells and maybe the branches were like those of a tree that drops its fruit as the harvest ripens. Anything was possible, because it was new. It was possible that this cell, like the great maestro of a symphony, directed the production of sperm in the testis. Maybe spermatozoa developed directly from the branched cell. Sertoli and the other histology students on the campus, such as Golgi, may have discussed the work of Professor Waldeyer in Berlin. Professor Waldeyer had extracted a substance from logwood (Haematoxylon campechianum) that someone had collected from Central America. Apparently, the substance, now called hematoxylin, was a nonquinonoid that was soluble in water and, when readily oxidized, would stain leather. Waldeyer was the first to propose this solution for staining histological tissues, too. However, it was not

Chapter 1 History of the Sertoli Cell Discovery

until Ehrlich formulated the extract with alum that the method worked efficiently as a counter stain to eosin [18]. Thus, Sertoli had little to work with in 1863 but he must have tried whatever solutions he could find in the laboratory or borrow from other labs. He mentioned in the paper that he tried clearing the tissue with nitric and acetic acids, and even tried staining with iodine. He wrote, as translated by Setchell [10], “Nitric acid shrivels and deforms the cells and turns the nuclei yellow. Tincture of iodine stains these structures yellow, but the nuclei even more intensely than the cells.” Such testing of different methods may have improved the observations, because in some drawings, Sertoli included round germ cells, “seminiferous cells,” embedded within the branched cell limbs (Fig. 1.4). Sertoli drew with intricate detail what he observed. He was the first to recognize and report lipid droplets in this cell, and he mentioned several times that lipid could exert very important functions in the cell, a function that we still know little about today. He also drew the cell as appearing syncytial or as a branched multinucleated cell, which surely raised many questions from students such as Golgi, because such ideas were common among those who studied the brain. Sometimes he simply looked at fresh tissue (Fig. 1.5) and observed what appeared to be an epithelial lining of the branched cells surrounding germ cells. His drawings indicated that the united branched cells were protecting or extending a hand to the germ cells (Fig. 1.4). Even without the privilege of seeing Sertoli’s notebook, we can imagine that it contained numerous pages of drawings and descriptions, many of which he used as the basis for writing the following narrative, as translated by Setchell from Sertoli’s original 1865 paper [10]:

FIGURE 1.4 Drawings taken from the original paper (Figs. II a–d and III a–e) of Sertoli [15].

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FIGURE 1.5 Drawings taken from the original paper (Figs. IV and V a–b) of Sertoli [15].

IV. Finally, some special cells, which I saw in moderate number in the preparations and which, to my knowledge, have not previously been observed and described by anyone. They appear in the form of irregularly cylindrical or conical cells, with indistinct margins, provided with a nucleus, always containing a nucleolus. Their contents comprise fine droplets of fat in a substance that is reasonably transparent because it is homogeneous. These cells almost always have quite transparent extensions, in the interior of which fine droplets of fat can frequently be seen. They have an irregularly shaped body from which often protrude one or more extensions, and two extremities of which the upper is usually large and bounded by a well-marked margin that sometimes appears double (Fig. I a,c,d). Lower down the cell often is contracted somewhat, formed like a sort of collar (Fig. I a,d, Fig. III b). The other, luminal extremity, becomes narrower and forms an extension, which often ends abruptly in a rounded off tip with delicate outlines (Fig. III c,d). Often, the tip is torn and it is not possible to determine how it ends normally. I have observed that other cells bifurcate and send out secondary extensions (Fig. I b,d).

Such detailed descriptions by Sertoli are amazing, considering the crude equipment and conditions that he worked under at the time. To put his work in perspective, it is helpful to consider the time frame of events in microscopy and histology in the 19th century that is shown in Table 1.1. Thus, by the time Sertoli published his first paper in 1865, most of the accepted standard methods of histology were lacking: fixation of tissue, embedding, sectioning with microtomes, and routine histological stains. In the year that Sertoli published his last manuscript as a professor, 1886 [19], major breakthroughs in

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Rex A. Hess and Luiz R. França TABLE 1.1 Significant Scientific Discoveries Surrounding the Period of Sertoli

1839

Theodor Schwann presented his famous “cell theory.”

1839

The 3- × 1-inch glass microscope slide was established as a standard by the Microscopical Society of London.

1840

The first commercial glass coverslips were used.

1841

Kölliker reported that spermatozoa arose from cells within the testis.

1850

Leydig’s article on interstitial cells is published.

1850s Clark’s alcohol-acetic solution and Müller’s solution for fixation were revealed. 1855

Water immersion lens first displayed at the Paris Exposition.

1858

Carmine stain first used by Joseph Gerlach.

1859

Butlerov discovered formalin.

1860s Wilhelm Waldeyer first used aniline dyes, one called Paris blue. 1863

Waldeyer was first to stain histological sections with an extract that became hematoxylin.

1864

Fromman first demonstrated the use of silver for the identification of axons.

1865

Sertoli’s paper on “branched cells” of the testis is published.

1869

Theodor Klebs first used melted paraffin to support a tissue block while sectioning.

1870s First standard microscopical slides used for teaching. 1873

Ernst Abbe published his work on the theory of the microscope, which explained the difference between magnification and resolution. His formula was used to calculate resolution.

1873

Ernst Leitz microscope is introduced with a revolving mount (turret) for five objectives.

1879

Carl Zeiss Jena produced its first oil immersion objective in 1880, designed by Ernst Abbe.

1879

Walther Flemming discovered mitosis.

1880

August Kohler determined the optimum spacing for the light source and the condenser, which would produce sharper images; thus, was born Kohler illumination.

1880s First electrical illumination, rather than sunlight, is reflected off a substage mirror. 1885

The first sliding microtome was developed.

1886

The first rotary microtome was developed.

1886

The substage condenser was developed.

1886

Ernst Abbe introduces the apochromatic objective lens, bringing the red, yellow, and blue colors into one focus, and requiring numerous lens elements.

1886

Ehrlich invented a stable solution of hematoxylin with a long shelf life.

1886

Benda introduced iron-hematoxylin techniques.

1893

Blum tested formalin as a fixative, and Carl Weigert introduced it as routine preservative for tissues.

1894

Zenker’s fixative became available.

1896

“Sudan” stains for lipids introduced by Daddi.

1897

Bouin’s fixative became available.

microscopic technique and instrumentation were just coming on the scene, such as Kohler illumination and Ernst Abbe’s apochromatic lenses, which became available just before Sertoli retired. Despite these handicaps in technology, Sertoli worked with whatever was available and carried the observations to the limits of current microscopic resolution. Thus, we must conclude that Sertoli was an exceptional and determined student, with a keen skill for observation and capable of having original thoughts that others of his day were not giving consideration.

VI. COMPLETING THE MANUSCRIPT Before Sertoli completed the first draft of his manuscript, he probably had numerous discussions with his adviser, Professor Oehl. Professor Eusebio Oehl had just founded the first Institute of Physiology at the University of Pavia in 1861. His research was not focused on the testis and, at the time that he mentored Sertoli and Golgi, he was studying extracted human saliva and salivary ducts. Thus, we must assume that Sertoli’s focus on the testis may not have been top priority for his major adviser. On the other hand, in the late 1800s, every observation under the microscope was something new and it is not inconceivable to think that the adviser would recommend that each new student study a different organ. Regardless of the reason why he studied the testis, Sertoli was making major discoveries that would contradict the published literature and this must surely have made his professor either very excited or very worried. The last figure in Sertoli’s manuscript was a low magnification of a seminiferous tubule cross section showing germ cells and even spermatozoa associated with the branched cells that contained lipid droplets (Fig. 1.6). He had a great desire to observe the borders of the branched cell, and in his final experiment he tried something new [10]: “… in some sections treated with a weak solution of ammonia, I have seen that quite definite dark borders, limit the basal extremity of the branched cells ….” After these remarks, he showed restraint by pointing out the limits of his observations. He ended the paper with his conclusions from numerous observations. One of the most important of which was his conclusion that “it is not likely that these cells produce the spermatozoa …”[10], for which he gave three arguments: (1) Spermatozoa have been observed in the extension of only a very few cells. (2) The spermatozoa are found only in the extensions, where it would be possible for them to have

Chapter 1 History of the Sertoli Cell Discovery

FIGURE 1.6 Drawing taken from the original paper (Fig. VI) of Sertoli [15].

entered accidentally, and I have never seen spermatozoa inside the cells. (3) The formation of the spermatozoa would be consistent neither with the form of the branched cells, which are different from the seminiferous cells, the real progenitors of the spermatozoa, nor with their constant position inside the tubule, nor their tendency to enclose the seminiferous cells among their branches, nor finally their communication with one another through the extensions.

Yet, in the manner of an honorable scientist, he said he could not categorically deny the possibility, but that he did not think the branched cells produced spermatozoa. Nevertheless, with the insight of a keen scientist, Sertoli ended the paper with a suggestion that the “function of the branched cells is linked to the formation of spermatozoa.” This comment along with that on page 1 of the manuscript regarding “mother cells” indicates that Sertoli was indeed the first to suggest that the “branched cell” served as a “cellule madri” or “mother cell” or “sustentacular cell.” It is likely that Sertoli made significant progress in writing the manuscript during 1864. The entire process of manuscript preparation, rewrite, submission, review, resubmission, and printing sometimes takes nearly 1 year nowadays, so it is reasonable to picture the same process taking more than 1 year in the 1800s. However, the time from submission to publication may have been considerably shorter than today, because every observation in the 1800s was publishable and the number of capable reviewers for any subject was limited. Therefore, it is likely that Sertoli completed his major observations and writing in late 1863 or early 1864 and submitted the manuscript for publication prior to graduation in 1865. There has been no mention of why Sertoli was the single author on the paper, even though it is reasonable to assume that his research professor approved of the work in his laboratory. Until about 1950, it was common to see single-author papers or papers with just two authors. This may reflect the fact that much of

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the research performed during the late 1800s and early 1900s did not require collaboration and involved the use of simple tools of investigation. Research efforts back then required a tremendous personal endeavor. Furthermore, until the late 20th century, the evaluation of professorships did not depend on counting the total number of publications. The first manuscript is always very special. A young scientist will dream of the day when his or her own name appears beneath the title of a manuscript. Sertoli may have written the title several times, but the published title had a very specific focus: “Dell’ esistenza di particolari cellule ramificate nei canalicoli seminiferi del testicolo umano,” or as interpreted: “On the existence of special branched cells in the seminiferous tubules of the human testis.” He restricted the study to just one cell type, although he had observed all of the germ cells, had noted what is now called stages of the seminiferous epithelium [20], and even recognized the beginnings of a “wave” of spermatogenesis.

