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Müllerian ducts give rise to the oviducts, uterus, cervix, and vagina. During female reproductive tract development in mice, the bilateral Müllerian duct epithelium grows caudally until reaching the urogenital sinus epithelium. This is followed by further caudal growth with the reduction of the urogenital sinus epithelium. Finally, the vaginal epithelium of adult mice is entirely derived from the Müllerian duct epithelium. Here, we explored the mechanisms underlying mouse vaginal development via cell proliferation, apoptosis, and lineage analyses. We found that at the late embryonic stages, apoptosis occurred at the attachment site of bilateral Müllerian duct epithelia below the cervix, resulting in bilateral lumen traffic. The Müllerian duct epithelium was enclosed by the urogenital sinus epithelium at their boundary region on embryonic day (E) 16.5, whereas the Müllerian duct epithelium encased the urogenital sinus epithelium at postnatal day (P) 0 through lateral enlargement. Lateral Müllerian duct enlargement was accompanied by focal ERK activation within the curved epithelial tips and the specific localization of mitotic nuclei on the luminal side of the Müllerian duct epithelial layer at E17.5. Descent of the Müllerian duct epithelium and shortening of the urogenital sinus epithelium occurred rapidly after birth, accompanied by cell proliferation in the Müllerian duct epithelium and its peripheral mesenchymal tissues as well as intense apoptosis in the urogenital sinus epithelium around their boundary region. Urogenital sinus epithelium was localized at the base of the vagina at P7. In conclusion, the mouse vagina develops laterally at the late embryonic stages and caudally after birth., (© 2022 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.)

The vertebrate female reproductive tract has undergone considerable diversification over evolution, having become physiologically adapted to different reproductive strategies. This review considers the female reproductive tract from the perspective of evolutionary developmental biology (evo-devo). Very little is known about how the evolution of this organ system has been driven at the molecular level. In most vertebrates, the female reproductive tract develops from paired embryonic tubes, the Müllerian ducts. We propose that formation of the Müllerian duct is a conserved process that has involved co-option of genes and molecular pathways involved in tubulogenesis in the adjacent mesonephric kidney and Wolffian duct. Downstream of this conservation, genetic regulatory divergence has occurred, generating diversity in duct structure. Plasticity of the Hox gene code and wnt signaling, in particular, may underlie morphological variation of the uterus in mammals, and evolution of the vagina. This developmental plasticity in Hox and Wnt activity may also apply to other vertebrates, generating the morphological diversity of female reproductive tracts evident today., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) enzymes are secreted metalloproteinases with major roles in development, morphogenesis, and tissue repair via the assembly and degradation of extracellular matrix (ECM). In this study, we investigated the role of ADAMTS18 in the development of the reproductive tract in female mice by phenotyping Adamts18 knockout (Adamts18 -/- ) mice. The results showed that Adamst18 mRNAs were abundantly expressed in vaginal epithelial cells and muscularis cells of the developing vagina. At the time of vaginal opening (5 weeks of age), about 41 % of Adamts18 -/- females showed enlarged protrusions in the upper and middle parts of the vagina, reduced vaginal length, and simultaneously exhibited vaginal atresia. 6% Adamts18 -/- females exhibited vaginal septum. Histological analyses revealed that the paired Mullerian ducts in ∼33 % female Adamts18 -/- embryos failed to fuse at embryonic day 15.5 (E15.5) resulting in the formation of two vaginal cavities. Results of TUNEL assay and immunohistochemistry for caspase-3 showed that the number of apoptotic cells in the terminal portion of the vagina of 5-week-old Adamts18 -/- females with vaginal atresia was significantly decreased. Adamts18 -/- females also showed a significant decrease in serum estradiol E2 compared to age-matched Adamts18 +/+ females. Results of qRT-PCR showed that the expression level of the anti-apoptosis gene Bcl-2 was significantly increased and that of the apoptosis-related gene Epha1 was decreased in the vagina of 5-week-old Adamts18 -/- females. These results suggest that ADAMTS18 regulates vaginal opening through influencing the fusion of Mullerian ducts and apoptosis of vaginal cells in mice., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The development of the female reproductive tract can be divided into three parts consisting of Müllerian duct organogenesis, pre-sexual maturation organ development, and post-sexual maturation hormonal regulation. In primates, Müllerian duct organogenesis proceeds in an estrogen independent fashion based on transcriptional pathways that are suppressed in males by the presence of AMH and SRY. However, clinical experience indicates that exposure to xenoestrogens such as diethylstilbestrol (DES) during critical periods including late organogenesis and pre-sexual maturational development can have substantial effects on uterine morphology, and confer increased risk of disease states later in life. Recent evidence has demonstrated that these effects are in part due to epigenetic regulation of gene expression, both in the form of aberrant CpG methylation, and accompanying histone modifications. While xenoestrogens and selective estrogen receptor modulators (SERMS) both can induce non-canonical binding confirmations in estrogen receptors, the primate specific fetal estrogens Estriol and Estetrol may act in a similar fashion to alter gene expression through tissue specific epigenetic modulation., (Copyright © 2021 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The study of male and female reproductive tract development requires expertise in two separate disciplines, developmental biology and endocrinology. For ease of experimentation and economy, the mouse has been used extensively as a model for human development and pathogenesis, and for the most part similarities in developmental processes and hormone action provide ample justification for the relevance of mouse models for human reproductive tract development. Indeed, there are many examples describing the phenotype of human genetic disorders that have a reasonably comparable phenotype in mice, attesting to the congruence between mouse and human development. However, anatomic, developmental and endocrinologic differences exist between mice and humans that (1) must be appreciated and (2) considered with caution when extrapolating information between all animal models and humans. It is critical that the investigator be aware of both the similarities and differences in organogenesis and hormone action within male and female reproductive tracts so as to focus on those features of mouse models with clear relevance to human development/pathology. This review, written by a team with extensive expertise in the anatomy, developmental biology and endocrinology of both mouse and human urogenital tracts, focusses upon the significant human/mouse differences, and when appropriate voices a cautionary note regarding extrapolation of mouse models for understanding development of human male and female reproductive tracts., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish ( Danio rerio ) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh To discover the roles of Amh in the absence of Müllerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes., (Copyright © 2019 by the Genetics Society of America.)

