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In nonmammalian vertebrates, steroids have been hypothesized to induce somatic sex differentiation, since manipulations of the steroidal environment of gonads have led to various degrees of sex reversal. Whereas the critical role of estrogens in ovarian differentiation is well documented, studies on androgens have produced a perplexing variety of results depending upon species variations and nature of androgens used. In this way, testosterone induces masculinization of females in some species but provokes paradoxical feminization of males in many other species such as the urodelan Pleurodeles waltl. In reptiles this phenomenon could be interpreted by conversion of exogenous testosterone to estradiol by aromatase. Treatments of Pleurodeles larvae with nonaromatizable androgens bring support to this hypothesis and suggest a role of androgens in sex differentiation. Dihydrotestosterone (DHT) could not induce the paradoxical feminization of ZZ larvae. In addition, DHT as well as 11β-hydroxy-androstenedione could drive a functional male differentiation of ZW larvae. Moreover, other 5α reduced androgens also induced sex reversal of female larvae. Yet, the 5α reductase inhibitor CGP 53133 and antiandrogens such as flutamide or cyproterone acetate did not exert any effect on male sex differentiation of ZZ larvae. Though the precise role of androgens is still unknown, especially for 11-oxygenated androgens, our results suggest an implication in male sex differentiation. In this way, testosterone could play a pivotal role in being metabolized either into other androgens during testis differentiation or into estradiol during ovarian differentiation. J. Exp. Zool. 296A:46–55, 2003. © 2003 Wiley-Liss, Inc.

Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance, is a member of the transforming growth factor beta superfamily of growth and differentiation factors. In contrast to other members of the family, which exert a broad range of functions in multiple tissues, the principal function of AMH is to induce regression of the Mullerian ducts during male sex differentiation. However, the patterns of expression of AMH and its type II receptor in the postnatal ovary indicate that AMH may play an important role in ovarian folliculogenesis. This review describes several in vivo and in vitro studies showing that AMH participates in two critical selection points of follicle development: it inhibits the recruitment of primordial follicles into the pool of growing follicles and also decreases the responsiveness of growing follicles to FSH.

Mammalian sex differentiation involves the action of a cascade of genes. Discovery of the sex-determining region of the Y chromosome (SRY) marked the beginning of the delineation of the genes in the cascade. Studies of the genetics of mammalian sex reversal and the embryogenesis of the mice are essential in this endeavor. A number of genes involved in the pathway have been identified and all except one of these genes have a putative role in male sex differentiation. Besides SRY being the master switch in male sex differentiation the hierarchical relationship of the genes identified are far from being understood. Similarly, our knowledge of the genetic regulation of female sex differentiation is minimal. Differential screening and gene expression profiling bring a new dimension to the pursuit with the identification of a number of genes previously unknown to be involved in sex differentiation. Wider application of functional genomic techniques and introduction of proteomic analyses are expected to shed light to our understanding of this complicated developmental process.

The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site. The E. coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.

A region of homology, containing the contiguous SF3A2 (formerly called SAP62) and AMH genes, exists between human chromosome 19 (HSA19p) and mouse chromosome 10 (MMU10). In a previous study it was shown that SF3A2/Sf3a2 is very highly conserved between the two species and that AMH/Amh is somewhat less conserved although both human and mouse genes encode a protein (AMH) playing the same critical role during early male sex differentiation. The close association between SF3a2/Sf3a2 and AMH/Amh was thought to maintain open chromatin in the AMH/Amh promoter region, thus facilitating the necessary precise timing of AMH/Amh expression following that of SRY/Sry at the onset of testis differentiation. Further investigation of DNA upstream of Amh has revealed that there is another gene, in close association (about 400 bp) with Sf3a2, which has significant similarities to the N-terminus of a known guanine nucleotide releasing protein (GNRP) and consequently is provisionally named GNRPx/Gnrpx. The Gnrpx-Sf3a2-Amh (GSA) locus of the mouse (MMU10) is conserved in the human (HSA19p). Mapping the Sf3a2 transcription start site eventually led us to locate and characterize its promoter. We found that Sf3a2 and Gnrpx share a bi-directional promoter, with the latter being transcribed in an antisense direction. It has now been shown by RT-PCR analysis that both Sf3a2 and Gnrpx are widely expressed and therefore are likely to be ‘housekeeping’ genes. GNRPx/Gnrpx messenger RNA codes for a C-terminally truncated protein (149/164 aa), which contains an as yet uncharacterized domain common to GNRPs (and related proteins) and which may therefore act as a specific antagonist of a complete GNRP protein (>1200 aa) involved in the regulation of the GTPase (G-protein/Ras) cycle.