VII. AFTER GRADUATION Sertoli graduated in 1865 and traveled to the University of Vienna to study with Ernst Wilhelm von Brücke, a physiologist [3]. In 1866 he returned home briefly to defend his country in the war between Prussia and Austria. After the war was over, he stayed in the army for a while before returning to science. In 1867 he went to work in the laboratory of Ernst Felix Immanuel Hoppe-Seyler in Tübingen (not yet part of Germany). Throughout his career, Sertoli’s research studies focused on many different organ systems other than male reproduction. At different times, his studies included the following: the lymphatic system, lungs, coccygeal gland, nutrition, kidney, tactile hairs, and smooth muscle [12]. After a series of lectures at the Politecnico in Milan in 1870, he was given a professorship at the Advanced Royal School of Veterinary Medicine in Milan [3]. From 1871 to 1880, Sertoli was professor and chair of anatomy and physiology, and then from 1880 to 1907, he was chair of physiology. It was reported by Negrini [3] that in 1900 at the Anatomy Congress in Pavia, the works of numerous eminent Italian and foreign scientists were presented on a series of microscopes, and one microscope held the label “Cells of Sertoli.” By this date, others were also referring to the branched cells in association with Sertoli’s name [21, 22]. In the fall of 1906, a year before Sertoli retired as professor, we can easily imagine that Golgi’s name

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Rex A. Hess and Luiz R. França

was being mentioned rather frequently in the cafés surrounding the University of Milano. Golgi had been nominated every year since 1901 for the Nobel Prize in Physiology and Medicine. Although there is no documentation that Sertoli and Golgi were long-term friends or anything more than fellow students in the same laboratory, we can imagine that Sertoli may have discussed this important event with his colleagues in Milano. We have no record that Sertoli and Golgi corresponded, but it is easy to imagine that Sertoli congratulated Golgi after he was awarded the Nobel Prize in 1906. Golgi went on to become one of the most famous scientists in neurobiology and cell structure. Every student of histology will remember the name “Golgi” even if they do not always remember the name “Sertoli.” Yet Golgi surely thought about the fact that von Ebner [22] had named the branched cells the “branched cells of Sertoli,” whereas his own name was only associated with a cytoplasmic organelle. Sertoli retired the year after Golgi received the Nobel Prize. He returned to his hometown of Sondrio, due to an illness, and there he lived until his death on January 28, 1910. Sertoli never married and devoted his adult life to teaching and his research.

a primary role in preparing spermatozoa to reach and fertilize the egg [23]. Other misconceptions were prevalent during this transition period. For example, Bardeleben in 1897 thought that the branched cells were derived from Leydig cells [24] and it was not until the 1940s that this idea was replaced [25]. Also, Sertoli implied [15] and von Ebner [26] agreed that the branched cells were syncytial. La Vallette, however, insisted that they were individual [27], and it was several decades before this argument was settled. Lonnie Russell [28] pointed out, in the first edition of this book, that early electron microscopy did not detect the cell boundary, but in 1956, Don Fawcett was able to demonstrate the Sertoli cell membrane with high resolution [29]. In 1878 Sertoli published a statement that the branched cells, which he now called cellule fisse or fixed cells (Table 1.2), no longer divided in the adult testis [30]. It was not until 1963 that this observation was actually accepted [31], and a more detailed understanding of Sertoli cell proliferation and maturation did not come until the 1980s [32]. It is amazing that more than a century was necessary for new and solid information on Sertoli cell function to be published. In this regard, only at the end of the 1960s did scientists in the field show clearly the existence of the blood–testis barrier (now called the Sertoli

VIII. TRANSITION TO THE MODERN SERTOLI CELL Because it would have been difficult to characterize Sertoli and germ cells morphologically, just as it is difficult even today if the tissues are not well fixed and embedded, several decades after the discovery of the Sertoli cell, scientists were still debating the origin and function of the cell [21, 23]. The only common point was that the Sertoli cell had an important role in supporting germ cells, which was based on the strong evidence provided by Sertoli in his 1865 publication. In this regard, although the cyclical changes in the cell and spermatogenesis were being noted at the end of the 19th century, it was strongly claimed that Sertoli cells originated from germ cells or that part of the Sertoli cell population gave rise to germ cells and formed other Sertoli cells, in an amitotic process. In this way, it appears that Sertoli cells were originally defined as what we now characterize functionally as stem cells. Among other functions that they were ascribing for Sertoli cells at that time, we can cite nutrition, secretions, dehydration of germ cells, shaping of the sperm nucleus, formation of the spermatid bundles, and spermiation of germ cells. During this period, many researchers strongly suggested that the Sertoli cell had

TABLE 1.2 Significant Discoveries by Enrico Sertoli that Are Associated with Spermatogenesis Cellule ramificate (branched cells) of the seminiferous tubules, later called cellule fisse or fixed cells [30, 42]; later called branched cells of Sertoli by von Ebner [22], and finally Sertoli cells by Hanes in 1910 [43]. Cellule mobili (germ cells) that form spermatozoa; the first to note “mobility” of these germ cells within the seminiferous epithelium. Noted maturation of germ cells was not uniform in the seminiferous epithelium [2], thus hinting of stages; however, Sertoli always gave credit to von Ebner for discovery of the cycle of the seminiferous epithelium [2]. Cellule germinative or spermatogonia. Spermatozoa were derived from “nematoblasti” or spermatids. Noted “branched cells” were highly resistant to chemical digestion compared to germ cells. Recognized spermatid differentiation, tail formation, nuclear elongation, formation of head, and the cytoplasmic droplet. Three stages of spermatocyte (cellule seminiferi) development [30]. Two types of spermatogonia. A spermatogenic wave [30]. Cytoplasmic bridges between germ cells [30]. Sertoli cells do not divide in the adult testis, in contrast to germ cells [19].

Chapter 1 History of the Sertoli Cell Discovery

11

cell barrier) [33–35]. This barrier appears only after the formation of tight junctions between adjacent Sertoli cells and is responsible for the formation of the basal and luminal compartments of the seminiferous epithelium, the former being essential for the development of young spermatocytes and spermatids. Sertoli’s publication in 1865 was so convincing that scientists rather quickly gave the cell his name. This acceptance probably allowed others to continue their focus on spermatozoa and their origin in the testis, the real interest of those studying male reproduction at that time. As such, other topics became more important to establish (Table 1.2), such as the development of germ cells and their association in stages, as well as the duration of the cycle of the seminiferous epithelium [20, 36–38]. During this post-Sertoli period, endocrine aspects of testis function and diseases involving the Sertoli cell became topics of serious inquiry [10]. From 1865 until 1955, the average number of papers published with Sertoli as a key word was approximately one per year, with most of the growth starting in 1952 with the classical publication by Leblond and Clermont defining the stages of the cycle of the seminiferous epithelium [20] (Fig. 1.7). This transition period included two world wars, which of course reduced the ability to perform research, but at the same time allowed the development of significantly improved technology, for example, the transmission electron microscope, which promoted a burst in new research involving the Sertoli cell. For that reason, from 1966 to 1970 the number of papers per year more than doubled to 31 per year. It is interesting to note that the rate doubled again from 1971 to 1975, the period during which Lonnie Russell began publishing his research. From the time of his first publication in 1973, the number of Sertoli cell publications per year has grown by more than threefold. The number of Sertoli cell papers published per year has continued to increase and as of 2002 had reached more than 400 papers per year.

IX. POSTSCRIPT FIGURE 1.7 (A) Graph of the total number of publications retrieved from the National Library of Medicine via PubMed that use the term Sertoli cell in the title, abstract, or as a mesh term. (B) Graph showing the mean number of Sertoli cell papers per year for each period shown.

It was reported by Brian Setchell [12] that Sertoli was a quiet man, “… speaking little in the laboratory, sometimes passing whole days in silence.” Sertoli was a stout man [12], of “medium height, stocky, robust, and with a large head.” Such a personal description provides a striking contrast to the modern-day person we now call “Mr. Sertoli Cell.” Lonnie Russell was never quiet. He was a conversationalist and without doubt would have died much younger if he had to spend even one day without speaking a word.

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Rex A. Hess and Luiz R. França

Lonnie was neither short nor stocky, but we might label him robust with a large head. Lonnie detested bureaucracy and departmental business, whereas Sertoli became department chair early in his career and served until shortly before his death. Furthermore, whereas Sertoli never married, Lonnie crossed that bridge numerous times. Lonnie was tall and a very large man, whose stature was a command performance at every scientific meeting. He loved to debate and never shirked from raising a touchy question following the presentation of a paper at a meeting. So, it is interesting that the modern-day scientist who truly immortalized the name Sertoli was personally a contrast in many ways to the real Professor Sertoli. However, the two men did share many wonderful traits, not the least of which was that they studied the same cell. Both scientists loved microscopy, which is noted in the detailed drawings by Sertoli [15, 30] and the exceptional transmission electron microscopy by Russell [1, 39–41]. If there is an afterworld, surely the two “Sertologists” will still be searching for the holy grail, to prove the omnipotent theory [1] that Sertoli is the “mother cell.” The following is a poem written by Luiz Renato de França and dedicated to the memory of Lonnie D. Russell: From Sertoli to Lonnie: “Let my children go” Since the beginning of your life I took care of you Knowing your needs and my limits I grew up and developed as much as I could At some critical moments I took you in my arms And you deeply touched my soul Now, my dear friend It is time for you to go Good luck on your Journey And I will always be here for you

References 1. Russell, L. D., and Griswold, M. D., eds. (1993). “The Sertoli Cell.” Cache River Press: Clearwater, FL. 2. Ober, W. B., and Sciagura, C., (1981). Leydig, Sertoli and Reinke: Three anatomists who were on the ball. Pathol. Ann. 16, 1–13. 3. Negrini, F. (1910). Enrico Sertoli. Commemorazione del Prof. Francesco Negrini. Clinica Verterinaria 33, 146–161. 4. Negrini, F., et al. (1908). Onoranze al Prof. Enrico Sertoli. Clinica Veterinaria 31, 49–62. 5. Usuelli, F. (1934/1935). Enrico Sertoli (1842–1910). Ann. Veterinario Italiano 13, 455–461. 6. Taddia, C. (1985/1986). Istofisiologica dell cellule del Sertoli nei mamiferi. In “Facolta di Medicina Veterinaria.” Universita degli Studi di Milano, Italy.

7. Belloni, L. (1915). Enrico Sertoli in la medicina a Milano dal settecento al 1915. Storia Milano. Fondazione Treccani degli Alfieri, 1862 16, 1028. 8. Zanobio, B. (1970). Sertoli, Enrico. In “Dictionary of Scientific Biographies. American Council of Learned Societies” (C. C. Gillispie, ed.), pp. 319–320. Charles Scribner’s Sons, New York. 9. Internet websites used in the search for information on Sertoli: http://www.ibmsscience.org/history_zone/histology.htm http://www.geocities.com/hotsprings/2615/medhist/micro.html http://www.cas.muohio.edu/%7Embi-ws/microscopes/ index.html http://www.whonamedit.com/doctor.cfm/556.html http://www.sertoli.com/pages/568089/index.htm http://lsvl.la.asu.edu/paperproject/Microsopehistory/ http://www.az-microscope.on.ca/history.htm http://www.omni-optical.com/micro/sm101.htm http://www.nobel.se/medicine/articles/golgi/ http://www.arsmachina.com/micromenu.htm 2003. 10. Setchell, B. P. (1984). Male reproduction. In “Benchmark Papers in Human Physiology Series” (L. L. Langley, ed.), pp. 10–20. Van Nostrand Reinhold Company, New York. 11. Setchell, B. P. (1986). Some important contributors to our understanding of the male reproductive system: Monesi, Sertoli, Spallanzani and Aubry. Exerpta Medica Int. Cong. 716, 1–12. 12. Setchell, B. P. (1993). Foreword. In “The Sertoli Cell” (L. D. Russell and M. D. Griswold, eds.), pp. v–vi. Cache River Press, Clearwater, FL. 13. Andraos, J. (2002). “Nobel Prizes in Physiology & Medicine,” p. 12. Department of Chemistry, York University, Toronto. 14. Kölliker, A. (1863). “Handbuch der Gewebelehre des Menschen,” 4th ed. W. Logier, Berlin. 15. Sertoli, E. (1865). Dell’esistenza di particolari cellule ramificate nei canalicoli seminiferi del testicolo umano. Morgagni 7, 31–40. 16. Gernsheim, H. (1986). “The Concise History of Photography.” Thames & Hudson, London. 17. Kölliker, A. (1841). “Die samenfaden entwickeln sich aus oder in zellen, die sich zur zeit der geschlechtsreife oder der brunst in den hoden bilden, durch vorgänge, die den bei der entwicklung der thierischen elementartheile statt fidenden analog sind, von der gewöhnlichen entwicklung der thiere aus eiern dagegen bedeutend abweichen, in Beitrage zure Kenntnis der Geschlechtverhältnisse und der Samenflussigkeit wirbelloser Thiere.” W. Logier, Berlin. 18. Ehrlich, P. (1886). Fragekasten. Zeit Mikroskopie 3, 150. 19. Sertoli, E. (1886). Sur la caryokinése dans la spermatogénese. Arch. Italiennes Biologie 7, 369–375. 20. Leblond, C., and Clermont, Y. (1952). Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. NY Acad. Sci. 55, 548–573. 21. Regaud, C. (1897). Sur la morphologie de la cellule de Sertoli et sur son role dans la spermatogénese chez les mammiféres. Compt. Rend. Anat. Paris 1, 21–31. 22. Ebner, V. V. (1888). Zur spermatogenese bei den säugethieren. Arch. Mikrosk. Anat. 31, 236–292. 23. Loisel, G. (1901). Origine et role de la cellule de Sertoli dans la spermatogenese. Compt. Rend. Soc. Biol. Paris IIs(iii), 974–977. 24. Bardleben, K. V. (1897). Die zwischenzellen des säugtierhodens. Anat. Anz. 13, 529–536. 25. Gillman, J. (1948). The development of the gonads in man, with a consideration of the role of fetal endocrines and the histogenesis of ovarian tumors. Contrib. Embryol. 210, 11–131. 26. Ebner, V. V. (1902). Mannliche geschlechtsorgne. In “Handbunch der Gewebelchre des Menschen” (A. Kolliker, ed.), p. 402. W. Engelman, Leipzig, Germany.