Study Question: Is there an increased prevalence of male microchimerism in women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, as evidence of fetal exposure to blood and anti-Müllerian hormone (AMH) from a (vanished) male co-twin resulting in regression of the Müllerian duct derivatives?, Summary Answer: Predominant absence of male microchimerism in adult women with MRKH syndrome does not support our hypothesis that intrauterine blood exchange with a (vanished) male co-twin is the pathophysiological mechanism., What Is Known Already: The etiology of MRKH is unclear. Research on the phenotype analogous condition in cattle (freemartinism) has yielded the hypothesis that Müllerian duct development is inhibited by exposure to AMH in utero. In cattle, the male co-twin has been identified as the source for AMH, which is transferred via placental blood exchange. In human twins, a similar exchange of cellular material has been documented by detection of chimerism, but it is unknown whether this has clinical consequences., Study Design, Size, Duration: An observational case-control study was performed to compare the presence of male microchimerism in women with MRKH syndrome and control women. Through recruitment via the Dutch patients' association of women with MRKH (comprising 300 members who were informed by email or regular mail), we enrolled 96 patients between January 2017 and July 2017. The control group consisted of 100 women who reported never having been pregnant., Participants/materials, Setting, Methods: After written informed consent, peripheral blood samples were obtained by venipuncture, and genomic DNA was extracted. Male microchimerism was detected by Y-chromosome-specific real-time quantitative PCR, with use of DYS14 marker. Possible other sources for microchimerism, for example older brothers, were evaluated using questionnaire data., Main Results and the Role of Chance: The final analysis included 194 women: 95 women with MRKH syndrome with a mean age of 40.9 years and 99 control women with a mean age of 30.2 years. In total, 54 women (56.8%) were identified as having typical MRKH syndrome, and 41 women (43.2%) were identified as having atypical MRKH syndrome (when extra-genital malformations were present). The prevalence of male microchimerism was significantly higher in the control group than in the MRKH group (17.2% versus 5.3%, P = 0.009). After correcting for age, women in the control group were 5.8 times more likely to have male microchimerism (odds ratio 5.84 (CI 1.59-21.47), P = 0.008). The mean concentration of male microchimerism in the positive samples was 56.0 male genome equivalent per 1 000 000 cells. The prevalence of male microchimerism was similar in women with typical MRKH syndrome and atypical MRKH syndrome (5.6% versus 4.9%, P = 0.884). There were no differences between women with or without microchimerism in occurrence of alternative sources of XY cells, such as older brothers, previous blood transfusion, or history of sexual intercourse., Limitations, Reason for Caution: We are not able to draw definitive conclusions regarding the occurrence of AMH exchange during embryologic development in women with MRKH syndrome. Our subject population includes all adult women and therefore is reliant on long-term prevalence of microchimerism. Moreover, we have only tested blood, and, theoretically, the cells may have grafted anywhere in the body during development. It must also be considered that the exchange of AMH may occur without the transfusion of XY cells and therefore cannot be discovered by chimerism detection., Wider Implications of the Findings: This is the first study to test the theory that freemartinism causes the MRKH syndrome in humans. The study aimed to test the presence of male microchimerism in women with MRKH syndrome as a reflection of early fetal exposure to blood and AMH from a male (vanished) co-twin. We found that male microchimerism was only present in 5.3% of the women with MRKH syndrome, a significantly lower percentage than in the control group (17.2%). Our results do not provide evidence for an increased male microchimerism in adult women with MRKH as a product of intrauterine blood exchange. However, the significant difference in favor of the control group is of interest to the ongoing discussion on microchimeric cell transfer and the possible sources of XY cells., Study Funding/competing Interest(s): None., Trial Registration Number: Dutch trial register, NTR5961., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In female mice, the Müllerian duct develops into the oviduct, uterus and vagina. The fate of epithelia is determined by factors secreted from the mesenchyme. Retinoic acid (RA) and its receptors are present in the mesenchyme of cranial Müllerian duct. RA induces Müllerian duct to uterine epithelial differentiation whereas inhibition of RA receptors induces vaginal epithelial differentiation. Thus, RA signaling in the Müllerian duct is required to promote differentiation of the mesenchyme into the future uterus. Perinatal estrogen exposure induces various abnormalities in Müllerian duct-derived organs. These include a cranial shift of the border among oviduct, uterus and vagina as well as precancerous lesions suppressed by co-treatment with RA and estrogen. Since RA synthesis enzymes and receptors are expressed both in the epithelium and stroma after birth, RA signaling may act in the epithelia to maintain adult epithelial homeostasis and to prevent irreversible lesions induced by perinatal estrogen exposure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The reproductive cycle encompasses processes such as sex organ differentiation and development in the early life stages and maturation of the gametes in the adult organism. During the early life stages, critical developmental programming of the endocrine and reproductive systems occurs, and exposure to chemicals during these critical developmental windows can result in impaired reproductive function later in life. It is therefore important to evaluate long-term consequences of early life stage exposure to endocrine-disrupting chemicals. The African clawed frog Xenopus tropicalis has several characteristics that facilitate studies of developmental and reproductive toxicity. Here I present a X. tropicalis life cycle test protocol including study design, exposure regimes, and endpoints for chemical disruption of sex differentiation, gonadal and Müllerian duct development, the thyroxin-regulated metamorphosis, estrogen synthesis (activity of the CYP19 aromatase enzyme), spermatogenesis, oogenesis, puberty and fertility.

