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The first giant step for males
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Society of Reproductive~ll~9(;ooologists
FERTILITY AND STERILITY Copyright
:0
Vol. 65, No.4, April 1996 Printed on acid-free paper in U. S. A.
1996 American Society for Reproductive Medicine
The first giant step for males
Paul G. McDonough, M.D. * Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia
Male development is anomalous in that it is not constitutive, but must be induced. During early development there are common gonadal accessory structures in both sexes. To progress from this stage of "neutrality" to male development requires the prenatal regression ofthe Mullerian system and the virilization ofWolffian structures. For decades, conventional wisdom held that T performed this dual function. In the late 1940s, a 32-year-old French physiologist suggested that a substance other than T might be responsible for Mullerian duct regression in male fetuses. He suspected complexity when simplifying assumptions were the norm. In a series of technically difficult experiments, Jost (1) was able to perform intrauterine castrations of fetal rabbits and conclude that a second, unidentified testicular hormone had the unique ability to regress the system that normally gives rise to the oviducts, uterus, and proximal vagina. Jost (1) named the putative factor "l'horReceived December 4, 1995. * Reprint requests: Paul G. McDonough, M.D., Department of Obstetrics and Gynecology, 1120 Fifteenth Street, Augusta, Georgia 30912-3360 (FAX: 706-721-6830). "Moments in Reproductive Medicine" is a quarterly section of Fertility and Sterility designed to recognize individuals or important milestones in the field of reproductive medicine. Each section will be generated by one of the four affiliated societies of the American Society for Reproductive Medicine: Society of Reproductive Endocrinologists, Society of Reproductive Surgeons, Society for Assisted Reproductive Technology, and Society for Male Reproduction and Urology.
704
McDonough Moments in reproductive medicine
mone inhibitrice" or Mullerian-inhibiting substance (MIS). In recent years it has been called Mullerian inhibiting factor or, simply, anti-Mullerian hormone (AMH). The data from Jost's experiments built a draw bridge for all future investigators of mammalian sex development. His experiments form the basis for what have been called "Jostian Principles." Jost himself would have rebelled at the phraseology because he was inherently skeptical of "absolute truths." In Jost's mind, principles are for physicists not biologists. Ifhe were alive today he would probably repeat his own experiments using the purified MIS. Except for a small coterie of individuals in the American scientific community, Jost's conclusions were not accepted universally. His champions were largely physicians who found his data to be remarkably consistent with nature's own experiments in this area. The technical difficulties involved in the intrauterine surgery and the small number of experimental subjects raised serious questions about reproducibility. Rabbits were scarce in post-World War II France and had a more important place on the kitchen table than in the laboratory. Jost had to make every rabbit count and this sometimes required attending to his subjects at home. At the experimental level one major objection to Jost's conclusions was that the injection of testicular secretions did not prevent the formation ofthe Mullerian derivatives in female fetuses. Subsequent attempts to study MIS were clouded by cumbersome bioassays with indistinct end points and by the small
Fertility and Sterility
quantities of MIS protein in tissue. These problems deterred most investigators from entering this field. Others simply doubted the existence of such a substance. In recent years the refinement of protein purification techniques and development of nucleic acid technology have yielded pure MIS protein and isolated the MIS gene. Gene-targeting technology has created female transgenic mice overexpressing MIS and male mice in whom the gene for MIS "is knocked out" (2, 3). The females do not develop a uterus and the males retain their Mullerian system. This is exactly as Jost's physiologic experiments almost 50 years ago predicted. To this day, Jost's experiments are a model for all students of science because they demonstrate the importance of careful advance planning, meticulous execution, and critical analysis of experimental data. Important research always generates more questions and Jost's work was the trigger for the next important question-what causes the testis to develop? The answer to this question required the development of a new technology-recombinant DNA. Deletion mapping of the Y chromosome in sex-reversed human mutants (46,XX males