Chapter 1 History of the Sertoli Cell Discovery 27. La Vallette, S. V. (1878). Über die genese der samenkörper. Arch. Mikrosk. Anat. 15, 261–314. 28. Russell, L. D. (1993). Form, dimensions, and cytology of mammalian Sertoli cells. In “The Sertoli Cell” (L. D. Russell and M. D. Griswold, eds.), pp. 1–37. Cache River Press, Clearwater, FL. 29. Fawcett, D. W., and Burgos, M. (1956). The fine structure of the Sertoli cells in the human testis. Anat. Rec. 124, 401–402. 30. Sertoli, E. (1878). Sulla sturttura dei canalicoli seminiferi dei testicolo. Arch. Sci. Med. 2, 107–146, 267–295. 31. Attal, J., and Courot, M. (1963). Développement testiculaire et établissement de la spermatogenése chez le taureau. Ann. Biol. Animale Biochim. Biophys. 3, 219–241. 32. Orth, J. (1982). Proliferation of Sertoli cells in fetal and postnatal rats: A quantitative autoradiographic study. Anat. Rec. 203, 485–492. 33. Kormano, M. (1967). Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat. Histochemie 9, 327–338. 34. Setchell, B. P., Voglmayr, J. K., and Waites, G. M. (1969). A blood–testis barrier restricting passage from blood into rete testis fluid but not into lymph. J. Physiol. (Lond.) 200(1), 73–85.

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35. Dym, M., and Fawcett, D. W. (1970). The blood–testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3(3), 308–326. 36. Brown, H. H. (1885). On spermatogenesis in the rat. J. Microsc. Sci. 25, 343–370. 37. Roosen-Runge, E. C., and Giesel, Jr., L. O. (1950). Quantitative studies on spermatogenesis in the albino rat. Am. J. Anat. 87, 1–23. 38. Ortavant, R. (1954). Contribution à l’étude de la durée du processus spermatogénétique du Bélier à l’aide du 32P. Soc. Biol. Paris CR 148, 37–55. 39. Russell, L., and Clermont, Y. (1976). Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules. Anat. Rec. 185(3), 259–278. 40. Russell, L. D. (1979). l Further observations on tubulobulbar complexes formed by late spermatids and Sertoli cells in the rat testis. Anat. Rec. 194(2), 213–232. 41. Russell, L. D. (1980). Sertoli–germ cell interactions: A review. Gamete Res. 3, 179–202. 42. Walker, C. E., and Embleton, A. L. (1906). On the origin of the Sertoli or foot-cells of the tesis. Proc. Royal Soc. Lond. 1(xxviii), 50–52. 43. Hanes, F. M. (1910). The biological significance of the Sertoli cells. Proc. Soc. Exp. Biol. Med. 7, 136–137.

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C H A P T E R

2 Perspective on the Function of Sertoli Cells MICHAEL D. GRISWOLD Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington

I. INTRODUCTION II. THE VIEW IN 1993 III. CHANGES TO OUR VIEW IN THE LAST TEN YEARS IV. IMPORTANT QUESTIONS REMAIN V. THE “PERMISSIVE” VIEW VI. THE NEXT TEN YEARS References

II. THE VIEW IN 1993 We started our discussion in 1993 with the assumption originally stated by Enrico Sertoli that Sertoli cells are “linked to the production of spermatozoa.” The extent to which that link is mandatory was unknown. Clearly, both testosterone and follicle-stimulating hormone (FSH) acted on the Sertoli cells and the result of those actions had measurable impacts on spermatogenesis. At the time, some investigators believed that Sertoli cells regulated virtually all aspects of germ cell development (the dogmatic Sertologists), whereas others believed that germ cell development was affected by Sertoli cell functions but that most germ cell development appeared to be autonomous (germ cell worshiping cult).

I. INTRODUCTION More than 10 years have passed since Lonnie Russell and I sat down with a few beers at my home in Idaho and wrote the Preface to The Sertoli Cell [1]. This book was the first major work dedicated to our favorite cell. We pointed out that there are “dogmatic, religious Sertologists” who believed in the so-called “omnipotent” Sertoli cell theory and that there was a “germ cell worshiping cult” who believed in only minimal involvement by Sertoli cells. The reaction of my colleagues to that irreverent and impertinent preface was interestingly mixed. Some thought that it was outside of the bounds of serious science, whereas others thought it was the most useful part of the book. Despite the light tone of our writing, we did raise serious questions relating to the function of Sertoli cells as we understood them in 1993. Have any or all of those questions been answered in the intervening decade? Have new questions surfaced with the new research? SERTOLI CELL BIOLOGY Edited by M. K. Skinner and M. D. Griswold

III. CHANGES TO OUR VIEW IN THE LAST TEN YEARS The last decade has not been kind to the league of dogmatic Sertologists of which both Lonnie and I were charter members. Two general categories of experiments including germ cell culture and germ cell transplantation have established that most germ cell development is quite autonomous. In 2002 the Dym laboratory published convincing evidence that they had established a cell line that underwent meiosis and spermatogenesis in vitro in the absence of Sertoli cells [2].

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Michael D. Griswold

They reported the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. Meiosis was apparent but the round spermatids never developed tails. Further studies will be necessary to determine if these cells can function as “normal” haploid germ cells, but the work clearly illustrates the potential for germ cell development in the absence of somatic cell input. Several other studies on invertebrates and lower vertebrates such as teleosts have also shown successful spermatogenesis in vitro in the absence of somatic cells [3]. Germ cell transplantation has been used to demonstrate that spermatogonial stem cells can be maintained in culture in the absence of Sertoli cells for several months [4]. Transplantation of the cultured germ cells into recipients demonstrated that functional stem cells were present in the cultures. Germ cell transplantation has also provided evidence that the germ cells in the testis are in control of their own fate. The transplantation of rat germ cells into the mouse testis showed that rat stem cells can develop and go through meiosis and form relatively normal elongated spermatids even though they interacted with mouse Sertoli cells [5]. In addition, the time required for the completion of this process in each mammalian species is unique and fixed. Franca et al. [5] used the technique of spermatogonial transplantation to ask the question as to which cell type(s) determined the rate at which germ cells proceeded through spermatogenesis. Rat germ cells were transplanted into a mouse testis, and the mouse was euthanized 12.9–13 days after administration of a single dose of [3H]thymidine. The investigators found that two separate timing programs existed for germ cell development in the recipient mouse testis: one of rat and one of mouse duration. Rat germ cells that were supported by mouse Sertoli cells always differentiated with the timing characteristic of the rat and generated the spermatogenic structural pattern of the rat, demonstrating that the timing of the cell differentiation process of spermatogenesis was regulated by germ cells alone.

IV. IMPORTANT QUESTIONS REMAIN Taken together the experiments described above show that much of the development of germ cells can occur relatively independent of Sertoli cells and that the timing of the process is intrinsic to the germ cells. So where does that leave the views of the “dogmatic Sertologist”? Successful and complete spermatogenesis resulting in sperm capable of fertilization in the absence of Sertoli cells still has not been demonstrated

in mammals. So, although the germ cells apparently have a great deal of developmental autonomy, there still appears to be a Sertoli cell requirement for complete and successful spermatogenesis. In the models for germ cell–Sertoli cell interactions that Lonnie Russell and I proposed in 1993 we suggested that important molecular communications occurred between germ cells and Sertoli cells but we pointed out that not one signal from either of these cell types had been identified with certainty. Although a number of growth factors have been shown to affect germ cell–Sertoli cell interactions in vitro, and growth factors that affect stem cells in vivo have been found, the identification of a distinct signaling system between Sertoli cells and germ cells undergoing spermatogenesis remains elusive.

V. THE “PERMISSIVE” VIEW We also suggested in 1993 that the action of hormones on spermatogenesis may be primarily permissive in nature. This concept of the Sertoli cell acting in a permissive way fits well with the “support cell” or “nurse cell” view and it may be useful to view the major function(s) of Sertoli cells as “permissive.” In his 1994 review article, Sharpe [6] suggested that we view the function of Sertoli cells and their response to hormones as affecting the efficiency of spermatogenesis. Adapting these views to current understanding would be to suggest that Sertoli cells provide the environment, structural organization, and biochemical milieu that allow the efficient but autonomous development of germ cells into spermatozoa. The knockout of several genes that are unique to Sertoli cells in the testis has supported the view of the “germ cell worshiping cult” or the modified “permissive” view. In the past few years, the knockout of the FSH receptor and the FSHβ gene gave emphasis to the permissive nature of FSH actions. The FSH receptor and the FSHβ knockout males were fertile but produced reduced numbers of sperm in a smaller than normal testis [7–9]. In Table 2.1 a number of known Sertoli cell products whose genes have been knocked out by naturally occurring mutations or by homologous recombination are listed. Note that there is consistency in the description of the spermatogenic phenotypes. The elimination of these genes associated with the function of Sertoli cells, in general, leads to lowered sperm counts but does not totally block spermatogenesis. The spermatogonial phenotype of the androgen insensitivity or insufficiency models still provides the “dogmatic Sertologists” some solace. The action of testosterone on spermatogenesis is more difficult to visualize as “permissive” because, in the absence of

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Chapter 2 Perspective on the Function of Sertoli Cells TABLE 2.1 Examples of Naturally Occurring and Engineered Knockout of Genes Expressed in Sertoli Cells and the Resulting Spermatogenic Phenotype Gene

Phenotype

Reference

FSH receptor

Fertile, underdeveloped testis with 50% reduction in Sertoli cells

[16]

Inhibin α

Initially develops normally, but ultimately develops gonadal stromal tumor

[16]

Transferrin

Fertile but with abnormalities in spermatogenesis

[17]

Clusterin or SGP-2

Fertile with minor abnormalities in spermatogenesis

[18]

Cyclic protein 2 or cathepsin L

Fertile with increased numbers of atrophic tubules

[19]

Pem- androgen regulated homeobox

Fertile and normal

[20]

Phosphatidylserine synthase 2

Lowered fertility

[21]

Fyn tyrosine kinase

Fertile, a significant reduction in testis weight and degenerated germ cells were observed at 3 and 4 wk of age

[22]

Desert hedgehog

Anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells

[23]

Bclw

Initially fertile with progressive degeneration

[24]

Tyro 3, Axl, and Mer tyrosine kinases

Mice lacking any single receptor, or any combination of two receptors, are viable and fertile, but male animals that lack all three receptors progressively produce no mature sperm

[25]

Gata 1

Tissue specific knockout, no altered phenotype, normal spermatogenesis

[26]

response to testosterone, a condition such as is seen in the testicular feminized mutant (tfm) mice and humans, spermatogenesis is clearly blocked at meiosis [10]. Note that the interpretation of the action of androgens in the tfm condition is complicated by cryptorchidism. The phenotype of the leuteinizing hormone (LH) receptor knockout differs in that spermatogenesis is blocked at the round spermatid stage [11]. In other models blocking androgen action such as EDS-treated rats, or hypophysectomy, some spermatids are found [12]. The abundance of evidence can be interpreted to indicate that a testosterone-dependent signal or product from Sertoli cells is mandatory for successful spermatogenesis. Sertoli cells may be essential for several processes but the clearest effect is on the elongation of spermatids and the formation of sperm tails. A recent publication using gene chip technology looked at the action of testosterone on the GnRH-deficient mutant mouse (hypogonadal; hpg). Hpg mice age 35–45 days were injected subcutaneously with 25 mg testosterone propionate, and the animals were sacrificed 4, 8, 12, and 24 hr after treatment. Although the expression of many genes was changed, at early time points the expression of more genes was depressed rather than stimulated, suggesting that a primary effect of androgens may be to inhibit the expression of genes that maintain the prepubertal condition [13]. The role of the Sertoli cells in testis formation is much easier to reconcile with the “dogmatic Sertologists” view. The initial expression of Sry and other genes involved

in testis formation is clearly an important function of Sertoli cells and a prerequisite for the formation of a testis [14]. Other potential functions of Sertoli cells in early testis development could include the inhibition of meiosis and the regulation of the mitosis of gonocytes. The fetal testis of the mouse (presumably the Sertoli cells) produces a putative inhibitor of meiosis, and germ cells that are exposed to it develop as prospermatogonia [15]. None of the agents that allow Sertoli cells to regulate meiosis and mitosis have been identified as yet.