Development of the human female reproductive tract is reviewed from the ambisexual stage to advanced development of the uterine tube, uterine corpus, uterine cervix and vagina at 22 weeks. Historically this topic has been under-represented in the literature, and for the most part is based upon hematoxylin and eosin stained sections. Recent immunohistochemical studies for PAX2 (reactive with Müllerian epithelium) and FOXA1 (reactive with urogenital sinus epithelium and its known pelvic derivatives) shed light on an age-old debate on the derivation of vaginal epithelium supporting the idea that human vaginal epithelium derives solely from urogenital sinus epithelium. Aside for the vagina, most of the female reproductive tract is derived from the Müllerian ducts, which fuse in the midline to form the uterovaginal canal, the precursor of uterine corpus and uterine cervix an important player in vaginal development as well. Epithelial and mesenchymal differentiation markers are described during human female reproductive tract development (keratins, homeobox proteins (HOXA11 and ISL1), steroid receptors (estrogen receptor alpha and progesterone receptor), transcription factors and signaling molecules (TP63 and RUNX1), which are expressed in a temporally and spatially dynamic fashion. The utility of xenografts and epithelial-mesenchymal tissue recombination studies are reviewed., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Background/aim: Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance (MIS), is glycoprotein hormone secreted by the fetal Sertoli cells to regulate regression of the Müllerian ducts, the anlagen of the uterus, fallopian tubes, and upper vagina. After its existence was predicted in 1946 and its isolation and purification in the 1970's, a huge amount of information has been gathered on its molecular biology and function in the last 30-40years. Once thought to be a locally acting factor in the male fetus during sexual differentiation, it is now recognized as an endocrine hormone present in both sexes and with functions throughout life. One of the remaining controversies is the possible role of AMH during fetal testicular descent. In the human with aberrant AMH function, the boy has cryptorchidism with persistent Müllerian duct syndrome (PMDS), where the testes are often intraabdominal and on an abnormally long gubernacular cord. By contrast, in rodent models knockout of the AMH gene does not cause cryptorchidism., Methods/results: In this review we examined the evidence in the literature for and against a role for AMH in testicular descent and considered the implications of the different anatomy of the gubernacular cord in rodents versus children., Conclusion: We conclude that AMH may have a role in shortening the gubernacular cord in humans which is concealed in rodent models by differences in anatomy of the gubernacular cord in rodents. The controversy could be resolved by re-examination of the gubernacular cord in boys with PMDS and mice with AMHKO., Type of Study: Review., Level of Evidence: V., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We present a detailed review of the embryonic and fetal development of the human female reproductive tract utilizing specimens from the 5th through the 22nd gestational week. Hematoxylin and eosin (H&E) as well as immunohistochemical stains were used to study the development of the human uterine tube, endometrium, myometrium, uterine cervix and vagina. Our study revisits and updates the classical reports of Koff (1933) and Bulmer (1957) and presents new data on development of human vaginal epithelium. Koff proposed that the upper 4/5ths of the vagina is derived from Müllerian epithelium and the lower 1/5th derived from urogenital sinus epithelium, while Bulmer proposed that vaginal epithelium derives solely from urogenital sinus epithelium. These conclusions were based entirely upon H&E stained sections. A central player in human vaginal epithelial development is the solid vaginal plate, which arises from the uterovaginal canal (fused Müllerian ducts) cranially and squamous epithelium of urogenital sinus caudally. Since Müllerian and urogenital sinus epithelium cannot be unequivocally identified in H&E stained sections, we used immunostaining for PAX2 (reactive with Müllerian epithelium) and FOXA1 (reactive with urogenital sinus epithelium). By this technique, the PAX2/FOXA1 boundary was located at the extreme caudal aspect of the vaginal plate at 12 weeks. During the ensuing weeks, the PAX2/FOXA1 boundary progressively extended cranially such that by 21 weeks the entire vaginal epithelium was FOXA1-reactive and PAX2-negative. This observation supports Bulmer's proposal that human vaginal epithelium derives solely from urogenital sinus epithelium. Clearly, the development of the human vagina is far more complex than previously envisioned and appears to be distinctly different in many respects from mouse vaginal development., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Human female reproductive tract development rests mostly upon hematoxilyn and eosin stained sections despite recent advances on molecular mechanisms in mouse studies. We report application of immunohistochemical methods to explore the ontogeny of epithelial and mesenchymal differentiation markers (keratins, homobox proteins, steroid receptors), transcription factors and signaling molecules (TP63 and RUNX1) during human female reproductive tract development. Keratins 6, 7, 8, 10, 14 and 19 (KRT6, KRT7, KRT8, KRT10, KRT14, KRT19) were expressed in a temporally and spatially dynamic fashion. The undifferentiated Müllerian duct and uterovaginal canal, lined by simple columnar epithelia, expressed KRT7, KRT8 and KRT19. Glandular derivatives of the Müllerian duct (uterine tube, uterine corpus and endocervix) maintained expression of these keratins, while tissues that undergo stratified squamous differentiation (exocervix and vagina) expressed KRT6, KRT14 and KRT10 during development in an age-dependent fashion. TP63 and RUNX1 were expressed prior to KRT14, as these two transcription factors are known to be upstream from KRT14 in developing Müllerian epithelium. In the vagina, KRT10, a marker of terminal differentiation, appeared after endogenous estrogens transformed the epithelium to a thick glycogenated squamous epithelium. Uroplakin, a protein unique to urothelium, was expressed only in the bladder, urethra and vaginal introitus, but not in the female reproductive tract itself. Mesenchymal differentiation was examined through immunostaining for HOXA11 (expressed in uterine mesenchyme) and ISL1 (expressed in vaginal mesenchyme). A detailed ontogeny of estrogen receptor alpha (ESR1), progesterone receptor (PGR) and the androgen receptor (AR) provides the mechanistic underpinning for the teratogenicity of estrogens, progestins and androgens on female reproductive tract development. Immunohistochemical analysis of differentiation markers and signaling molecules advance our understanding of normal development of the human female reproductive tract. These observations demonstrate remarkable similarities in mouse and human female reproductive tract development, but also highlight some key differences., (Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17β-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l -1 ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l -1 ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l -1 ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l -1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Congenital reproductive tract anomalies could impair fertility. Female and male reproductive tracts are developed from Müllerian ducts and Wolffian ducts, respectively, involving initiation, elongation and differentiation. Genetic basis solely for distal reproductive tract development is largely unknown. Lhfpl2 (lipoma HMGIC fusion partner-like 2) encodes a tetra-transmembrane protein with unknown functions. It is expressed in follicle cells of ovary and epithelial cells of reproductive tracts. A spontaneous point mutation of Lhfpl2 (LHFPL2(G102E)) leads to infertility in 100% female mice, which have normal ovarian development, ovulation, uterine development, and uterine response to exogenous estrogen stimulation, but abnormal upper longitudinal vaginal septum and lower vaginal agenesis. Infertility is also observed in ~70% mutant males, which have normal mating behavior and sperm counts, but abnormal distal vas deferens convolution resulting in complete and incomplete blockage of reproductive tract in infertile and fertile males, respectively. On embryonic day 15.5, mutant Müllerian ducts and Wolffian ducts have elongated but their duct tips are enlarged and fail to merge with the urogenital sinus. These findings provide a novel function of LHFPL2 and a novel genetic basis for distal reproductive tract development; they also emphasize the importance of an additional merging phase for proper reproductive tract development.