and 46,XY females) isolated sex-related-Y (SRY) in 1990 (4). The protein encoded by the SRY gene appears to be the primary sex-determining signal for mammalian testis development. The current challenge is to understand how SRY expression initiates the biosynthesis of androgens in fetal Leydig cells and MIS in fetal Sertoli cells. The gene that appears to be capable of this dual activity goes by the moniker offushi tarazu factor-I (FTZ-Fl). Targeted homozygous disruption of the Ftz-Fl gene in mice interferes with adrenal and gonadal development (5). Fushi-tarazu factor-l (Ftz-Fl) is expressed in fetal Sertoli and Leydig cells. It is a gene that appears to be required absolutely for embryonic differentiation of steroidogenic tissue. Fushi-tarazu factor-l encodes a nuclear receptor protein that is designated as steroidogenic factor 1 (SF-I). Recent evidence from a young American Gynecological and Obstetrical Society funded investigator, Wen-hui Shen and her colleagues (6) strongly suggests that SF-I, in addition to being a global regulator of all steroid enzyme genes, binds to the regulatory region of MIS and directly regulates this important gene. The activity of Jost's putative l'hormone inhibitrice (AMH) comes on soon after the expression of SRY and SF1. This suggests that SRY up-regulates SF-l directly or indirectly to stimulate T production by
Vol. 65, No.4, April 1996
Leydig cells and SF-l in turn regulates MIS gene expression leading to Mullerian duct regression. SF-l appears to be the key stimulus for the biosynthesis of androgens in Leydig cells and activation of MIS expression in Sertoli cells. The complete characterization of the SF-l protein as a hormonally regulated transcription activator awaits the identification of its ligand and other cofactors. The other genes known to be involved in the male sexdetermining pathway are all involved in transcriptional control; however, their target genes have not been defined yet. The link between those genes expressed in Sertoli cells (SRY, SF-I, MIS) and others that are more related to the developing mesenchyme Wilms tumor gene (Wt-l) remain elusive. Regardless of the ultimate complexity of this unique developmental system, Jost will be remembered as the first to challenge the simplifying assumptions that handicapped the progress of science in this area. He was a one-man patriarch and pioneer in developmental biology, fetal endocrinology, and the experimental method. Sex determination has become a model for understanding development because it is one ofthe few systems in which there is a clear, distinct choice between two developmental fates. Jost recognized that the elimination of the Mullerian system was a critical first step in the pathway to maleness. Jost's work came at a very appropriate time in the evolution of thought about mammalian sex determination. At the same time, a young Canadian anatomist by the name of Murray Barr and his graduate student E.G. Bertrand (7) made a serendipitous observation while studying the responses of cells in the central nervous systems of cats to various nerve stimuli: central nervous system cells in female cats had a distinct nuclear mass that was absent in males. The discovery of MIS by Jost (1) and the identification of sex chromatin by Barr and Bertrand (7) were scientific twists of fate that opened the door for all future investigators to walk through. These discoveries were the launching pad or perhaps space station for all future studies of mammalian sex determination.
REFERENCES 1. Jost A. Recherches sur la differenciation sexuelle de l'embryon de lapin III. Role des gonades foetales dans la differenciation sexuelle somatique. Arch Anat Microsc Morphol Exp 1947;36:271-315. 2. Behringer RR, Cate RL, Froelick GJ, Palmiter RD, Brinster
McDonough Moments in reproductive medicine
705
RL. Abnormal sexual development in transgenic mice chronically expressing Mullerian inhibiting substance. Nature 1990;345: 167 -70. 3. Behringer RR, Finegold MJ, Cate RL. Mullerian inhibiting substance is required for normal male development and fertility. Cell 1994;79:415-25. 4. Gubbay J, Collignon J, Koopman P, Capel B, Economou A, Munsterberg A, et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990;346: 245-50.
706
McDonough
Moments in reproductive medicine
5. Luo X, Ikeda Y, Parker KL. A cell specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994;77:481-90. 6. Shen WH, Moore CD, Ikeda Y, Parker KL, Ingraham HA. Nuclear receptor steroidogenic factor 1 regulates the mullerian inhibiting substance gene: a link to the sex determination cascade. Cell 1994;77:651-61. 7. Barr ML, Bertrand EG. A morphological distinction between neurones of the male and female, and the behavior of the nucleolar satellite during accelerated nucleoprotein synthesis. Nature 1949;163:676-7.
Fertility and Sterility
medlc.lne..