VI. THE NEXT TEN YEARS Predictions about the contents of a third book dedicated to the Sertoli cells are almost entirely speculation, but some areas of progress can be anticipated based on current technology. If there is a third book dedicated to Sertoli cells and if it is published about 10 years from now, we will have a nearly complete knowledge of the genes expressed in Sertoli cells throughout their development and we will know what many of these genes do (at least at the physiological level). We will probably have candidates for a complete picture of genes that influence testis determination, suppression of meiosis in the embryonic and prepubertal mammals, stimulation of meiosis during puberty, and for influencing the morphological development of gonocyte to spermatozoa. This knowledge will provide for much

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Michael D. Griswold

greater insight into genetic diseases that affect fertility. We will probably have the capability to maintain most germ cells in culture in the absences of Sertoli cells and then to transition them to haploid cells in vitro. We will have in vitro systems that will produce gametes capable of successful fertilization. These systems will enable the efficient and routine genetic manipulation of gametes. What will we still lack in 10 years? It is unlikely that we will have a contraceptive that is targeted to the Sertoli cells. Several factors have led to this prediction including the evidence presented in Table 2.1 that interfering with Sertoli cell functions may impede the efficiency of spermatogenesis but may not cause complete aspermatogenesis. It is likely that the predictions made here will be about as accurate as my annual picks in the NCAA basketball pool, where I usually end up last. However, one prediction can be made with relative assurance of accuracy. Investigators will continue to be fascinated by the beauty and complexity of Sertoli cells, and there will be further pursuit of the original premise of Enrico Sertoli that, the cells are somehow linked to the production of spermatozoa.

References 1. Russell, L. D., and Griswold, M. D., eds. (1993). Preface. In “The Sertoli Cell,” pp. 365–390. Cache River Press, Clearwater, FL. 2. Feng, L. X., et al. (2002). Generation and in vitro differentiation of a spermatogonial cell line Science 297, 392–395. 3. Saiki, A., et al. (1997). Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes. Dev. Growth Differ. 39(3), 337–344. 4. Nagano, M., et al. (1998). Culture of mouse spermatogonial stem cells. Tissue Cell 30(4), 389–397. 5. Franca, L. R., et al. (1998). Germ cell genotype controls cell cycle during spermatogenesis in the rat. Biol. Reprod. 59(6), 1371–1377. 6. Sharpe, R. M. (1994). Regulation of spermatogenesis. In “The Physiology of Reproduction,” 2nd ed. (E. Knobil and J. D. Neill, eds.), pp. 1363–1433. Raven Press, New York. 7. Kumar, T. R., et al. (1997). Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat. Genet. 15(2), 201–204. 8. Dierich, A., et al. (1998). Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor

9.

10.

11.

12.

13. 14. 15. 16. 17.

18.

19.

20.

21.

22. 23.

24. 25. 26.

leads to aberrant gametogenesis and hormonal imbalance. Proc. Natl. Acad. Sci. USA 95(23), 13612–13617. Wreford, N. G., et al. (2001). Analysis of the testicular phenotype of the follicle-stimulating hormone beta-subunit knockout and the activin type II receptor knockout mice by stereological analysis. Endocrinology 142(7), 2916–2920. Fritz, I. (1978). Sites of actions of androgens and follicle stimulating hormone on cells of the seminiferous tubule. In “Biochemical Actions of Hormones” (G. Litwack, ed.), pp. 249–278. Academic Press, New York. Zhang, F. P., et al. (2001). Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol. Endocrinol. 15(1), 172–183. Kerr, J. B., Maddocks, S., and Sharpe, R. M. (1992). Testosterone and FSH have independent, synergistic and stage-dependent effects upon spermatogenesis in the rat testis. Cell Tissue Res. 268(1), 179–189. Sadat-Ngatchou, P., McLean, D. J., and Griswold, M. D. (in press). Biology of Reproduction. Lovell-Badge, R. (1992). The role of Sry in mammalian sex determination. Ciba Found. Symp. 165, 162–182. McLaren, A. (1995). Germ cells and germ cell sex. Philos. Trans. R. Soc. Lond. B Biol. Sci. 350(1333), 229–233. Matzuk, M. M., et al. (1992). Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 360(6402), 313–319. Sylvester, S. R., and Griswold, M. D. (1994). The testicular iron shuttle: A “nurse” function of the Sertoli cells. J. Androl. 15(5), 381–385. Bailey, R. W., et al. (2002). Heat shock-initiated apoptosis is accelerated and removal of damaged cells is delayed in the testis of clusterin/ApoJ knock-out mice. Biol. Reprod. 66(4), 1042–1053. Wright, W. W., et al. (2003). Mice that express enzymatically inactive cathepsin L exhibit abnormal spermatogenesis. Biol. Reprod. 68(2), 680–687. Pitman, J. L., et al. (1998). Normal reproductive and macrophage function in Pem homeobox gene-deficient mice. Dev. Biol. 202(2), 196–214. Bergo, M. O., et al. (2002). Defining the importance of phosphatidylserine synthase 2 in mice. J. Biol. Chem. 277(49), 47701–47708. Maekawa, M., et al. (2002). Fyn tyrosine kinase in Sertoli cells is involved in mouse spermatogenesis. Biol. Reprod. 66(1), 211–221. Pierucci-Alves, F., Clark, A. M., and Russell, L. D. (2001). A developmental study of the Desert hedgehog-null mouse testis. Biol. Reprod. 65(5), 1392–1402. Russell, L. D., et al. (2001). Spermatogenesis in Bclw-deficient mice. Biol. Reprod. 65(1), 318–332. Lu, Q., et al. (1999). Tyro-3 family receptors are essential regulators of mammalian spermatogenesis. Nature 398(6729), 723–728. Lindeboom, F., et al. (2003). A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse. Nucleic Acids Res. 31(18), 5405–5412.

C H A P T E R

3 Structure of the Sertoli Cell

I. II. III. IV. V. VI.

REX A. HESS

LUIZ R. FRANÇA

Reproductive Biology & Toxicology, Veterinary Biosciences, University of Illinois, Urbana, Illinois

Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil

likely the work of Don Fawcett at Harvard in the 1950s [3, 4] that firmly established the importance of studying Sertoli cell morphology, because from 1951 until 1973, the year that Lonnie Russell published his first manuscript on testicular morphology [5], the number of publications dealing with the Sertoli cell jumped to approximately 440. From 1973 to 1990, about 4000 Sertoli cell papers were published, which is an indication of the recognition of the importance of Sertoli cells in testicular function. Such growth in Sertoli cell interest is a direct result of the tremendous impact that morphological studies, especially those of Dr. Russell, have had on the reproductive sciences.

BRIEF HISTORY FORM AND FUNCTION NUCLEUS AND NUCLEOLUS CYTOPLASM THE SERTOLI (BLOOD–TESTIS) BARRIER MISCELLANEOUS OBSERVATIONS References

I. BRIEF HISTORY The Sertoli cell received its family name in a paper published by von Ebner [1] in which he described the cells as “the cells of Sertoli.” It is amazing that the early scientists were able to deduce the cell’s basic structure so well, especially when consideration is given to the poor resolution of microscopes in the 1800s (see Chapter 1) and the lack of proper fixation, embedding, sectioning, and staining. In fact, Sertoli’s original observations were so intuitive that few scientists at that time bothered to study the cell in great depth, because most everyone accepted his descriptions and went on to other more important topics of the day. Most scientists waited for the improved resolution provided by the electron microscope before returning to the study of the Sertoli cell. From 1865, when Sertoli published his famous observations, until 1950, approximately 85 manuscripts were published with Sertoli cell or other descriptive names for this cell in the title or as key words. Then in 1953 the first paper to observe the testis with the transmission electron microscope was published [2]. However, it was most SERTOLI CELL BIOLOGY Edited by M. K. Skinner and M. D. Griswold

II. FORM AND FUNCTION The total number of Sertoli cells establishes the upper limit of sperm production by the testis [6–11], and spermatogenic efficiency is highly correlated with Sertoli cell support capacity, which is the best indication of Sertoli cell function [12]. Sertoli cell individual volume shows high variation in mammals, varying from approximately 2000–3000 μm3 to 6000–7000 μm3 in mammals already investigated [11–13]. However, paradoxically, it is generally recognized that species in which Sertoli cell volume is high are those with the lowest spermatogenic efficiency. The volume density of Sertoli cells in the seminiferous epithelium also changes considerably in mammals, and the mean figures range from approximately 15% in mice and rabbit

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Copyright 2005, Elsevier Science (USA). All rights reserved.

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Rex A. Hess and Luiz R. França

FIGURE 3.1 (A) Drawing of a reconstructed rat Sertoli cell surface (stage V). Numerous indentations and sheet-like cytoplasmic processes are noted to provide crypts for developing spermatids and recesses for spermatocytes and spermatogonia. (Modified and reprinted from Wong and Russell [139], Copyright 1983 Am. J. Anat., by permission of Wiley Liss, Inc., a subsidiary of John Wiley & Sons, Inc.). (B) Drawing of a Sertoli cell cytoplasm showing the inclusion of elongate spermatid nuclei within deep crypts. (Modified and reprinted from Fawcett [140] by permission of the American Physiological Society.)

to 40% in the woodchuck and humans [11, 13]. As stated for the cell volume, it is also generally noted that species with lower Sertoli cell occupancy in the seminiferous tubule epithelium, such as mice, rabbits, rats, and hamsters, are those with higher Sertoli cell and spermatogenic efficiencies [11]. The Sertoli cell, which is columnar in height (Fig. 3.1) and assumed to always extend from the basement membrane of the seminiferous epithelium to the lumen [13], performs its nurse-like cell functions by extending its cytoplasm in thin arm-like processes in two dimensions and sheet-like or cylindrical processes in three dimensions around the developing germ cells (Fig. 3.2) and by forming specialized junctional complexes that consist of gap and tight junctions, actin filaments, and smooth endoplasmic reticulum [14–20]. Approximately 40% of the Sertoli cell surface (Fig. 3.2) contacts the surface of elongated spermatids [21, 22], which illustrates the extent to which the Sertoli cell stretches its cytoplasm to communicate directly with the developing germ cells. Enrico Sertoli used the term mother cells on the first page of his publication [23], suggesting that this cell type served a unique function in its relationship to the developing germ cells. Indeed, the Sertoli cell cytoplasm is indented by the germ cells (Figs. 3.1–3.4) in every stage of the cycle of the seminiferous epithelium, with certain stages showing tremendous indentation

FIGURE 3.2 Ultrastructural section through a single rat Sertoli cell at a level where the cell surrounds nine step 9 elongating spermatids (GC; 1–9). The Sertoli cell edge is enhanced with black ink to demonstrate the tremendous volume density of its plasma membrane.

Chapter 3 Structure of the Sertoli Cell

21

FIGURE 3.3 Sertoli cell variation within the cycle of the seminiferous epithelium is illustrated with sections from the rat stage V (Type A cell) and late stage VI (Type B cell). In stage V, the elongate spermatid (ES) heads are found deep within crypts of the Sertoli cell (SC), adjacent to round spermatids (RS) and pachytene spermatocytes (PS), with the movement being toward the basement membrane in the Type A cell (arrow). In late stage VI, the elongate spermatids (ES) are found near the tubule lumen, with round spermatids and pachytene spermatocytes. The drawing of a Type B cell shows the Sertoli cell membrane encapsulating spermatid heads near the lumen, with movement of the spermatids in a proluminal direction (arrow).

or deep crypts, as seen in stage V in the rat, and often referred to as Type A Sertoli cells [13]. Type B Sertoli cells are those that support the movement of elongate spermatids toward the lumen, as seen starting in stages VI to VIII in the rat (Fig. 3.3). Thus, spermiation appears to separate these two basic structural features of the Sertoli cell. The cycle of the seminiferous epithelium is also associated with other Sertoli cell changes, the most notable being its relative volume, with volume density being smallest in stages VII and VIII in the rat and greatest in stages XII through XIV [11, 24, 25], which reflects the unique ability of this cell to alter its form and function, without noticeable alteration in its volume [26], throughout the cycle.