The Müllerian duct gives rise to female reproductive organs, such as the oviduct and uterus. During gestation, the Wolffian duct, which generates male reproductive organs and the kidney, is formed, and the Müllerian duct then elongates caudally along the preformed Wolffian duct. Anatomical separation of these two ducts in chick embryos demonstrated that the Wolffian duct is required for Müllerian duct formation. Likewise, a few reports supported this notion in mice, including studies on Wnt9b mutant mice and Wolffian duct-specific Lhx1 deletion. However, anatomical ablation of the Wolffian duct has not been established in mice. In this study, we addressed the importance of the interaction between these two reproductive ducts, by generating mice that specifically expressed a diphtheria toxin subunit in the Wolffian duct. While this genetic ablation of the Wolffian duct resulted in kidney hypoplasia/agenesis in both male and female mutant mice, the female mutant mice lacked the uterus, which is derived from the Müllerian duct. At mid-gestation, the Müllerian duct was truncated at the level where the mutant Wolffian duct was prematurely terminated, meaning that Müllerian duct elongation was dependent on the preformed Wolffian duct. However, Wnt9b expression in the Wolffian duct and the resultant canonical Wnt activity, as well as Lhx1 expression, were not affected in the mutant mice. These results suggest that the Wolffian duct regulates Müllerian duct elongation by currently unidentified mechanisms that are independent of canonical Wnt signaling or Lhx1 expression.

Marsupials differ from eutherian mammals in their reproductive strategy of delivering a highly altricial young after a short gestation. The young, with its undeveloped organ systems completes its development post-natally, usually within a pouch. The young is dependent on milk with a composition that varies through lactation to support its growth and changing needs as it matures over a lengthy period. Gonadal differentiation occurs after birth, providing a unique opportunity to examine the effects of hormonal manipulations on its sexual differentiation of the highly accessible young. In marsupials a difference in the migration of the urinary ducts around the genital ducts from eutherian mammals results in the unique tammar reproductive tract which has three vaginae and two cervices, and two distinctly separate uteri. In the tammar wallaby, a small member of the kangaroo family, we showed that virilisation of the Wolffian duct, prostate and phallus depends on an alternate androgen pathway, which has now been shown to be important for virilisation in humans. Through hormonal manipulations over differing time periods we have achieved sex reversal of both ovaries and testes, germ cells, genital ducts, prostate and phallus. Whilst we understand many of the mechanisms behind sexual differentiation there are still many lessons to be learned from understanding how sex reversal is achieved by using a model such as the tammar wallaby. This will help guide investigations into the major questions of how and why sex determination is achieved in other species. This review discusses the control and development of the marsupial urogenital system, largely drawn from our studies in the tammar wallaby and our ability to manipulate this system to induce sex reversal., (Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Objective: To determine whether the process of vitrification affects the development of the female Müllerian duct., Study Design: We examined the difference in morphology and gene expression of the developing uteri of 30 female mice from transferred vitrified embryos and 30 female mice from transferred fresh embryos., Results: By employing histology, the data showed that there were no significant differences between the 2 groups of the same age. With the use of reverse transcription polymerase chain reaction and Western blotting, the data showed that there was no significant change in the expression of Wnt genes (Wnt4, Wnt5a, Wnt7a), beta-catenin/TCF target genes, and homeobox A10 (HOXA10) gene during uterine development in the vitrified group as compared with the control group., Conclusion: These data suggest that vitrifying preimplantation embryos may have no effects on morphology and gene expression of the uterus of offspring.

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-β family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.

Müllerian duct differentiation and development into the female reproductive tract is essential for fertility, but mechanisms regulating these processes are poorly understood. WNT signaling is critical for proper development of the female reproductive tract as evident by the phenotypes of Wnt4, Wnt5a, Wnt7a, and β-catenin (Ctnnb1) mutant mice. Here we extend these findings by determining the effects of constitutive CTNNB1 activation within the mesenchyme of the developing Müllerian duct and its differentiated derivatives. This was accomplished by crossing Amhr2-Cre knock-in mice with Ctnnb1 exon (ex) 3(f/f) mice. Amhr2-Cre(Δ/+); Ctnnb1 ex3(f/+) females did not form an oviduct, had smaller uteri, endometrial gland defects, and were infertile. At the cellular level, stabilization of CTNNB1 in the mesenchyme caused alterations within the epithelium, including less proliferation, delayed uterine gland formation, and induction of an epithelial-mesenchymal transition (EMT) event. This EMT event is observed before birth and is complete within 5 days after birth. Misexpression of estrogen receptor α in the epithelia correlated with the EMT before birth, but not after. These studies indicate that regulated CTNNB1 in mesenchyme is important for epithelial cell differentiation during female reproductive tract development.

Müllerian inhibiting substance (MIS) has been discovered by Alfred Jost at the beginning of the fifties. MIS is a glycoprotein belonging to the TGF-beta family. Its various functions differ between males and females and according to the age of the individual. In male, the protein is synthesized by Sertoli's cells and induces the disappearance of Müllerian's ducts, the development of the male genital tract. Its role in adult males remains quite unknown. In female, the protein is secreted by granulosa cells and plays a role during folliculogenesis as it regulates the initial and cyclic recruitment of ovarian follicles. MIS is also a good marker of follicular reserve and ovarian function. Therefore, it plays a role in different areas such as assisted medical reproduction and oncology. This protein represents a potential major diagnosis as well as prognostic tool in reproduction., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

In developing mammalian males, conversion of the Wolffian ducts into the epididymides and vasa deferentia depends on androgen secretion by the testes, whereas in females these ducts remain in a vestigial form or regress. However, there is continuing uncertainty whether the androgen needs to be delivered locally, either by diffusion from the adjacent testis or, by secretion into the lumen of the duct, or whether circulating androgens maintain and virilize the Wolffian ducts. To resolve this uncertainty, we transplanted either day 0-2 or day 8-9 post-partum testes beneath the flank skin of three groups of neonatal (days 0-1) female tammar wallabies, where they developed and secreted physiological levels of hormones. The Wolffian ducts of all these females were retained and had formed extensive epididymides when examined at days 25, 34 and 87 after birth. In the two older groups of females, sampled after the time of prostatic bud formation, the urogenital sinus was virilized and there was extensive prostatic development similar to that of normal males of the same age, showing that androgen secretion had occurred. Virilization of the Wolffian ducts occurred during an early but short-lived window of sensitivity. This study provides the first clear evidence that under physiological conditions virilization can be mediated by circulating androgen.