The genetics of sex determination is a child of the twentieth century, which over- turned the previously held view that sex was determined by the environment. The last quarter of the century witnessed an active search for sex-determining genes in mammals. Although success- ful, the modus operandi of these genes remained unknown, and the relationship between the sex- determining systems of mammals and other vertebrates remained enigmatic. To overcome these problems, scientists in the 21st century should heed William Bateson's counsel to treasure excep- tions, for they point the way to progress. One exception to conventional concepts of sex determina- tion is the bilaterally asymmetrical distribution of ovaries and testes in true hermaphroditism. Ovaries favour the left side in humans and the right side in mice. Observations suggesting that a reversal of asymmetry may occur with increasing organ size may point to a possible explanation. A reevaluation is also required regarding the beginning of sex differentiation, in view of mounting evidence of a sex difference in growth rates of early embryos. Another question to be settled is whether the function of SRY is confined to the fetal gonad. The recent demonstration that Sry induces cell proliferation in the fetal mouse gonad (Schmahl et al., 2000) further emphasizes the importance of differential growth in sex determination and differentiation. It is suggested that SRY represents an additional growth-promoting gene sequestered by mammals to enable the XY embryo to undergo male sex differentiation in the female hormonal environment of the uterus. An increased awareness of the relationship between growth and gonadal differentia- tion should lead to a better understanding of sex determination in mammals and an ability to relate the function of sex-determining genes to the effects of environmental factors. J. Exp. Zool. 290:484-489, 2001. © 2001 Wiley-Liss, Inc. LAYING THE FOUNDATIONS The genetics of sex determination is a child of the twentieth century, which overturned the pre- viously held view that sex was determined by en- vironmental conditions (Mittwoch, '73, '85). The century began with the rediscovery of Mendel's laws by three independent European investiga- tors (Correns, 1900; de Vries, 1900; Tschermak, 1900). In the years that followed, scientists in the USA discovered sex chromosomes in insects. An earlier finding by Henking (1891) in Gottingen, Germany, had established that as a result of meio- sis in the plant bug, Pyrrhocoris apterus, two kinds of spermatocytes were formed, one of which con- tained a large chromatin element, labeled X, which was absent in the other. This led McClung (1901), of Kansas University, to suggest that the odd chromosome was connected with the deter

Mammalian sex differentiation is a hormone-dependent process in the male following the determination of a testis from the indifferent gonad through a cascade of genetic events. Female sex differentiation is not dependent on ovarian hormones, yet there is evidence that members of the Wnt family of developmental signaling molecules play a role in Müllerian duct development and in suppressing Leydig cell differentiation in the ovary. The testis induces male sex differentiation (including testis descent) through a time-dependent production of optimal concentrations of anti-Müllerian hormone, insulin-like factor(s) and androgens. Observations in several human syndromes of disordered fetal sex development corroborate findings in murine embryo studies, although there are exceptions in some gene knockout models. The ubiquitously expressed AR interacts in a ligand-dependent manner with coregulators to control the expression of androgen-responsive genes. Preliminary studies suggest the possibility of hormone resistance syndromes associated with coregulator dysfunction. Polymorphic variants in genes controlling androgen synthesis and action may modulate androgenic effects on sex differentiation.