III. NUCLEUS AND NUCLEOLUS One of the first features of the Sertoli cell that is taught to new students is the unique appearance of the nucleus and its tripartite nucleolus (Fig. 3.5). In most species, the nucleus is found very near to the base of the cell, if not sometimes appearing to rest on the basement membrane. Fixation and embedding techniques make huge differences in the appearance of the nucleus, with neutral buffered formalin fixation and

paraffin embedding resulting in the most distorted appearance, even making it difficult to recognize some Sertoli cells (Fig. 3.5). Glutaraldehyde provides the optimum fixation, and epoxy resin embedment provides the highest resolution of nuclear detail, even compared to glycol methacrylate. The Sertoli cell nucleus is large (250–850 μm3) and can take on several different shapes depending on the stage of the seminiferous cycle [11] and age of development (Fig. 3.6). From birth to adulthood, the nucleus is often elongated, extending toward the lumen, but there are definite changes in appearance as the testis matures. The early support cells of the seminiferous tubule are often called pre-Sertoli cells because the nuclear appearance differs from the adult form, yet in some species the pre-Sertoli cells still have the typical nuclear features of irregular shape and a prominent nucleolus [27]. The large tripartite nucleolus and the numerous satellite karyosomes or chromocenters [28], which are prominent in the adult Sertoli cell nucleus, are smaller or less abundant in the developing testis (Fig. 3.6). For instance, on day 5 postbirth it is easy to recognize rat Sertoli cell nuclei, because they are the only nuclei located along the basement membrane. Between day 5 and 10 postbirth, however, germ cells migrate to the basement membrane and some Sertoli cell nuclei

FIGURE 3.4 A montage of the typical Sertoli cell. The organelles depicted are for illustrative purposes and not necessarily photographed from the cell shown at lower magnification. In the base of the Sertoli cell, the nucleus (Nu) contains a large nucleolus (Nuc) and satellite karyosomes (Sk). In this region, lipid droplets (Li) are often noted and the Sertoli cell–Sertoli cell tight junctional complexes are found (Jct). The body of the Sertoli cell cytoplasm contains the typical epithelial cell organelles and inclusion bodies: a Golgi apparatus (Go) that is small compared to most secretory cells; an abundance of rough endoplasmic reticulum (RER); smooth endoplasmic reticulum (SER) that is often found adjacent to mitochondria (Mit); Mit are either elongate parallel to microtubules (Mt) or circular with a donut-like shape; glycogen granules can be seen near the nucleus but other inclusions such as multivesicular bodies (MVB) are seen scattered throughout the cytoplasm; lysosomes (Ly) are usually seen near the MVB and typically near the apical border in stages where spermiation has occurred; unique structures called ectoplasmic specializations (Eps) are found adjacent to the spermatid heads; and tubulobulbar complexes (Tub) are seen with higher magnification within the curvature of the late spermatid head and represent a Sertoli cell invagination or penetration by spermatid cytoplasm, which participates in attachment of elongating spermatids to Sertoli cells and in the elimination of excess cytoplasm prior to spermiation.

Chapter 3 Structure of the Sertoli Cell

23

FIGURE 3.5 Recognition of Sertoli cells in histological sections depends on several factors including quality of fixation, the embedding material, and thickness of the section. Shown here are different Sertoli cell nuclei taken from different stages of spermatogenesis in adult mouse testes embedded in different media. (A–E) Mouse testis fixed with Bouin’s, embedded in paraffin, and stained with PAS/hematoxylin. Most nuclei are smaller in size than in the plastic resins. The euchromatin appear washed out, and satellite nucleoli (S) are difficult to recognize next to the nucleolus (Nuc). (F–J) Mouse testis fixed by vascular perfusion with 4% glutaraldehyde, embedded in glycol methacrylate (GMA), and stained with PAS/hematoxylin. The deep nuclear indentations (arrows) are visible, and prominent nucleoli (Nuc) are easily recognized along with the satellite karyosomes (S). (K–O) Mouse testis fixed by vascular perfusion with 4% glutaraldehyde, embedded in epoxy resin for electron microscopy, sectioned 1 μm for light microscopy and stained with toluidine blue. Nuclear indentations are prominent (arrow), and the euchromatin (E) show greater detail than with the GMA embedding.

become oval in shape (Fig. 3.6), which makes their recognition without special markers rather difficult. By day 25, nearly all Sertoli cell nuclei are displaced from the basement membrane by germ cells, and it is not until the mature testis is formed that the nuclei begin to appear along the base again in association with specific stages of spermatogenesis. Pituitary gonadotrophins have been shown to increase nuclear diameter [29].

In the adult testis, nearly all Sertoli nuclei contain deep indentations of their nuclear envelope (Fig. 3.7), which gives the nucleus irregular shapes that have been called pyramidal, triangular and even “planoconvex” [13]. Such invaginations of the nuclear envelop are invested with vimentin intermediate filaments, but it is not known if the filaments help to maintain the structure or if the filaments are better anchored in such

FIGURE 3.6 Sertoli cell nuclei change in appearance and density from early development to adulthood. Shown here are rat testes from day 5 to day 100. SC, Sertoli cell; GC, germ cells. (A–B) Day 5 postnatal SC nuclei (box and arrows) are packed tightly along the basement membrane, with considerable variation in the shape, ranging from triangular to oval. A single prominent nucleolus is present as are small satellite karyosomes that are usually found along the nuclear envelope. Gonocytes GC are located in the center of the seminiferous cords. (C) Day 15 SC nuclei are larger in size and appear more angular than on day 5. Some are displaced from the basement membrane by the migration of GC. Satellite chromocenters (arrow) are more prominent in the SC nuclei. (D) Day 25 SC nuclei are completely displaced from the basement membrane by GC. The nuclei now show deep invaginations and several satellite karyosomes (arrow). (E) Day 35 SC nuclei still contain an abundance of satellite karyosomes and most remain displaced away from the base. (F) Day 100 SC nuclei show the typical adult form lying along the basement membrane, with euchromatic appearance and a prominent tripartite nucleolus.

Chapter 3 Structure of the Sertoli Cell

25

FIGURE 3.7 Sertoli cell nucleus showing typical indentations (arrows) and prominent nucleolus, lying near the basal lamina (Bl). Lateral Sertoli cell–Sertoli cell junctions (Jct) are seen on both sides of the cell. Peritubular myoid and Leydig cells are noted beneath the seminiferous tubule.

structures. Information regarding the function of these nuclear indentations is limited; however, one study has shown that some proteins can be found in higher concentrations at nuclear invagination areas [30]. The nucleoplasm is typically euchromatic and stains very light blue with hematoxylin due to the relatively small amount of heterochromatin (Fig. 3.7) that is scattered finely in the nucleus and along the membrane (Fig. 3.8), particularly in the developing testis. Unusual formations, such as crystalloids, have been reported in the Sertoli cell nuclei of large mammals, such as the bull [31], but in most species the Sertoli cell nucleus has evenly distributed chromatin with only the nucleolus standing out as a distinct intranuclear structure. Using cytochemistry, however, it has been possible to observe other components, such as calcium, which were described as “large round-shaped” precipitates [32]. The nucleolus is large and easily recognized (Figs. 3.5–3.7), and the entire complex is often found in three distinct parts (tripartite; Figs. 3.9 and 3.10) [33, 34] in many species. With hematoxylin staining, the nucleolus and satellite structures are strongly basophilic. Only the main nucleolar body is seen in most histological and ultrastructural sections (Fig. 3.5), and although this could be due to the thickness of sections, it has also been found that the number of large chromocenters varies between 1 and 10 or more [28]. The large satellite chromocenters are usually considered part of the tripartite nucleolus structure (Figs. 3.5, 3.7, 3.9, 3.10), but have limited participation in RNA synthesis [35];

nevertheless, the chromocenters contain nearly all of the heterochromatin found in the Sertoli cell nucleus [28]. Others have localized decondensed DNA in the fibrillar components and other regions of the nucleolus [33, 36],

FIGURE 3.8 Sertoli cell nucleus showing a deep indentation containing intermediate filaments (Fi). Nuclear pores (Np) are seen along the nuclear envelope (Ne). A thin proteinaceous band crosses between the nuclear envelope at the opening of the pores. Heterochromatin (Ht) forms a very thin line attached to the nuclear envelope.

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Rex A. Hess and Luiz R. França

FIGURE 3.9 Sertoli cell nucleus sectioned through the two prominent satellite chromocenters, with the nucleolus out of the plane of section. Nuclear pores are barely seen because the chromatin is so fine with few clumps along the nuclear membrane. The Golgi complex is seen adjacent to the nucleus in three small sections.

FIGURE 3.10 The tripartite nucleolus often seen in the Sertoli cell nucleus. The vesicular nucleolus body is flanked by more dense satellite chromocenters that are attached to the nucleolus by thin filaments (arrowheads).

suggesting that transcriptional activity may be active in this region, but it remains to be determined whether this activity has overall significance. The chromocenters are smaller and more numerous in the developing testis and there is a continuous decrease in numbers with age, but the number was not shown to be correlated with androgen receptor presence, although there were distinct mouse strain differences [28]. It is interesting that the X chromosome was found near the center of one of the chromocenters, whereas the Y chromosome was found at the nuclear periphery [33]. Telomeric sequences were shown by in situ hybridization to give a strong signal in the chromocenters [33]. The Sertoli cell nucleus has a high density of nuclear pores (Fig. 3.8), the number of which has been shown to vary depending on the stage of the spermatogenic cycle, with the highest density appearing to occur in stages XIII–I of the rat [37]. It was suggested that this increase in density corresponded with the apparent increased metabolic needs of the Sertoli cell. Species differences are found in Sertoli cell morphology, particularly the nucleus. To illustrate this aspect, note that the nucleolus in ruminant Sertoli cells is heavily vacuolated (Fig. 3.11) or multivesiculated [38, 39]. In the monkey testis, Sertoli cell nuclei are typically located approximately in the middle of the seminiferous epithelium, in contrast to most other species, including rodents, where the nuclei are typically near the basement membrane (Fig. 3.12), except during spermiation [40].

FIGURE 3.11 Nucleus of the bovine Sertoli cell showing indentations of the nuclear envelope and prominent vesiculation of the nucleolus. (Used with the permission of Dr. Karl-Heinz Wrobel, University of Regensburg, Germany.)

Chapter 3 Structure of the Sertoli Cell

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FIGURE 3.12 Seminiferous tubules from monkey and mouse testes at similar stages of spermatogenesis. Sertoli cell nuclei are encircled to illustrate species differences in numerical density of Sertoli cells per tubule cross section. Also, note that the nuclei are located midway between basement membrane and lumen in the monkey tubule, but along the basement membrane in the mouse.

IV. CYTOPLASM A. Mitochondria Sertoli cell cytoplasm contains an abundance of mitochondria, which is indicative of its high metabolic activity [13]. In the body of the cell, mitochondria are more numerous (Fig. 3.13) and scattered among the other organelles. Mitochondrial shape seems to vary more in the Sertoli cell than in most other epithelial cell types, and these morphological appearances are species specific and can be used to differentiate between Sertoli and germ cells [41, 42]. The mitochondria can be rather long (Fig. 3.14), even reaching 2–3 μm [13] or more, but they can also appear to be “cup shaped” or even shaped like “donuts” (Figs. 3.4, 3.13, 3.15). Mitochondrial cristae of the Sertoli cell are predominantly tubular, but the orthodox foliate forms are also present [13]. The number of cristae, however, is less than that typically found in steroid-producing cells such as the Leydig cell [13]. Cytoplasmic organelles of the Sertoli cell are usually polarized within the cell body by the extensive tracts of microtubules that travel from base to apex (Fig. 3.16). When the testis is exposed to a microtubule inhibitor, such as the fungicide carbendazim [43, 44], the organelles collapse into a perinuclear region, particularly the mitochondria (Fig. 3.16).