Follicle-stimulating hormone (FSH) regulation of aromatase gene expression in vitro requires the transcriptional coactivator beta-catenin. To ascertain the physiological significance of beta-catenin in granulosa cells during folliculogenesis, mice homozygous for floxed alleles of beta-catenin were intercrossed with Amhr2cre mice. Conditional deletion of beta-catenin in 8-wk-old females occurred in derivatives of the Müllerian duct, granulosa cells and, surprisingly, in brain, pituitary, heart, liver, and tail. Female mice deficient for beta-catenin were infertile, despite reaching puberty and ovulating at the expected age, indications of apparently normal ovarian function. In contrast, their oviducts were grossly distended, with fewer but healthy oocytes. In addition, their uteri lacked implantation sites. Together, these two phenotypes could explain the complete loss of fertility. Nevertheless, although the ovary appeared normal, with serum estradiol concentrations in the normal range, there was marked animal-to-animal variation of mRNAs encoding beta-catenin and aromatase. Similarly, inhibin-alpha and luteinizing hormone receptor mRNAs varied considerably in whole ovaries, whereas pituitary Fshb mRNA was significantly reduced. Collectively, these features suggested cyclization recombination (CRE)-mediated recombination of beta-catenin may be unstable in proliferating granulosa cells, and therefore may mask the suspected steroidogenic requirement for beta-catenin. We tested this possibility by transducing primary cultures of granulosa cells from mice homozygous for floxed alleles of beta-catenin with a CRE-expressing adenovirus. Reduction of beta-catenin significantly compromised FSH stimulation of aromatase mRNA and subsequent production of estradiol. Collectively, these data suggest that FSH regulation of steroidogenesis requires beta-catenin, a role that remains hidden when tested through Amhr2cre-mediated recombination in vivo.

Members of the Wnt family of genes such as Wnt4, Wnt5a, and Wnt7a have been implicated in the formation and morphogenesis of the Müllerian duct into various parts of the female reproductive tract. These WNT ligands elicit their action via either the canonical WNT/beta-catenin or the non-canonical WNT/calcium pathway and could possibly function redundantly in Müllerian duct differentiation. By using the Müllerian duct-specific anti-Müllerian hormone receptor 2 cre (Amhr2-cre) mouse line, we established a conditional knockout model that removed beta-catenin specifically in the mesenchyme of the Müllerian duct. At birth, loss of beta-catenin in the Müllerian duct mesenchyme disrupted the normal coiling of the oviduct in the knockout embryo, resembling the phenotype of the Wnt7a knockout. The overall development of the female reproductive tract was stunted at birth with a decrease in proliferation in the mesenchyme and epithelium. We also discovered that Wnt5a and Wnt7a expression remained normal, excluding the possibility that the phenotypes resulted from a loss of these WNT ligands. We examined the expression of Frizzled (Fzd), the receptors for WNT, and found that Fzd1 is one receptor present in the Müllerian duct mesenchyme and could be the putative receptor for beta-catenin activation in the Müllerian duct. In summary, our findings suggest that mesenchymal beta-catenin is a downstream effector of Wnt7a that mediates the patterning of the oviduct and proper differentiation of the uterus.

Several recent publications have contributed to our understanding of the processes involved in development of the Müllerian ducts in both sexes and regression of these structures in male embryos. Additionally, new insights in the regulation of the anti-Müllerian hormone (AMH) signaling pathway, the pathway, which mediates the male specific degeneration of Müllerian ducts, have been gained. It has become clear that the Müllerian duct is formed by invagination of the coelomic epithelium and elongates primarily by proliferation. Later on cells of the coelomic epithelium perform epithelial to mesenchymal transition and move around the epithelium of the Müllerian duct to induce degeneration of this structure in male embryos. Besides AMH and its specific type II receptor AMHR2 two different type I receptors as well as different SMAD family members have been shown to be involved in the AMH signaling cascade. Other factors including WT1, WNT7a, beta-catenin and MMP2 act upstream and downstream of AMH signaling. Here we try to draw an overall picture of Müllerian duct formation and regression by integrating the recent literature in the field., (2007 S. Karger AG, Basel)

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.

The murine female reproductive tract is undifferentiated at birth and undergoes pronounced growth and cytodifferentiation during postnatal life. Postnatal reproductive tract development proceeds in the absence of high levels of circulating estrogens and is disrupted by precocious exposure to estrogens. The WNT gene family is critical in guiding the epithelial-mesenchymal interactions that direct postnatal uterine development. We have previously described a role for Wnt7a in controlling morphogenesis in the uterus. In addition to patterning defects, Wnt7a mutant uteri are atrophic in adults and do not show robust postnatal growth. In the present study, we examine immature female Wnt7a mutant and wild-type uteri to assess the cellular processes that underlie this failure in postnatal uterine growth. Levels of proliferation are higher in wild-type versus Wnt7a mutant uteri. Exposure to the potent estrogen-agonist diethylstilbestrol (DES) leads to an increase in cell proliferation in the uterus in wild-type as well as in mutant uteri, indicating that Wnt7a is not required in mediating cell proliferation. In contrast, we observe that Wnt7a mutant uteri display high levels of cell death in response to DES, whereas wild-type uteri display almost no cell death, revealing that Wnt7a plays a key role as a cell death suppressor. The expression pattern of other key regulatory genes that guide uterine development, including estrogen receptor (alpha), Hox, and other WNT genes, reveals either abnormal spatial distribution of transcripts or abnormal regulation in response to DES exposure. Taken together, the results of the present study demonstrate that Wnt7a coordinates a variety of cell and developmental pathways that guide postnatal uterine growth and hormonal responses and that disruption of these pathways leads to aberrant cell death.