Anti-Mullerian hormone (AMH), a member of the transforming growth factor-β family, is an important factor of male sex differentiation. It is produced by Sertoli cells from the time of fetal sex differentiation to puberty. AMH is also produced by granulosa cells from the time of birth to the end of ovarian activity. As other members of the transforming growth factor-β family, AMH signals through two related but distinct receptors, both serine/threonine kinases with a single transmembrane domain, called type II and type I. The type II receptor has been cloned in 1994 and is expressed solely in AMH target organs. Engagement of the type I receptor BMPR-IB and downstream effector Smad1 by AMH has recently been demonstrated, however, its role in AMH biological actions remains to be proven.

A histological study was performed on serially sectioned human and mouse embryos to study the influences of programmed cell death (PCD) during morphogenesis for clarifying the existing controversies on the morphology and basic processes involved in the embryonic development of the male anterior urethra. The following new insights into the development of the anterior urethra could be established. The formation of the urethra starts with the early adhesion of the arms of the genital tubercle. In this way an epithelial plate is formed, located in the ventral midline, that is in continuity with the cloacal membrane. Male sex differentiation takes place following rupture of this cloacal membrane through programmed cell death. Fusion of the urogenital swellings with primary luminization gives rise to the penile urethra, whereas the glandular part of the urethra is formed through secondary luminization of the epithelial cord that is formed during fusion of the arms of the genital tubercle, i.e., the glans. In both fusion processes, apoptosis plays a key role. The consequence of fusion of the urogenital swellings is that their mesodermal cores unite on the ventral aspect of the penile urethra, where they differentiate into the integumental structures. The prepuce starts to develop as a fold of ectoderm with a mesodermal core after complete fusion of the entire urethra. Finally, the scrotum was found to develop through merging of the labioscrotal swellings and not by fusion. (C) 2000 Wiley-Liss, Inc.

Müllerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS−269 and −192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1- and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.

Müllerian Inhibiting Substance (MIS) functions to promote regression of the Müllerian duct during male development. Maintaining the sexually dimorphic pattern of MIS expression is essential for proper mammalian reproductive tract development. Here, we show that the intricate spatial and temporal pattern of MIS expression is directed by a remarkably small proximal promoter of only 180 base pairs in length. Expression of the MIS-human growth hormone transgene (MIS/GH) is restricted to Sertoli cells in embryonic testis and to granulosa cells of postnatal ovary, consistent with the known MIS expression pattern. The proximal MIS promoter is therefore sufficient to direct the initiation and the maintenance of MIS gene expression in both sexes. Moreover, in vivo MIS promoter activity requires an intact binding site for the orphan nuclear receptor SF-1. Taken together, these data strongly suggest that SF-1 directly activates MIS in embryonic and postnatal gonads. Consistent with the proposed role of SF-1 in mammalian sex-determination, our study provides physiological evidence that a SF-1 binding site is essential for gene activation of an embryonic testis-specific marker.

Anti-mullerian hormone (AMH) induces degeneration of the mullerian ducts during male sex differentiation and may have additional functions concerning gonadal development. In the immature rat testis, there is a marked developmental increase in AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in Sertoli cells, concomitant with the initiation of spermatogenesis. AMHRII mRNA is also expressed at a high level in Sertoli cells in adult rats. To obtain information about the possible functions of AMH in the testis, we investigated the postnatal expression patterns of the genes encoding AMH and AMHRII in the rat testis in more detail. Using RNase protection assays, AMH and AMHRII mRNA expression was measured in total RNA preparations from testes or testicular tubule segments isolated from control rats and from rats that had received various treatments. The testicular level of AMHRII mRNA was found to be much higher than that of AMH mRNA in adult rats. AMH mRNA was detected at a maximal level at stage VII of the spermatogenic cycle and at a low level at the other stages. AMHRII mRNA increases from stage XIII, is highest at stages VI and VII, and then rapidly declines at stage VIII to almost undetectable levels at stages IX-XII. It was found that the increase in testicular AMHRII mRNA expression during the first 3 weeks of postnatal development also occurs in sterile rats (prenatally irradiated), and hence, is independent of the presence or absence of germ cells. Yet, the total testicular level of AMHRII mRNA was decreased in sterile adult rats (prenatally irradiated or experimental cryptorchidism), as compared with intact control rats. However, treatment of adult rats with methoxyacetic acid or hydroxyurea, which resulted in partial germ cell depletion, had no effect on total testicular AMHRII mRNA expression. We conclude that a combination of multiple spermatogenic cycle events, possibly involving changes of Sertoli cell structure and/or Sertoli cell-basal membrane interactions, regulate autocrine AMH action on Sertoli cells, in particular at stage VII of the spermatogenic cycle.