B. Endoplasmic Reticulum Rough endoplasmic reticulum is located in the basal region and is sparse [13], but smooth endoplasmic reticulum is a predominant organelle in adult Sertoli cells (Figs. 3.4 and 3.16), suggesting that the cell has a predominant function related to the metabolism of lipids or steroids. It is often found adjacent to mitochondria (Figs. 3.4 and 3.13–3.15) and in some species is found evenly dispersed between the elongate spermatid head near the tubule lumen or surrounding lipid droplets [13]. In the water buffalo, aggregates and whorls of smooth endoplasmic reticulum can be quite large in the base of the cell, as well as near the heads of elongate spermatids [38]. In the body of Sertoli cells from all species studied, microtubules help to maintain the linear appearance of the smooth endoplasmic reticulum and the alignment of mitochondria (Fig. 3.16). Apparently this reticulum is a continuous structure, which includes thin branches that surround the heads of elongate spermatids at the tubulobulbar complex, a spermatid projection into the Sertoli cell cytoplasm (Fig. 3.17). The smooth endoplasmic reticulum is also a component of the ectoplasmic specialization, which composes the unique Sertoli/germ cell junctional complex (Figs. 3.18 and 3.19). It has been suggested that the amount of smooth endoplasmic reticulum in Sertoli cells might be

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FIGURE 3.13 Sertoli cell cytoplasm in the body of the cell. Germ cells (GC) are outlined by the black lines. Mitochondria (M1) are aligned in the microtubule direction but some mitochondria have the unique donut shape (M2), whereas others have a C-shape (M3) and are closely aligned to smooth endoplasmic reticular structures (S). L, lysosome.

FIGURE 3.14 Sertoli cell mitochondria (Mi) near the basal lamina (Bl) are long, thin, vesiculated structures that are easily distinguished from the germ cell mitochondria that exhibit wide spaces in their lamellae. ER, endoplasmic reticulum; Ly, lysosome; Li, lipid droplet.

FIGURE 3.15 Sertoli cell base lying on the basement membrane (BM), which contains an amorphous layer next to the Sertoli cell plasmalemma and small collagen fibrils. Rare, small endocytotic or exocytotic vesicles (En) are found along the basal membrane. The thin peritubular myoid cell (PM) is found adjacent to the basement membrane. Mi, Sertoli cell mitochondrion; ER, endoplasmic reticulum.

Chapter 3 Structure of the Sertoli Cell

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FIGURE 3.16 (A) Sertoli cell cytoplasm in a control testis showing thin microtubules (arrows) oriented in the long axis from base to apical regions (double arrow). Organelles such as mitochondria (M) and endoplasmic reticulum (ER) are oriented in the direction of the microtubules. (B) Sertoli cell cytoplasm in a testis exposed to the fungicide carbendazim, a microtubule poison. The mitochondria are no longer oriented longitudinally along microtubule tracts, but are collapsed (circle) near the Sertoli cell nucleus (SC).

FIGURE 3.17 Tubulobulbar complexes are seen penetrating into the apical cytoplasm of the Sertoli cell from the adjacent germ cell. The complexes end with bulbous heads that contain a fuzzy outer layer on the Sertoli cell side. SER, smooth endoplasmic reticulum.

FIGURE 3.18 Sertoli cell cytoplasm adjacent to the developing spermatid nucleus is a highly specialized attachment site that is given the name ectoplasmic specialization. The ectoplasmic specialization consists of the spermatid plasmalemma over the acrosome, the Sertoli cell membrane, and a zone of actin filament bundles sandwiched between the Sertoli cell membrane and a thin layer of smooth endoplasmic reticulum cisternae.

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FIGURE 3.19 Early formation of the ectoplasmic specialization. The Sertoli cell cytoplasm adjacent to an early step elongating spermatid nucleus is highly specialized to begin the formation of the ectoplasmic specialization. The closeness of Sertoli and germ cell (GC) membranes is noted.

related to paracrine interactions with germ cells [45]. Morphometric studies comparing Sertoli cell structure in W/W (which lack virtually all germ cells) and control mice showed that although the testes of mutant animals were about eight times smaller than controls, the numbers of Sertoli cells in the two groups did not differ; nevertheless, Sertoli cell volume and surface area in the mutant animals were significantly smaller. Smooth endoplasmic reticulum was also significantly reduced in the mutant animals, whereas other organelle volumes and surface areas, expressed per cell, did not differ significantly in W/W and control animals.

C. Lysosomes and Multivesicular Bodies The Sertoli cell is phagocytotic and helps to remove spermatid cytoplasm through the tubulobulbar complexes and by phagocytosis of the residual bodies and degenerating germ cells. Such activities lead to the formation of lysosomes and multivesicular bodies that are located throughout the cytoplasm (Fig. 3.4), but particularly near ectoplasmic specializations and residual bodies (Fig. 3.20). As such, their presence will change depending on the stage of spermatogenesis. As a curiosity that is related to residual bodies, Sertoli cells phagocytose daily about 0.3% of the testis mass, meaning that in 1 year the total volume phagocytosed by this cell would correspond to the weight of both testes [46].

FIGURE 3.20 The residual body (RB) contains leftover cytoplasm from the developing germ cell (GC), which is phagocytosed by the Sertoli cell. Outlines of Sertoli cell cytoplasm are noted by the black lines, indicating the extensive ramification of its cellular arms or processes as it engulfs the residual bodies. Small collections of germ cell mitochondria can be seen within the residual bodies (arrows).

Chapter 3 Structure of the Sertoli Cell

D. Golgi The Golgi apparatus is relatively small in the Sertoli cell (Fig. 3.4), and the multiple components are typically dispersed in the supranuclear region (Figs. 3.9 and 3.13). Studies have shown that this is a single network of stacked saccules [47–49]. It is interesting that condensing vesicles that are associated with secretory pathways are not reported in the Sertoli cell [13]. However, small exocytotic or endocytotic vesicles are found on the basement membrane side of this cell (Fig. 3.15).

E. Cytoskeleton Primary elements of the cytoskeletal system include microtubules, actin filaments, and intermediate filaments (vimentin). The general roles of these elements in cellular structure and function are well known and include maintaining cell shape and polarity, moving or positioning of intracellular organelles, forming of pseudopodia and other cytoplasmic extensions such as microvilli, sorting and targeting of proteins, and anchoring of organelles to the plasmalemma. The Sertoli cell appears to carry these important cellular functions even further, because these elements are involved in a number of unique seminiferous epithelial activities associated with anchoring of germ cells, translocation of germ cells through the different stages of development, and nurturing and preparing the release of mature spermatids from the epithelium into the seminiferous tubule lumen [50–57]. For a complete overview of this unique set of Sertoli cell cytoplasmic components, the reader is encouraged to study the outstanding chapter by A. Wayne Vogl et al. presented in The Sertoli Cell [58]. Two of the major Sertoli cell components depend on the cytoskeletal elements for structure and function: the ectoplasmic specialization and the tubulobulbar complex. The ectoplasmic specialization is a cell junctional complex that forms between two Sertoli cells [59] and between Sertoli and germ cells [52], although the junctions are different between the two cell types [15]. It consists of the plasma membrane of the two adjacent cells, bundles of actin filaments on the Sertoli cell cytoplasmic side sandwiched between the Sertoli cell plasma membrane and a thin cistern of smooth endoplasmic reticulum (Figs. 3.18 and 3.19). Since 1993, considerable data have been published to support the original hypothesis that the ectoplasmic specialization functions as an anchor for the developing elongate spermatids and with this anchor the Sertoli cell is able to translocate the germ cell into deep crevices and then move the cell back out toward the lumen in preparation for spermiation

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[50–53, 55, 60–76]. The in vitro work of Vogl, in particular, has clearly shown that Sertoli cell microtubules are capable of moving isolated ectoplasmic specializations in both directions, suggesting that both a kinesin-type and dynein motor proteins are associated with the junctional complex [50, 53, 55, 77]. Tubulobulbar complexes are unique plasma membrane specializations that form as thin tubular indentations of Sertoli cell cytoplasm or protrusions of the adjacent germ cell cytoplasm. The complexes have bulbous regions at the end of a distal tubule that terminates as a bristle-coated pit. These structures form between adjacent Sertoli cells and between Sertoli cells and the head of late spermatids (Fig. 3.17), just prior to spermiation [17, 19, 59, 78–82]. They are found in numerous species, but only the Sertoli cell–germ cell complexes are reported in some species. Although these structures can reach up to 4 μm in length, they are recognizable only by transmission electron microscopy [59]. Their function appears to be at least threefold: (1) to help anchor germ cells to the Sertoli cell, (2) to help eliminate spermatid excess cytoplasm, and (3) to aid in the recycling or elimination of the ectoplasmic specialization, a function that is necessary for remodeling of the Sertoli cell barrier and required before the spermatid can be released in the act of spermiation. Disruption of tubulobulbar complex formation results in abnormal development of the spermatid head and larger than normal cytoplasmic droplets at the time of sperm release [59]. Cytoskeletal elements have also been visualized as participating in other important functions of the Sertoli cell. Microtubules are a major component of the long axis of the cell body, as seen by immunostaining for αtubulin (Fig. 3.21) and easily recognized by electron microscopy [58, 83, 84]. Several of the cytoplasmic organelles are aligned and transported by the microtubule tracts, as seen by their linear appearance in the cell body (Fig. 3.16). Vimentin intermediate filaments also appear along the long axis but more in the base, and a strong band is continuous around the Sertoli cell nucleus (Fig. 3.21). The importance of microtubules and intermediate filaments in helping to maintain Sertoli cell structure is best illustrated in the studies of chemical toxicant effects on the seminiferous epithelium [44, 84–96]. For example, the fungicide carbendazim, a mild microtubule poison that binds to αtubulin [44], disrupts Sertoli cell microtubule formation and induces sloughing of the elongate spermatids in a stage-specific manner (Fig. 3.22). The elongate spermatids are sloughed by breaking off from the epithelium with attached Sertoli cell cytoplasm, yet the microtubules still stain positive in the sloughed cytoplasm, whereas in the body of the

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FIGURE 3.21 Seminiferous epithelium stained immunohistochemically for αTubulin (A, C) and vimentin

intermediate filaments (B, D). αTubulin (arrows) extends from the base of the Sertoli cell to the apex in (A) rat and (C) ram testes. Vimentin staining (arrows) is reserved for the perinuclear region of the Sertoli cell in (B) rat and (D) ram testes, with the most intense staining located along the base, but wisps of vimentin filaments reaching out to the tips of elongating spermatids. (Ram testes photographs used with permission of Dr. Karl-Heinz Wrobel, University of Regensburg, Germany.)

Sertoli cell the microtubule staining is lost. This study supports the general hypothesis that is now well accepted that microtubule nucleation in the Sertoli cell is unique in that the nucleation centers are not found in the perinuclear centrosome, but rather at the apical cell periphery [57, 95]. Microtubules grow in the apical to basal direction and appear to anchor within the perinuclear intermediate filaments [57]. Loss of the microtubules within the Sertoli cell body, as shown after carbendazim treatment, is associated with a simultaneous collapse of the intermediate filaments (Fig. 3.22), which apparently weakens the apical cytoplasm and thus allows the spermatids, whose tails are dangling into the tubule lumen, to break away from the Sertoli cell. These data provide further proof that the microtubules extend into the nuclear crevasse and interact with the intermediate filaments for maintenance of the Sertoli cell structural scaffolding.

V. THE SERTOLI (BLOOD–TESTIS) BARRIER Sertoli cell–Sertoli cell tight junctions form the blood–testis barrier, which is now referred to as the Sertoli cell barrier. Several proteins, including actin, zonula occludens 1 (ZO-1), occludin, claudin, espin, and gelsolin, have been described as components of the Sertoli cell barrier or associated with the ectoplasmic specialization, and most of them belong to three classes of tight junction integral membrane proteins known as occludins, claudins, and junctional adhesion molecules [97]. However, species variations in tight junction organization are observed [72, 98], and a precise and rigidly organized actin–tight junction relationship is not absolutely mandatory for the presence or maintenance of tight junctions [72]. Although the mechanisms are not yet known, several molecules

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Chapter 3 Structure of the Sertoli Cell

FIGURE 3.22 Effects of disrupting αtubulin in Sertoli cells. (A) αTubulin staining of a control stage VII rat seminiferous tubule shows striations extending from the basement membrane to the lumen, where step 19 spermatids are lining up for spermiation. (B) The fungicide carbendazim is a microtubule poison that causes rapid sloughing of the late elongate spermatids [44]. Note the small strands of Sertoli cell cytoplasm clinging to the sloughing heads of sperm. (C) Immunostaining for αTubulin following carbendazim treatment for 2 hr. All staining is lost in the body of Sertoli cells, but staining is found in the sloughed cells in the lumen. (D) Control tubule stained for vimentin intermediate filaments. Most staining is found perinuclear but small filament strands extend into the body of Sertoli cells. (E) Carbendazim treatment causes sloughing of late spermatids into the lumen, denuding the apical cytoplasm of Sertoli cells. (F) Immunostaining for vimentin following carbendazim treatment for 2 hr. All staining is lost in the body of Sertoli cells, but staining is found perinuclear where vimentin has collapsed following treatment.

such as transforming growth factor 3 (TGF3), occludin, protein kinase A, protein kinase C, and signaling pathway (TGF3/p38 mitogen-activated protein kinase) have been shown to regulate Sertoli cell tight junctions [74, 99–102]. Also, recent in vitro studies in rats have shown that nitric oxide synthase (NOS) is an important physiological regulator of Sertoli cells tight junction dynamics, exerting its effects through the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway [103, 104]. The extracellular matrix is also able to regulate the Sertoli cell tight junction functions through mechanisms involving the participation of tumor necrosis factor α (TNFα) and its regulatory role on collagen α3 (IV) and other proteins that maintain the homeostasis of the extracellular matrix [75].