Ontogenetic expression of progesterone receptor (PR) and effect of estrogens on PR expression in the fetal female rat reproductive tract were investigated. To evaluate ontogenetic PR expression, female reproductive tract from untreated fetuses was examined on gestational days (GD) 15.5, 17.5, 19.5 and 21.5. To evaluate estrogen effects, pregnant rats were injected once per day with oil, 17beta-estradiol (E(2)) or diethylstilbestrol (DES) from GD 15 through 21. Female fetuses were prepared for real-time reverse-transcription polymerase chain reaction (RT-PCR) or immunohistochemistry for PR. Increase in PR mRNA expression was detected in the Müllerian duct on GD 21.5 compared to that on GDs 15.5 and 17.5 in untreated fetuses (P<0.05). Prenatal administration of E(2) or DES increased Müllerian PR mRNA levels by GD 21.5 compared with oil controls (P<0.01). To identify cell and region in which PR was expressed and up-regulated by E(2) and DES, localization was evaluated within three regions along the Müllerian duct axis which differentiate into oviduct, uterus and upper vagina in immunohistochemistry. In untreated fetuses, Müllerian epithelial PR immunoreactivity was weak on GDs 15.5 and 17.5, but then became moderate on GDs 19.5 and 21.5 in all three regions. These fetuses exhibited faint signals in Müllerian mesenchymal PR immunoreactivity during gestational monitoring. Critically, Müllerian mesenchymal PR staining became intense after E(2) exposure in all three regions by GD 21.5, but no change was observed in Müllerian epithelial PR. Similarly, DES dramatically induced Müllerian mesenchymal PR in all regions by GD 21.5, and also enhanced proximal epithelial PR. On the other hand, middle and caudal epithelial PRs were reduced by DES. These affected mesenchymal and epithelial cells by DES were ER alpha immunopositive in the Müllerian duct, except for middle Müllerian epithelium. These findings clearly demonstrate cell-specific PR localization and region-specific effect of DES on PR in the developing rat Müllerian duct, and provide fundamental information critical for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen receptor agonists.

Objective: To determine, in mice with disrupted Müllerian inhibiting substance (MIS) receptor genes, whether MIS affects gubernacular development; MIS causes Müllerian duct regression and is proposed to be involved in the first stage of testicular descent, because gubernacular development is abnormal in humans with persistent Müllerian duct syndrome., Materials and Methods: Ten wild-type, 11 heterozygotic and 12 homozygotic mice for MIS receptor mutations were killed at 17.5 or 18.5 days after conception or at birth, to provide serial sagittal sections of the pelvis. The amount of cremaster muscle, mitotic bodies in the gubernacular bulb, and gubernacular size were quantified by computer analysis (four mice/group)., Results: Müllerian ducts were present in the homozygous mutants, partially present in the heterozygotes and absent in the wild-type controls. All mice had descended testes. The cremaster muscle was significantly less developed in homozygous mutants than in wild-type controls (P < 0.001) and heterozygotes (P < 0.01) at birth. The mitotic index between the gubernacula of all groups was indistinguishable. There was no statistical difference in gubernacular area amongst the groups. Poor cremaster muscle development in homozygous mutants gave the muscle a loose mesenchymal appearance., Conclusions: Although there was an observable effect on cremaster muscle development in these mutant mice, gubernacular development and testicular descent were otherwise normal, and thus there must be other reasons for the observed differences in humans with persistent Müllerian duct syndrome.

Wnts are intercellular growth and differentiation factors that regulate several key developmental steps, such as gastrulation, neurulation, and organogenesis, including the development of the midbrain, central nervous system, kidney, and limbs. Wnts are also needed for a normal development of the reproductive system. Deficiency of Wnt-4, -5a, and -7a, for example, results in sex reversal, infertility, and/or malformation of the internal and external genitals. Here we focus on the importance of Wnts in the female reproductive system., (Copyright 2001 Wiley-Liss, Inc.)

Molecular genetic techniques were used to determine if mutations in the genes encoding anti-Müllerian hormone (AMH) (also known as Müllerian inhibiting substance (MIS)) and its receptor (AMHR) are commonly present in patients with congenital absence of the uterus and vagina (CAUV). Twenty-two CAUV patients and 96 control subjects from diverse ethnic groups were studied after obtaining informed consent. Genomic DNA samples prepared from leukocytes were digested separately with several different restriction enzymes, and the resultant fragments were analyzed for restriction fragment melting polymorphisms (RFMPs) by denaturing gradient gel electrophoresis (DGGE). Electrophoretic mobility of DNA fragments which were 200-700 base pairs in length was compared using polyacrylamide gels that included linear gradients of denaturing solvents designed to separate DNA fragments according to sequence-dependent variation in thermal stability. Two RFMPs were found in the AMH gene in both patients and normal control subjects. One RFMP in the AMHR gene was present at low frequencies in both patients and normal control subjects. No RFMPs specific to CAUV patients were found in either gene. Because no mutations or rare DNA sequence polymorphisms were detected in the AMH and the AMHR genes in this group of CAUV patients, it is unlikely that either gene commonly has an etiologic role in CAUV., (Copyright 2001 Wiley-Liss, Inc.)

To elucidate whether the differentiation of the Müllerian duct (MD) is mediated by c-Jun proteins, Western immunoblot with c-Jun/sc-45 antibody was used to investigate these proteins in female chick left and right MDs (LMD and RMD, respectively). The content of these proteins (e.g., the 66-kDa, 45-kDa, and 39-kDa forms) in the LMD or RMD of various stages of embryos was detected by measuring their density in autoradiograms by a Spot-denso-program with Alpha Ease software. In the LMD, the growing embryonic sex tract, the content of the 66-kDa and 39-kDa proteins increased to their highest level in 9th to 12th day embryos and then declined thereafter. In the RMD, the apoptotic embryonic sex tract, the content of these proteins also showed a linear increase from the 9th to 10th day and then declined at the 13th day. When the RMD entered the apoptosis stages (14th to 18th day of incubation), these proteins were persistently overexpressed. Another protein (45 kDa) was detected in both ducts only at the 9th to 13th days, and its content was higher in RMD than in the LMD. In parallel to this finding, high caspase-3 activity (determined by the measurement of the fragmented 85-kDa poly ADP-ribose polymerase) was found in the RMD during apoptosis. The apoptotic death of RMD was prevented by in vivo diethylstilbestrol treatment, which inhibited the overexpression of the 66-kDa and 45-kDa proteins, the fragmentation of DNA, and the activity of caspase-3. No inhibitory effect was found for the 39-kDa protein.