The androgen insensitivity syndromes comprise a spectrum of phenotypic abnormalities in male sex differentiation and development that result from target tissue resistance to androgen action due to molecular lesions in the X-chromosome-linked androgen receptor gene. The androgen receptor, like other members of the superfamily of steroid receptors, is characterized by the presence of three structural domains that function in transcriptional activation, DNA binding, and steroid binding, respectively. Missense mutations in the androgen receptor gene causing amino acid substitutions are the most common molecular lesions among affected subjects; deletions, mRNA splice site alterations, and nonsense mutations occur less frequently. Because of the large number and diverse array of these naturally occurring mutations and their associated clinical phenotypes, there is a great opportunity for understanding the structure-function relationships of the androgen receptor from in vitro and in vivo expression of the mutant receptors in various cell types.

I. Introduction IN 1953 lohn Morris, an obstetrician at Yale University, reported a series of 82 individuals (80 cases collated from the literature and two cases of his own) who had a female phenotype despite the presence of bilateral testes (1). Since his initial description, studies of the endocrinology, pathophysiology, biochemistry, and molecular biology of the androgen insensitivity syndrome (AIS) have provided insights into the role of androgens in male sex differentiation, the mechanisms of androgen action, and aspects of the structure/function relationships of the androgen receptor. AIS is an archetypal example of a hormone resistance disorder. Androgens are secreted by the testes of these 46,XY individuals in normal or increased amounts; however, due to defective androgen receptor (AR1) function, there is loss of target organ response to the hormone, and the effects of androgens are reduced or absent. Clinical disorders of the AR are reported far more commonly than resistance disorders of other m...

Induction of testis development in mammals requires the presence of the Y-chromosome gene SRY. This gene must exert its effect by interacting with other genes in the sex-determination pathway. Cloning of a translocation chromosome breakpoint from a sex-reversed patient with campomelic dysplasia, followed by mutation analysis of an adjacent gene, indicates that SOX9, an SRY-related gene, is involved in both bone formation and control of testis development.

Normal male sex differentiation requires that Sertoll cells in the embryonic testes produce Mullerian Inhibiting substance (MIS), a TGFβ-like hormone that causes Mullerian duct regression. In primary Sertoli cells, the orphan nuclear receptor, steroidogenic factor 1 (SF-1), regulates the MIS gene by binding to a conserved upstream regulatory element. In heterologous (HeLa) cells, MIS gene activation by SF-1 requires removal of the SF-1 ligand-binding domain, implicating a Sertoll cell-specific ligand or cofactor. Finally, the sexually dimorphic expression of SF-1 during development coincides with MIS expression and Mullerian duct regression. We propose that SF-1 regulates MIS in vivo and participates directly in the process of mammalian sex determination.

Studies in adrenocortical cells have implicated the orphan nuclear receptor SF-1 in the gene regulation of the steroid hydroxylases. We used targeted disruption of the Ftz-F1 gene, which encodes SF-1, to examine its role in intact mice. Despite normal survival in utero, all Ftz-F1 null animals died by postnatal day 8; these animals lacked adrenal glands and gonads and were severely deficient in corticosterone, supporting adrenocortical insufficiency as the probable cause of death. Male and female Ftz-F1 null mice had female internal genitalia, despite complete gonadal agenesis. These studies establish that the Ftz-F1 gene is essential for sexual differentiation and formation of the primary steroidogenic tissues.