This investigation also showed that TNFα inhibited occludin, which is known to associate with Sertoli cell tight junction barrier assembly, and induced the expression of Sertoli cell collagen type IV, gelatinase B [matrix metaloprotease 9 (MMP9)] and tissue inhibitor of metaloproteases 1, and promoted the activation of pro-MMP9 [105].

VI. MISCELLANEOUS OBSERVATIONS A. Sertoli Cell Cycle Germ cell associations in the seminiferous tubules in mammals are organized in cyclical patterns of

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renewal and development; therefore, several studies were developed that were aimed at correlating these cyclic changes with morphological alterations in Sertoli cells during spermatogenesis in rats. In this regard, Ye et al. [26] investigated the presence of cyclic differences in volumes and surface areas of several Sertoli cell parameters using a sampling technique at the electron microscope level that sampled Sertoli cells proportionally within the seminiferous tubule. Significant differences were found among different stages of the cycle for the surface area of the cell, the volume of lipid, and the volume and surface area of the rough endoplasmic reticulum (RER). The volume and surface area of RER peaked at midcycle, and lower values were observed near the end of the cycle, showing an approximately 15-fold difference between the minimum and maximum values. Apparently, this variation found for RER parameters correlated with known patterns of protein secretion within the epithelium and with the secretion of specific proteins and important factors that control protein secretion. Another morphometric study performed at different stages of the cycle correlated Sertoli cell surface relationships and the changing volumes of developing germ cells in adult rats [22]. As a reflection of germ cell development, cyclic variation was noted for the Sertoli cell surface area that faces the basal compartment germ cells, but not the basal lamina. No cyclic variation was observed in the amount of Sertoli cell contact with each other at the level of the Sertoli cell barrier. However, when the adluminal compartment area was studied, significantly less Sertoli cell–Sertoli cell contact was seen in some stages of the cycle. Surface contact of germ cells with Sertoli cells (Figs. 3.2 and 3.20) increased progressively as germ cells entered the intermediate compartment and progressed in spermatogenesis. Also, the area in which Sertoli ectoplasmic specializations faced germ cells changed dramatically during spermatogenesis, reaching its maximum in elongating spermatids (Figs. 3.1 and 3.18).

B. Sertoli Cell Aging Compared to other testicular cells, the Sertoli cell is relatively resistant to chemical insults. However, the cell is sensitive to certain chemical exposures (see Chapter 20) and to abnormal conditions such as the formation of tumors and specific mutations, which can result in morphological changes in the nucleus and cytoplasmic organelles [106–110]. Aging is another factor that appears to be detrimental to the Sertoli cell [28, 110–114]. In addition to aging changes in the shape

of the Sertoli cell nucleus, alterations occur in the endoplasmic reticulum, lysosomes, specific Sertoli cell molecular markers [113], and in the Sertoli cell barrier [114]. A study by the Robaire laboratory [114] clearly demonstrated that with aging in the Brown Norway rat there is a complete breakdown in the Sertoli cell barrier (the blood–testis barrier), because lanthanum nitrate was seen penetrating the basal and adluminal compartments and entering the seminiferous tubule lumen.

C. Other Cytoplasmic Inclusions Other less obvious Sertoli cell components are observed, such as lipid droplets (Fig. 3.4). It is interesting that lipid droplets were first reported in this cell by the original discover of the cell [23], and the droplets are typically found in the basal compartment of all Sertoli cells, but the amount varies considerably between stages of spermatogenesis, as well as between species [13]. Lipids were originally the focus of considerable speculation in the 1800s [1], as well as during more modern times [115, 116]. More recent studies showing a significant increase in lipids in the postspermiation stages, following cimetidine treatment [117], have supported the hypothesis that Sertoli cell lipids are evidence of an ability to recycle lipids from the breakdown of germ cell degeneration and from residual body phagocytosis [13]. However, others have shown that the Sertoli cell’s abilities of uptake and conversion of fatty acids in vitro are nearly equal to those of the Leydig cell, but the effects of diabetes are less severe in the Sertoli cell [118]. Thus, the question of lipid function and metabolism in this cell type remains open. Glycogen particles are also found in the Sertoli cell cytoplasm (Fig. 3.4) and glycogen metabolism is reported for this cell [119, 120]. Glycogen recognition requires histochemistry for light microscopy [121, 122] and special fixation, such as ferrocyanide-osmium [123], is required for optimal recognition by electron microscopy. The amount of glycogen present is stage and species dependent [124, 125], with the dog Sertoli cell containing an abundance [13]. Cryptorchidism causes a decrease in the amount of glycogen in Sertoli cells [126]. In men having the Sertoli cell–only syndrome, an increased concentration of glycogen and intermediate filaments in certain Sertoli cells results in these cells staining darker [127]. An accumulation of glycogen within the cell cytoplasm is associated with the formation of Sertoli cell tumors and other types of malignancies [128–131].

Chapter 3 Structure of the Sertoli Cell

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D. Other Sertoli Cell Membrane Modifications In addition to the unusual membrane adaptations seen between Sertoli cells and the heads of elongated spermatids, there are small protrusions of the Sertoli cell cytoplasm into the cytoplasm of the developing germ cells (Fig. 3.23). In some species, such as the water buffalo, adjacent plasmalemmas of two Sertoli cells can be rolled like the lamina of a myelin sheath (Fig. 3.24).

E. Seminiferous Tubule/Rete Testis Transitional Region

FIGURE 3.23 The Sertoli cell (SC) cytoplasm becomes very thin as it expands as a sheet between two germ cells (GC), as outlined by the black lines. In a small region of the spermatid, the Sertoli cell’s cytoplasm protrudes into the germ cell.

The region that connects rete testis to the seminiferous tubule is known by several names, including tubulus rectus, intermediate region, straight tubules, terminal segment, and transitional zone [13, 132–137]. The region is lined by Sertoli cells with few spermatogonia. The transition from Sertoli cells to rete testis epithelium is abrupt (Fig. 3.25). In all species studied to date, Sertoli cells lining this junction have an appearance that differs from that of the typical adult differentiated cell. There are differences in the concentration and organization of cytoplasmic organelles, height of the cells, and appearances of the Sertoli cell nuclei. The nuclei appear more similar to those seen in the developing testis (compare Figs. 3.6 and 3.25). The Sertoli cell cytoplasm forms stringy processes that

FIGURE 3.24 Sertoli cells from water buffalo testis showing the rolling of adjacent Sertoli cell cytoplasm (arrows) into a myelin-like formation. The space between the Sertoli cells is a thin tight junction (arrows), compared to the wider intercellular space between Sertoli and germ cells (GC; arrowheads). (From Pawar et al. [38], Fig. 3.8, p. 47.)

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FIGURE 3.25 (A) The Sertoli cell–rete testis junction in the rat. The transition is rapid and the cuboidal rete testis epithelial cells cannot be mistaken for the taller Sertoli cells that extend into the lumen and appear to be stacked on one another and usually do not surround germ cells. Sertoli cell nuclei at this junction (b) appear similar to those found in the developing testis (Fig. 3.6) and stain darker than those found within seminiferous tubules that are active in spermatogenesis (a). (B) The Sertoli cell–rete testis junction in the bull. The Sertoli cell cytoplasm extends into the lumen forming a plug-like valve. (Used with permission of Dr. Karl-Heinz Wrobel, University of Regensburg, Germany.)

appear to form a “plug” or “valve” in the tubule lumen and it is generally accepted that this junction plays a role in regulating the flow of fluid from the seminiferous tubule into the rete testis, a concept suggested in 1902 [138].

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rat as a model for man. Novartis Found. Symp. 242, 82–95; discussion 95–97. Syed, V., and Hecht, N. B. (2001). Selective loss of Sertoli cell and germ cell function leads to a disruption in Sertoli cell–germ cell communication during aging in the Brown Norway rat. Biol. Reprod. 64, 107–112. Levy, S., Serre, V., Hermo, L., and Robaire, B. (1999). The effects of aging on the seminiferous epithelium and the blood–testis barrier of the Brown Norway rat. J. Androl. 20, 356–365. Lacy, D. (1962). Certain aspects of testis structure. Br. Med. Bull. 18, 205–208. Lacy, D. (1960). Light and electron microscopy and its use in the study of factors influincing spermatogenesis in the rat. J. Roy. Microsc. Soc. 79, 209–225. Sasso-Cerri, E., Giovanoni, M., Hayashi, H., and Miraglia, S. M. (2001). Morphological alterations and intratubular lipid inclusions as indicative of spermatogenic damage in cimetidinetreated rats. Arch. Androl. 46, 5–13. Hurtado de Catalfo, G. E., and De Gomez Dumm, I. N. (1998). Lipid dismetabolism in Leydig and Sertoli cells isolated from streptozotocin-diabetic rats. Int. J. Biochem. Cell Biol. 30, 1001–1010. Slaughter, G. R., and Means, A. R. (1983). Follicle-stimulating hormone activation of glycogen phosphorylase in the Sertoli cell-enriched rat testis. Endocrinology 113, 1476–1485. Guo, T. B., Chan, K. C., Hakovirta, H., Xiao, Y., Toppari, J., Mitchell, A. P., and Salameh, W. A. (2003). Evidence for a role of glycogen synthase kinase-3beta in rodent spermatogenesis. J. Androl. 24, 332–342. Montagna, W., and Hamilton, J. B. (1952). Histological studies of the human testis. II. The distribution of glycogen and other HIO4-Xchiff reactive substances. Anat. Rec. 112, 237–249. Nicander, L. A. (1957). Histochemical study on glycogen in the testes of domestic and laboratory animals, with special reference to variations during the spermatogenic cycle. Acta. Neerl. Morph. 1, 233–240. Russell, L. D., and Burguet, S. (1977). Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue Cell 9, 751–766. Fouquet, J. P. (1968). Infrastructural study of the glycogen cycle in the Sertoli cells of the hamster. C. R. Acad. Sci. Hebd. Seances Acad. Sci. D. 267, 545–548. Erkan, M., and Sousa, M. (2002). Fine structural study of the spermatogenic cycle in Pitar rudis and Chamelea gallina (Mollusca, Bivalvia, Veneridae). Tissue Cell. 34, 262–272. Gotoh, M., Miyake, K., and Mitsuya, H. (1987). A study of cryptorchidism. III. The histochemistry of complex carbohydrates in the testes of cryptorchid patients. Hinyokika Kiyo. 33, 905–914. Tedde, G., Montella, A., Fiocca, D., and Delrio, A. N. (1993). The sertolian epithelium in the testis of men affected by “Sertoli-cell-only syndrome.” Ital. J. Anat. Embryol. 98, 105–117. Henley, J. D., Young, R. H., and Ulbright, T. M. (2002). Malignant Sertoli cell tumors of the testis: A study of 13 examples of a neoplasm frequently misinterpreted as seminoma. Am. J. Surg. Pathol. 26, 541–550. Lee, P. J. (2002). Glycogen storage disease type I: Pathophysiology of liver adenomas. Eur. J. Pediatr. 161, S46–S49. Gurbuz, Y., and Ozkara, S. K. (2003). Clear cell carcinoma of the breast with solid papillary pattern: A case report with immunohistochemical profile. J. Clin. Pathol. 56, 552–554. Kolligs, F. T., Bommer, G., and Goke, B. (2002). Wnt/betacatenin/tcf signaling: A critical pathway in gastrointestinal tumorigenesis. Digestion. 66, 131–144.