HOX genes are transcription factors that are essential for the proper development of the müllerian tract in the embryonic period. It has been discovered that HOX genes are expressed in the adult uterus. Two of them, Hoxa10 and Hoxa11, have been demonstrated to be necessary for uterine receptivity and implantation in mice. Recent evidence also suggests such a role for HOX genes in humans. They are likely to be essential regulators of endometrial development in preparation for implantation. This article reviews the role of the HOX genes in the reproductive tract, their patterns of expression and regulation, the outcome of deficient HOX gene expression, and their potential mechanisms of action. The process of implantation is complex, and many molecular markers have been found expressed at high levels in the endometrium in the peri-implantation window. Targeted disruption has revealed that most of these molecules are redundant and not essential for implantation. The importance of Hox genes in this process has been well documented, and they remain one of the few well-characterized molecules necessary for implantation.

In mammalian development, the signaling pathways that couple extracellular death signals with the apoptotic machinery are still poorly understood. We chose to examine Müllerian duct regression in the developing reproductive tract as a possible model of apoptosis during morphogenesis. The TGFbeta-like hormone, Müllerian inhibiting substance (MIS), initiates regression of the Müllerian duct or female reproductive tract anlagen; this event is essential for proper male sexual differentiation and occurs between embryonic days (E) 14 and 17 in the rat. Here, we show that apoptosis occurs during Müllerian duct regression in male embryos beginning at E15. Female Müllerian ducts exposed to MIS also exhibited prominent apoptosis within 13 h, which was blocked by a caspase inhibitor. In both males and females the MIS type-II receptor is expressed exclusively in the mesenchymal cell layer surrounding the duct, whereas apoptotic cells localize to the epithelium. In addition, tissue recombination experiments provide evidence that MIS does not act directly on the epithelium to induce apoptosis. Based on these data, we suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions., (Copyright 1999 Academic Press.)

Objective: Several lines of evidence suggest that expression of the WT1 transcription factor gene is necessary for normal development of the renal and male reproductive systems. Female patients with severe reproductive tract developmental defects were examined for WT1 gene mutations., Study Design: The WT1 gene was analyzed in 25 patients with congenital absence of the uterus and vagina for mutations. Genomic deoxyribonucleic acid prepared from blood leukocytes was subjected to Southern blot analysis and denaturing gradient gel electrophoresis., Results: Common WT1 gene deoxyribonucleic acid sequence polymorphisms were found in both normal control subjects and patients with congenital absence of the uterus and vagina. No deoxyribonucleic sequence differences or mutations likely to cause congenital absence of the uterus and vagina were detected in the patients., Conclusions: The absence of WT1 gene mutations in patients with congenital absence of the uterus and vagina supports the hypothesis that WT1 expression is required only for later urogenital development, after the mesonephric and paramesonephric ducts have already formed.

The mammalian female reproductive system arises from the uniform paramesonephric duct. The molecular mechanisms that establish differential development along this axis are unknown. We determined the pattern and timing of genes of the Hoxa axis in the development of the Müllerian tract. Hoxa-9, Hoxa-10, Hoxa-11, and Hoxa-13 are all expressed along the length of the paramesonephric duct in the embryonic mouse. After birth, a spatial Hox axis is established, corresponding to the postnatal differentiation of this organ system in the mouse. Hoxa-9 is expressed in the fallopian tubes, Hoxa-10 in the uterus, Hoxa-11 in the uterus and uterine cervix, and Hoxa-13 in the upper vagina. This expression pattern follows the paradigm of spatial colinearity but is a novel exception to temporal colinearity that has been considered typical of Hox genes. These genes remain expressed in the adult mouse and are expressed in the same pattern in the human. The female reproductive system undergoes dramatic structural and functional changes during the estrous cycle and in pregnancy, retaining a high degree of developmental plasticity. The late establishment of a Hox axis and persistent expression of Hox genes in the adult may play an important role in preserving this plasticity.

Immature tiger salamander larvae were treated with 12.5 or 25 micrograms of estradiol, testosterone, or dihydrotestosterone (DHT), or 12.5 micrograms of estradiol combined with 12.5 micrograms of either testosterone or DHT. Müllerian duct epithelium was more stimulated by combined steroid treatment than by any steroid alone. Estradiol antagonized the action of DHT in the Wolffian duct. Both of the androgens and estradiol when administered alone at the higher dose stimulated enlargement of connective tissue surrounding the ducts, but the combined 12.5 micrograms androgen/12.5 micrograms estrogen treatment was more effective even though the total steroid administered was the same. The effectiveness of DHT on müllerian cells of this species is evidence against a required aromatization of androgens to explain paradoxical steroid effects and suggests that fundamental differences may exist in steroid receptors of müllerian ducts, connective tissue, and Wolffian ducts. A possible role for the urodele duct system for assessing estrogenic activity of environmental contaminants is discussed.

The present study analysed gubernaculum development in mice that had been induced, through transgenesis, to express human anti-Müllerian hormone (h-AMH) throughout prenatal life. Growth and differentiation of the gubernacular primordia were assessed through the analysis of serial, transverse or sagittal, histological sections of the lower abdomen. Transgenic males and females expressed biologically active amounts of h-AMH as measured by sensitive and specific ELISA and evidenced through the regression, in females, of Müllerian ducts after day 13 of prenatal life. Gubernacular primordia became distinguishable at the same age in control and transgenic male and female fetuses on day 12 after coitus. In both groups gubernacular cords (inguinal folds of the genital mesenteries) increased in length more in females than in males while gubernacular cones showed larger growth in males. h-AMH thus appeared not to affect the sexually dimorphic pattern of growth and development of these structures. Growth and differentiation of the gubernacular primordia was further examined in 18-day-old control and h-AMH transgenic fetuses that had been exposed to testosterone propionate injected into their mothers on days 12 and 14 of pregnancy. Testosterone treatment affected, to a minor extent, the growth of the female gubernacular cords: these were reduced in length (but had a larger surface area) compared with controls. The gubernacular cones were slightly increased in length but male-like differentiation of the tissues of the cones into a muscular and mesenchymal component was not noticed to any extent. The observations thus add experimental support to the contention that AMH, even in combination with testosterone, is not effective in establishing the male pattern of gubernacular primordia development.