Temperate wrasse (Halichoeres poecilopterus) is known to undergo sex transition from female to male (protogyny). In order to detect factors related to male sex differentiation which appear during sex transition, we compared protein constituents between transitional and mature gonads by two-dimensional gel electrophoresis. Eight proteins were found to increase in amount considerably in the transitional gonads in comparison with the counterparts in ovaries. Five of the eight proteins were also found in mature testes. One of the proteins shared by testes and transitional gonads had an isoelectric point at pH 5.3 and an apparent molecular weight of 26 kDa. Thus we termed the protein p 26. It was abundant in degenerating ovary, transitional gonad, and mature testis and less abundant in mature ovary and brain. Therefore, the expression of p 26 seemed to start during sex transition and continue during maturation of the testis. Amino acid sequence analysis revealed high homology of p 26 to ubiquitin C-terminal hydrolase, which is supposed to cleave ubiquitin associated with nuclear proteins and a transcriptional repressor. Therefore, p 26 was supposed to regulate gene expression through the mediation of ubiquitin. Proteins which seemed to be counterparts of p 26 were detected in testes of two other fish species by an antiserum against p 26. These results suggest that p 26 is a possible candidate for the factor which is related to sex differentiation.

Publisher Summary The correlation between the presence of a Y chromosome and a male phenotype suggests that a gene on the Y governs male sex differentiation in mammals. One Y chromosome is sufficient for testicular development, even in the presence of supernumerary X chromosomes, as in human individuals with 47,XXY, 48,XXXY, or 49,XXXXY karyotypes. This chapter discusses the findings on the structure and function of the human Y chromosome. Results obtained by deletion mapping and pulsed-field gel electrophoresis are reviewed and the assignment of the testis-determining locus (TDF) to the distal short arm of the Y chromosome is deduced. Experimental findings have been reviewed. These findings suggest that SRY is the testis-determining gene in man and its mouse homolog Sry is pivotal in murine testicular induction. Findings have been reported in one exceptional (Y+) XX male of DNA sequences from distal Yp and proximal Yq. The findings in this unusual XX male are explained by an aberrant interchange between an X and a rearranged Y chromosome during paternal meiosis.

Testosterone (T) is synthesized and secreted by testicular Leydig cells. High levels of T must be maintained within the seminiferous tubules of the testes to promote and maintain the process of spermatogenesis, whereas relatively lower concentrations of T are available to peripheral tissues from the general circulation. Androgen-dependent target tissues concentrate androgens by means of the specific, high-affinity binding of T and its potent 5α-reduced metabolite, dihydrotestosterone (DHT), to intracellular androgen receptors (AR). The intranuclear binding of the androgen receptor-steroid complex to DNA leads to the regulation of androgen-dependent gene transcription. Early aspects of male sex differentiation and development during the first trimester of fetal life are dependent on the normal biological function of the AR in tissues of the internal male reproductive tract and the external genitalia.

Publisher Summary The critical factor in mammalian sex determination and differentiation is the development of the gonad. It has been shown that the testes produce testosterone and anti-Mullerian hormone (AMH), which are responsible for inducing the male secondary sex characteristics. Sex determination can be reduced to its essence: the choice between forming testis or ovary. Once gonadal sex is determined, the subsequent processes—sex differentiation—commence. In mammals, sex determination is chromosomally based: females have two X chromosomes and males have an X and a Y. These results implied that the Y chromosome carried a gene required for testis development. In humans, this gene was called the “testis determining factor” (TDF) because of the uncertain nature of the genetic material involved. The role of TDF as a “switch” in sex determination is misunderstood. Many genes are required in the pathway leading to testis formation. Some of these genes are autosomal and some, X-linked, but in the absence of TDF, the pathway would not produce testes. In the presence of TDF, the pathway is completed and testes are produced, leading to male sex differentiation.