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132. Roosen-Runge, E. C. (1961). The rete testis in the albino rat: Its structure, development and morphological significance. Acta. Anat. (Basel) 45, 1–30. 133. Nykanen, M. (1979). Fine structure of the transitional zone of the rat seminiferous tubule. Cell Tissue Res. 198, 441–454. 134. Lindner, S. G. (1982). On the morphology of the transitional zone of the seminiferous tubule and the rete testis in man. Andrologia 14, 352–362. 135. Ezeasor, D. N. (1986). Ultrastructural observations on the terminal segment epithelium of the seminiferous tubule of West African dwarf goats. J. Anat. 144, 167–179. 136. Wrobel, K. H., Schilling, E., and Zwack, M. (1986). Postnatal development of the connexion between tubulus seminiferous and tubulus rectus in the bovine testis. Cell Tissue Res. 246, 387–400.

137. Wrobel, K. H., Sinowatz, F., and Mademann, R. (1982). The fine structure of the terminal segment of the bovine seminiferous tubule. Cell Tissue Res. 225, 29–44. 138. Spangaro, S. (1902). Uber die Histologischen Veränderungen des Hodens, Nebenhodens, Und Samenleiters von Geburt an biz zum Griesalter, mit besonderer Berucksichtgung der Hodenatrophie, des elastischen Gewebes und des Vorkommens von Krystallen im Hoden. Anat. Record 18, 593–771. 139. Wong, V., and Russell, L. D. (1983). Three-dimensional reconstruction of a rat stage V Sertoli cell. I. Methods, basic configuration, and dimensions. Am. J. Anat. 167, 143–161. 140. Fawcett, D. W. (1975). Ultrastructure and function of Sertoli cells. In “Handbook of Physiology: The Male Reproductive System” (D. W. Hamilton and R. O. Greep, eds.), pp. 21–55. American Physiology Society, Washington, DC.

P A R T

II SERTOLI CELL DEVELOPMENT

This Page Intentionally Left Blank

C H A P T E R

4 Embryonic Sertoli Cell Differentiation

I. II. III. IV.

ANDREA S. CUPP

MICHAEL K. SKINNER

Department of Animal Science, University of Nebraska, Lincoln, Nebraska

Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington

INTRODUCTION OVERVIEW OF EMBRYONIC TESTIS DEVELOPMENT STAGES AND TIMING SUMMARY References

II. OVERVIEW OF EMBRYONIC TESTIS DEVELOPMENT The genital ridge is primarily composed of a single layer of coelomic epithelium from 9 to 9.5 days postcoitus (dpc) in the mouse (Fig. 4.1) [1]. At 9–10 dpc the primordial germ cells (PGCs) migrate from extragonadal sites within the yolk sac to colonize the urogenital ridge [2, 3]. The gonad is bipotential after germ cell migration and morphologically can be distinguished from the adjoining mesonephric tissue but cannot be identified as an ovary or a testis [4]. Two morphological events occur to alter the bipotential gonad at 11 dpc of development. First, Sertoli cells differentiate in part from the coelomic epithelium and start to proliferate, aggregating with the PGCs. Second, mesonephric cells (endothelial or preperitubular in origin) migrate from the mesonephros and enclose the pre–Sertoli-PGC aggregates to form seminiferous cords (Fig. 4.1) [5]. Both of these events rely on expression of Sry by the developing Sertoli cell [6]. As the Sertoli cell differentiates (11–13 dpc) it acquires the ability to produce Müllerian inhibiting substance (MIS), which inhibits Müllerian duct growth. The Müllerian duct is the precursor female reproductive tract that differentiates into the cervix, uterus, oviduct, and portions of the anterior vagina [7, 8]. After seminiferous cord formation, cells within the interstitium differentiate to form immature Leydig cells, while the preperitubular cells further differentiate to form a single layer of cells enclosing each seminiferous cord. In the rat, steroidogenesis occurs within

I. INTRODUCTION In the past 10 years, we have experienced a veritable explosion in the amount of information that has been uncovered on genes important in the regulation of testis differentiation. With the identification of the testis determining gene, Sry, and localization of its expression in the Sertoli cell, new information has been elucidated on factors regulating the male sex differentiation pathway. The Sertoli cell is the critical cell type that initiates development of testis-specific gene expression, induces testis morphology, and establishes crucial parameters for spermatogenic function and capacity. Therefore, proper differentiation of the Sertoli cell during embryonic testis differentiation is mandatory for normal adult testis development and function. In this chapter, we examine embryonic testis development and the crucial role of the Sertoli cell in both sex determination and morphological events that result in the formation of a testis. The events regulating testis development are discussed using primarily the mouse as a model; however, comparisons will be made to rats, domestic livestock, and humans where information is available. SERTOLI CELL BIOLOGY Edited by M. K. Skinner and M. D. Griswold

43

Copyright 2005, Elsevier Science (USA). All rights reserved.

44

Andrea S. Cupp and Michael K. Skinner Developing Testis Stage I: Urogenital Ridge Mouse

9 dpc

Stage II: Indifferent / Bipotential Gonad 10-10.5 dpc

Stage III: Sex Determination 10.5-11 dpc

Stage IV: Cord Formation 11-12 dpc

Stage V: Development of Functional Testis 12-13 dpc 12

Developing Gonad Mesonephros Genital Ridge

Germ cell Colonization Sertoli cell Differentiation

Sry Expression Sertoli Cell Migration Sertoli/Germ Cell Aggregation

Migration of Pre-peritubular And Endothelial cells to form seminiferous cords

Seminiferous cord formation complete Initiation of Leydig cell migration and differentiation

FIGURE 4.1 Morphological stages that occur during testis differentiation from genital ridge formation to cord formation and cell proliferation in the mouse with staging in number of tail somites (ts) and days postcoitus (dpc).

the testis at 14.5 dpc when 3β-hydroxysteroid dehydrogenase (3β-HSD) is first expressed in pre-Leydig cells [9, 10]. Testosterone produced by the Leydig cells maintains and stabilizes the Wolffian duct, which is the precursor of the male reproductive tract structures: the epididymis, vas deferens, and portions of the secondary sex glands [11]. The seminiferous cords develop lumen around puberty to become the seminiferous tubules. A major function of the Sertoli cell within the testis is to provide the appropriate environment (e.g., production of proteins and growth factors) and cytoarchitectural support for the developing germ cells [12]. The comparisons between events that occur in the mouse, rat, and pig during testis development and correlated time points in embryonic testis development are presented in Table 4.1.

III. STAGES AND TIMING There are at least five different morphological stages of testis development, which are depicted in Fig. 4.1: (1) development of a genital ridge, (2) formation of an indifferent or bipotential gonad, (3) sex determination, (4) induction of testicular cords in the testis, and (5) development of a functional testis. These stages are represented in Fig. 4.1 by timelines in the mouse and are compared to other species (human, pig, cattle, sheep, and rats) in Table 4.1.

The first two stages, genital ridge formation and formation of an indifferent gonad, occur whether the individual has XX or XY chromosomes and, thus, is independent of testis or ovarian development. The last three stages of gonadal development are dependent on genes that are expressed within the indifferent gonad to result in either a testis or ovary. Expression of the gene Sry (sex-determining region of the Y chromosome) by the Sertoli cell directs the indifferent gonad to become a testis [13–15], whereas the absence of Sry and expression of Wnt4 appear to regulate formation of an ovary [16]. Thus, formation of testis-specific morphogenic structures such as seminiferous cords is dependent on differentiation of the Sertoli cell, expression of Sertoli cell–specific genes, and proliferation of the Sertoli cell population to result in normal testis development (reviews [1, 17]).

A. Stage I: Genital Ridge Development The initial step in the development of gonads is the formation of the genital ridge (Fig. 4.1) and urogenital system from the intermediate mesoderm; this step begins at 9–9.5 dpc in the mouse. The Wolffian duct, which is the precursor of the male reproductive tract system, is derived from lateral mesoderm and runs the length of the urogenital system and develops from the mesonephric duct [18]. The Müllerian duct, which is the precursor of the female reproductive system, appears between 11.5 and 12.5 dpc from invaginations

45

Chapter 4 Embryonic Sertoli Cell Differentiation TABLE 4.1 Gestational Age at Each Stage of Testis Development in Humans, Pig, Cattle, Mice, Rats, and Sheep Species

Genital ridge

Bipotential gonad

Testis cord formation

Reference

Human Gestational age

5 weeks

6 weeks

7–8 weeks

Sinisi et al., 2003

Gestational age Crown rump length

18–20 days 8–10 mm

21 days 10–12 mm

26 days 15 mm

McCoard et al., 2001

Cattle Gestational age Crown rump length

27–31 days 19 mm

Wrobel and Sub, 1998

Mouse Gestational age Total no. of somites No. of tail somites

9–10 dpc 13–28 s N

?N

[24, 25]

↓

↑

Sg, Sp survival ↑

Infertile

[30, 31, 33, 40]

↓

No change

Sg, Sc, Sp survival ↑

Infertile

[32, 35] [52, 241]

FSH

T

Neonatal hemicastration

↑

N

↑

Adult hemicastration

↑

N

Neonatal + FSH

↑

↓−↑ ?

Immature HPX + FSH

?

↓

Adult HPX + FSH

?

↓

Sertoli numbersa

Rat

?

↓

↓

?

Sg, Sc, Sp survival ↑

?

Fetal + FSH Ab

↓?

?

?

↓

?

?

[14]

Adult + FSH Ab

↓

N

N

?

Sg, Sc, Sp < N

?

[43, 44]

Immunized to FSHR

?

↑

N

?

Sp < N

?

[53]

Immunized to GnRH + FSH

Neonatal + GnRH-A

↓

↓

↓

↓

Sp < N

Reduced

[37, 159, 242, 243]

Adult + GnRH-A

↓

↓

↓

N

Sc, Sp < N

Infertile

[38]

Adult + GnRH-A + FSH

?

↓

↓

N

Sg, Sc, Sp survival ↑

?

[39]

[72]

Mouse Gain of FSH activity Neonatal hpg +FSH

?

–

↑

↑

Sg, Sc, Sp ↑

Infertile

Immature hpg +FSH

?

–

↑

No change

Sg, Sc, Sp ↑

Infertile

[73]

Pituitary tgFSH

↑

↑

↑

?

?

N

[92]

Ectopic tgFSH

↑

↑

N

?

?

N-infertile

[91]

hpg + tgFSH or tgFSHR

–

–

↑

↑

Sg, Sc, Sp ↑

Infertile

[67, 71, 96]

LHR–/–

↑

↓

↓

N

Sg, Sc, Sp ↓

Infertile

[97, 98]

Loss of FSH activity Immunized to FSHR

?

N

?

?

?

Reduced

[88, 244]

FSHβ–/–

–

N

↓

↓

Sg, Sc, Sp ↓ (Leydig N)

N

[80, 83]

FSHR–/–

↑

↓

↓

↓

Sg, Sc, Sp ↓ (Leydig ↓)

N-reduced?

[81, 82, 84]

Monkey Bonnet M. radiata Adult immunized to FSH

↓

N

?

?

Sg ↑-N; Sc, Sp ↓-N

Reduced-N

[165, 168, 169]

Adult immunized to FSHR

?

?

?

?

Sc ↓?, Sp ↓

Reduced

[169]

Adult hemicastration

↑

N

↑

?

Sp, Sc, Sp ↑

?

[163, 245]

Adult hemicast. + FSH Ab

↓?

N

N

?

Sc ↓?

?

[163]

Adult + GnRH-A + T

↓

N

?

?

Sg ↑, Sp ↓

?

[168]

Rhesus M. mulatta ?↑

N

↑

↑

Sg ↑?

?

[156]

Juvenile + GnRH

?

?

↑

↑

Sg ↑

?

[155]

Adult HPX

↓

↓

↓N

N

?

?Infertile

[246]

Juvenile + FSH

↑d

Adult HPX + T + FSH

?

↓

↓N

N

Sg

Adult hemicastration

↑

N

↑

N

Sg, Sc, Sp ↑

?

[246]

?

[162]

Adult immunized to FSH

?

N

↓

?

Sp ↓-n

?

[167]

Adult + FSH Abs

?

N

↓

?

Sp ↓-N

?

[166]

Adult + GnRH-A

↓

↓