. > . .•. . . . . ........<..
•..•......
Society of Reproductive~ll~9(;ooologists
FERTILITY AND STERILITY Copyright
:0
Vol. 65, No.4, April 1996 Printed on acid-free paper in U. S. A.
1996 American Society for Reproductive Medicine
The first giant step for males
Paul G. McDonough, M.D. * Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia
Male development is anomalous in that it is not constitutive, but must be induced. During early development there are common gonadal accessory structures in both sexes. To progress from this stage of "neutrality" to male development requires the prenatal regression ofthe Mullerian system and the virilization ofWolffian structures. For decades, conventional wisdom held that T performed this dual function. In the late 1940s, a 32-year-old French physiologist suggested that a substance other than T might be responsible for Mullerian duct regression in male fetuses. He suspected complexity when simplifying assumptions were the norm. In a series of technically difficult experiments, Jost (1) was able to perform intrauterine castrations of fetal rabbits and conclude that a second, unidentified testicular hormone had the unique ability to regress the system that normally gives rise to the oviducts, uterus, and proximal vagina. Jost (1) named the putative factor "l'horReceived December 4, 1995. * Reprint requests: Paul G. McDonough, M.D., Department of Obstetrics and Gynecology, 1120 Fifteenth Street, Augusta, Georgia 30912-3360 (FAX: 706-721-6830). "Moments in Reproductive Medicine" is a quarterly section of Fertility and Sterility designed to recognize individuals or important milestones in the field of reproductive medicine. Each section will be generated by one of the four affiliated societies of the American Society for Reproductive Medicine: Society of Reproductive Endocrinologists, Society of Reproductive Surgeons, Society for Assisted Reproductive Technology, and Society for Male Reproduction and Urology.
704
McDonough Moments in reproductive medicine
mone inhibitrice" or Mullerian-inhibiting substance (MIS). In recent years it has been called Mullerian inhibiting factor or, simply, anti-Mullerian hormone (AMH). The data from Jost's experiments built a draw bridge for all future investigators of mammalian sex development. His experiments form the basis for what have been called "Jostian Principles." Jost himself would have rebelled at the phraseology because he was inherently skeptical of "absolute truths." In Jost's mind, principles are for physicists not biologists. Ifhe were alive today he would probably repeat his own experiments using the purified MIS. Except for a small coterie of individuals in the American scientific community, Jost's conclusions were not accepted universally. His champions were largely physicians who found his data to be remarkably consistent with nature's own experiments in this area. The technical difficulties involved in the intrauterine surgery and the small number of experimental subjects raised serious questions about reproducibility. Rabbits were scarce in post-World War II France and had a more important place on the kitchen table than in the laboratory. Jost had to make every rabbit count and this sometimes required attending to his subjects at home. At the experimental level one major objection to Jost's conclusions was that the injection of testicular secretions did not prevent the formation ofthe Mullerian derivatives in female fetuses. Subsequent attempts to study MIS were clouded by cumbersome bioassays with indistinct end points and by the small
Fertility and Sterility
quantities of MIS protein in tissue. These problems deterred most investigators from entering this field. Others simply doubted the existence of such a substance. In recent years the refinement of protein purification techniques and development of nucleic acid technology have yielded pure MIS protein and isolated the MIS gene. Gene-targeting technology has created female transgenic mice overexpressing MIS and male mice in whom the gene for MIS "is knocked out" (2, 3). The females do not develop a uterus and the males retain their Mullerian system. This is exactly as Jost's physiologic experiments almost 50 years ago predicted. To this day, Jost's experiments are a model for all students of science because they demonstrate the importance of careful advance planning, meticulous execution, and critical analysis of experimental data. Important research always generates more questions and Jost's work was the trigger for the next important question-what causes the testis to develop? The answer to this question required the development of a new technology-recombinant DNA. Deletion mapping of the Y chromosome in sex-reversed human mutants (46,XX males and 46,XY females) isolated sex-related-Y (SRY) in 1990 (4). The protein encoded by the SRY gene appears to be the primary sex-determining signal for mammalian testis development. The current challenge is to understand how SRY expression initiates the biosynthesis of androgens in fetal Leydig cells and MIS in fetal Sertoli cells. The gene that appears to be capable of this dual activity goes by the moniker offushi tarazu factor-I (FTZ-Fl). Targeted homozygous disruption of the Ftz-Fl gene in mice interferes with adrenal and gonadal development (5). Fushi-tarazu factor-l (Ftz-Fl) is expressed in fetal Sertoli and Leydig cells. It is a gene that appears to be required absolutely for embryonic differentiation of steroidogenic tissue. Fushi-tarazu factor-l encodes a nuclear receptor protein that is designated as steroidogenic factor 1 (SF-I). Recent evidence from a young American Gynecological and Obstetrical Society funded investigator, Wen-hui Shen and her colleagues (6) strongly suggests that SF-I, in addition to being a global regulator of all steroid enzyme genes, binds to the regulatory region of MIS and directly regulates this important gene. The activity of Jost's putative l'hormone inhibitrice (AMH) comes on soon after the expression of SRY and SF1. This suggests that SRY up-regulates SF-l directly or indirectly to stimulate T production by
Vol. 65, No.4, April 1996
Leydig cells and SF-l in turn regulates MIS gene expression leading to Mullerian duct regression. SF-l appears to be the key stimulus for the biosynthesis of androgens in Leydig cells and activation of MIS expression in Sertoli cells. The complete characterization of the SF-l protein as a hormonally regulated transcription activator awaits the identification of its ligand and other cofactors. The other genes known to be involved in the male sexdetermining pathway are all involved in transcriptional control; however, their target genes have not been defined yet. The link between those genes expressed in Sertoli cells (SRY, SF-I, MIS) and others that are more related to the developing mesenchyme Wilms tumor gene (Wt-l) remain elusive. Regardless of the ultimate complexity of this unique developmental system, Jost will be remembered as the first to challenge the simplifying assumptions that handicapped the progress of science in this area. He was a one-man patriarch and pioneer in developmental biology, fetal endocrinology, and the experimental method. Sex determination has become a model for understanding development because it is one ofthe few systems in which there is a clear, distinct choice between two developmental fates. Jost recognized that the elimination of the Mullerian system was a critical first step in the pathway to maleness. Jost's work came at a very appropriate time in the evolution of thought about mammalian sex determination. At the same time, a young Canadian anatomist by the name of Murray Barr and his graduate student E.G. Bertrand (7) made a serendipitous observation while studying the responses of cells in the central nervous systems of cats to various nerve stimuli: central nervous system cells in female cats had a distinct nuclear mass that was absent in males. The discovery of MIS by Jost (1) and the identification of sex chromatin by Barr and Bertrand (7) were scientific twists of fate that opened the door for all future investigators to walk through. These discoveries were the launching pad or perhaps space station for all future studies of mammalian sex determination.
REFERENCES 1. Jost A. Recherches sur la differenciation sexuelle de l'embryon de lapin III. Role des gonades foetales dans la differenciation sexuelle somatique. Arch Anat Microsc Morphol Exp 1947;36:271-315. 2. Behringer RR, Cate RL, Froelick GJ, Palmiter RD, Brinster
McDonough Moments in reproductive medicine
705
RL. Abnormal sexual development in transgenic mice chronically expressing Mullerian inhibiting substance. Nature 1990;345: 167 -70. 3. Behringer RR, Finegold MJ, Cate RL. Mullerian inhibiting substance is required for normal male development and fertility. Cell 1994;79:415-25. 4. Gubbay J, Collignon J, Koopman P, Capel B, Economou A, Munsterberg A, et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990;346: 245-50.
706
McDonough
Moments in reproductive medicine
5. Luo X, Ikeda Y, Parker KL. A cell specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994;77:481-90. 6. Shen WH, Moore CD, Ikeda Y, Parker KL, Ingraham HA. Nuclear receptor steroidogenic factor 1 regulates the mullerian inhibiting substance gene: a link to the sex determination cascade. Cell 1994;77:651-61. 7. Barr ML, Bertrand EG. A morphological distinction between neurones of the male and female, and the behavior of the nucleolar satellite during accelerated nucleoprotein synthesis. Nature 1949;163:676-7.
Fertility and Sterility