Publisher Summary This chapter describes anti-Mullerian hormone. It discusses the molecular mechanisms of gene expression and protein bioactivity and the clinical applications of AMH research. In Jost's laboratory, the factor responsible for Mullerian regression was called hormone inhibitrice. The homozygosity of the mutated allele was established by the results of allele-specific hybridization, which showed that the patient's DNA hybridized only with the mutant oligonucleotide probe and not with the wild-type one. Identical results were obtained with DNA from the two other affected siblings. Although involved in male sex differentiation, the AMH gene is not located on a sex chromosome, in keeping with the tenet that only the genetic trigger for gonadal sex determination needs to be sex specific. Recent data on the location of the AMH gene in other species are available at present. No association to genes located on human chromosome 19 has been detected, indicating complex rearrangements in chromosomal structure during evolution.

The genetic terminology of sex determination and sex differentiation is examined in relation to its underlying biological basis. On the assumption that the function of the testis is to produce hormones and spermatozoa, the hypothesis of a single Y-chromosomal testis-determining gene with a dominant effect is shown to run counter to the following observed facts: a lowering in testosterone levels and an increase in the incidence of undescended testes, in addition to sterility, in males with multiple X chromosomes; abnormalities of the testes in autosomal trisomies; phenotypic abnormalities of XX males apparently increasing with decreasing amounts of Y-chromosomal material; the occurrence of patients with gonadal dysgenesis and XY males with ambiguous genitalia in the same sibship; the occurrence of identical SRY mutations in patients with gonadal dysgenesis and fertile males in the same pedigree; and the development of XY female and hermaphrodite mice having the same genetic constitution. The role of X inactivation in the production of males, females and hermaphrodites in T(X;16)16H mice has previously been suggested but not unequivocally demonstrated; moreover, X inactivation cannot account for the observed bilateral asymmetry of gonadal differentiation in XY hermaphrodites in humans and mice. There is evidence for a delay in development of the supporting cells in XY mice with ovarian formation. Once testicular differentiation and male hormone secretion have begun, other Y-chromosomal genes are required to maintain spermatogenesis and to complete spermiogenesis, but these genes do not function effectively in the presence of more than one X chromosome. The impairment of spermatogenesis by many other chromosome abnormalities seems to be more severe than that of oogenesis. It is concluded that the notion of a single testis-determining gene being responsible for male sex differentiation lacks biological validity, and that the genotype of a functional, i.e. fertile, male differs from that of a functional female by the presence of multiple Y-chromosomal genes in association with but a single X chromosome. Male sex differentiation in XY individuals can be further impaired by a euploid, but inappropriate, genetic background. The genes involved in testis development may function as growth regulators in the tissues in which they are active.

The initiation of male development in mammals requires one or more genes on the Y chromosome. A recently isolated gene, termed SRY in humans and Sry in mouse, has many of the genetic and biological properties expected of a Y-located testis-determining gene. It is now shown that Sry on a 14-kilobase genomic DNA fragment is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.

The basic nature of the greatly diversified sexualities in fish can be considered from the aspects of the control mechanisms, physiological and genetic sexes. The potencies of sex steroids to induce sex reversal are different for each steroid. Functional sex reversal from female to male is carried out by using 17α-methyltestosterone, 19-norethynyltesto-sterone or methylandrosterone. 11-ketotestosterone or androsterone have also been used but the dosage required was higher than those of synthetic androgens. The effective range of methyltestosterone required to induce masculinization in medaka, goldfish and tilapia is 20–30 mg/kg diet and rather wide, in the range 1–100 mg/kg diet, in zebrafish. In rainbow trout, the optimum concentration is quite low, in the range 0.1–3 mg/kg diet. In this species, treatment with more than 10 mg/kg diet induces sterile or hermaphroditic gonads and rarely induces sex reversal. Sex differentiation commences after hatching, either before or after the initiation of feeding. In viviparous fish such as guppy, the female is differentiated prior to parturition, and so the administration of methyltesto-sterone is started before parturition and continued until birth. In rainbow trout the first 8 weeks of the oral treatment are important. Treatment for longer than 60 days may be meaningless. Phenotypical feminization is induced successfully by using estradiol-17β. Estrone, diethylstilbestrol or ethynylestradiol are also used. Treatment with steroids is usually carried out by feeding. In salmon, where male sex differentiation is initiated before feeding commences, other procedures are used, such as immersion of alevins. Chromosome manipulation also has great possibilities for sex control. Artificial gynogenesis in species having female homogamety produces all female fry. Triploidy produces sterile fish in rainbow trout. This can be achieved easily by applying hydrostatic pressure or temperature shock. Particularly fruitful techniques for control of sex in aquacultured fish will involve the combination of steroid treatment with chromosome manipulation.

A female rabbit was immunized against purified bovine AMH and mated. Booster injections were given at Day 8 of pregnancy to ensure a high titer of anti-AMH antibodies at the time the rabbit fetal testis begins to produce AMH. In three consecutive litters, the immunized female produced a total of 12 males, 9 of which had persistent Müllerian duct derivatives. No other significant abnormalities were detected in these animals, which were compared to the offspring of a control saline-injected female. In particular, testicular morphology was normal in most animals, and serum FSH levels did not differ from controls. This experimental model lends no support to the hypothesis that AMH controls extra-Müllerian events of male sex differentiation, nor that of the existence of a regulatory mechanism for synthesis of AMH by Sertoli cells, but it does not definitely exclude these possibilities, inasmuch as our tentative conclusions are based upon study of only one immunized female.

The relative binding affinity of serum proteins for testosterone (BAPT) has been evaluated in 5 normal men, 3 normal women, 4 patients with testicular feminization syndrome and 3 young hypogonadal men. A “coefficient of cutaneous testosterone 5α-reductase activity” (T5αR) was also calculated in the same patients by means of an in vivo method which consisted of the combined administration of radioactive testosterone differently labeled via percutaneous and intravenous routes and then of the comparison of the isotopic ratios of urinary 5α- and 5β-androstane-3α,17β-diols. From the results obtained, it appears likely that the proportions of BAPT and T5αR in normal men are in versed both in patients with feminizing testes and in hypogonadal men. In addition, after treatment of one hypogonadal man with dihydrotestosterone (DHT), a male sex differentiation was observed and both BAPT and T5αR became equal to those of normal men. On the contrary, in the case of a castrated patient with testicular feminiza...

Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.

SEVERAL DIFFERENT KARYOTYPES, some with mosaicism, have now been demonstrated in cells from patients with the disorder of male sex differentiation known as Klinefelter's syndrome. The XXY sex chromosome pattern was the first aberration described in Klinefelter's syndrome1and has been the most frequent finding in cases subsequently studied. It has been suggested that those patients with Klinefelter's syndrome, but with karyotypes other than XXY, be considered as variants.2The sex chromosome pattern in the variants thus far recognized include XXXY, XXXXY, XXYY, XXXYY, and the mosaic karyotypes of XX/XXY, XY/XXY, XY/XXXY, XXXY/XXXXY, XX/XXY/ XXYYY. Although patients having these different karyotypes cannot be distinguished unequivocally on clinical grounds alone, it appears that the severity of mental retardation and disturbances of sex differentiation increase as the number of extra X chromosomes increases. This case of Klinefelter's syndrome is reported to provide clinical and cytological findings of a patient with

Mature female rabbits immunized with T-3-BSA developed highly specific antisera against T. Animals with the highest antibody titres revealed elevated plasma T-levels up to 2 000 ng/dl (normal: 15 ng/dl). Nevertheless, mating was successful and pregnancy uncomplicated. Immediately after normal delivery, the newborns were bled and fixed in Bouin's solution. In all neonatal plasma samples high concentrations of antibodies against T were found with identical characteristics as in the maternal plasma. T-levels were excessively elevated: up to 2 500 ng/dl in females and up to 25 000 ng/dl in males (normal: 20 ng/dl). Histological examination, however, revealed normal sex differentiation in the females, but inhibited male sex differentiation in the males: partial retrogression of Wolffian duct derivatives, absence of prostatic glands, feminized phallus with reduced corpus fibrosum, and marked hypospadias; testicular differentiation was normal, Mullerian ducts were absent. Our experiments indicate that hormones essential for normal fetal development can be neutralized in the fetus by their respective antibodies raised in the mother.