In male tammar wallabies, the scrotum is the first organ to become sexually differentiated, 4-5 days before birth (day 22 of gestation). This is followed by enlargement of the gubernaculum and processus vaginalis one day before birth. However the indifferent gonad does not show any signs of testicular cord formation or androgen production until later, at around the time of birth; this is more pronounced at 2 days post-partum (p.p.), when the testis takes on a characteristic rounded appearance. Primordial germ cells proliferate throughout the testis at this time, although the testis does not become significantly heavier than the ovary until around 80 days p.p.. In females, the appearance of the mammary glands is the first sign of sexual differentiation 4-5 days before birth. The indifferent gonad first shows signs of developing an ovarian cortex and medulla 7 days after birth. The migrating germ cells are confined to the cortex, and first start to enter meiosis about 25 days after birth. The Wolffian (mesonephric) ducts are patent to the urogenital sinus in fetuses at day 21 of gestation. In the female they have started to regress by 10 days p.p. and only rudiments remain by day 25 p.p.. The Müllerian (paramesonephric) ducts develop adjacent to the cranial pole of the mesonephros at about day 25 of gestation and grow caudally to meet the urogenital sinus between days 2 and 7 p.p.. The Müllerian duct of the female develops a prominent ostium abdominale by day 9 p.p., but this structure has completely regressed in males by day 13 p.p.. The testis and ovary both migrate caudally, together with the adjacent mesonephros, at about day 10 p.p.. The ovaries remain around the level of lumbar vertebra 4 after about day 7 p.p., while the testes continue to descend. The testes enter the internal inguinal ring at about day 25 p.p., about the time that prostatic buds first appear in the urogenital sinus, and are in the inguinal canal from days 25 to 36 p.p.. They enter the scrotum at around day 36 p.p., and testicular descent is complete by days 65-72 p.p.. Melanin develops in the tunica vaginalis 72 days after birth. The overall development of the urogenital system in this marsupial is similar to that of eutherians but the sequence of events differs, with some aspects of genital differentiation preceding gonadal differentiation, apparently because they are directly controlled by X-linked genes, rather than indirectly controlled by gonadal steroids.

Anti-müllerian hormone, normally responsible for the regression of müllerian ducts in male fetuses, induces stunting, germ cell loss, and seminiferous tubule formation in ovaries of bovine freemartin fetuses and of transgenic mice, which express the human anti müllerian hormone gene under the control of the metallothionein promoter. Because the latter have been studied only after birth, we undertook a detailed chronological study of their reproductive organs. Müllerian ducts of transgenic female fetuses regressed at the same time as those of normal or transgenic males. Maximal reduction of germ cell number occurred between 16 days postcoitus and birth, when most transgenic oocytes were still in the leptotene stage of the meiotic prophase, whereas normal oocytes had already reached the pachytene phase. Interference with progression of the meiotic prophase and germ cell loss in the fetal ovary are probably responsible for subsequent ovarian regression and retardation of follicle growth. Seminiferous tubule formation was not detectable prior to birth and occurred only rarely in postnatal ovaries. Aromatase activity of fetal transgenic ovaries was decreased, as well as serum concentration of testosterone in adult transgenic males, suggesting that high levels of anti-müllerian hormone may impair Leydig cell steroidogenesis.

Anti-müllerian hormone (AMH) is a member of the superfamily of peptide growth/differentiation factors which includes the activins and TGF-beta s. The putative AMH type II receptor, which was cloned recently (Baarends et al., 1994), is a member of the superfamily of transmembrane serine/threonine kinase receptors. In hypothetical evolutionary relationship dendrograms, both AMH and its putative receptor take isolated positions relative to their respective family members. The prenatal expression pattern of this putative AMH receptor is in accordance with the expected endocrine action of AMH on the mesenchymal cells located adjacent to the müllerian duct, and with known effects of AMH on gonadal differentiation. Postnatal expression of mRNA encoding this receptor in granulosa and Sertoli cells provides a new stimulus to study possible functions of AMH in the gonads.

The fetal testis functions as the sex differentiator by imposing a masculine pattern of development upon a genetic program that is inherently female. Two hormones produced by the fetal testis mediate the differentiation of the müllerian and Wolffian ducts (Figs. 1 and 4). MIS actively inhibits the development of the müllerian ducts, and testosterone induces the differentiation of the Wolffian ducts. The absence of these two hormones during fetal development in the female (the hormonal equivalent of no testes) permits müllerian duct differentiation and does not induce Wolffian duct development. The in vivo outcomes of ectopic MIS exposure or MIS deficiency illustrate the balance required to coordinately differentiate and cause regression of the respective male and female genital ducts. The observations made in the MIS-deficient mice demonstrate that codevelopment of both genital duct systems interferes with normal development of both systems and ultimately interferes with reproduction and fertility. Thus, reproduction and fertility in mammals appear to be most efficient if only one type of genital duct system develops. The phenotypes of the MIS-overexpressing transgenic mice and the MIS-deficient mice are similar yet different. Some of the explanations that might reconcile these differences probably lie with the receptor for MIS. Since the MIS-overexpressing transgenic mice are exposed to pharmacological levels of MIS during development, it seems possible that this may lead to productive interactions with other related receptors. Candidate genes have been isolated for the MIS receptor that are membrane-bound serine/threonine kinases (Baarends et al., 1994; di Clemente et al., 1994) similar to those cloned for the TGF-beta (Lin et al., 1992) and activin (Mathews and Vale, 1991) type II receptors. Interestingly, expression of these putative MIS receptor genes is localized by in situ hybridization to the mesenchymal cells adjacent to the müllerian ducts, suggesting that MIS most likely alters the surrounding mesenchyme to elicit müllerian duct regression. Experiments are underway to isolate the mouse MIS receptor gene to thereby generate MIS receptor-deficient mice and to compare the phenotype with the MIS gain-of-function and loss-of-function animals. Isolation of the human MIS receptor gene will facilitate the identification of human PMDS patients with normal levels of MIS that have mutations in the MIS receptor gene. Finally, studies of the MIS receptor gene will open up avenues for the molecular characterization of signal transduction pathways that mediate müllerian duct regression and Leydig cell proliferation control.

The epididymis is one of two organs which during the process of development in certain mammalian species descends from the dorsal abdominal wall into the scrotum. This phenomenon may or may not be accompanied by the testis during the descent, and it requires androgens. Androgen deficiency experimentally achieved induced cryptoepididymis. Both, experimentally with E2B induced as well as naturally occurring cryptoepididymis can be successfully treated with either HCG or LH-RH or a combination of both.

To determine whether mammalian Müllerian-inhibiting substance (MIS) is active in birds, Müllerian ducts from 7- to 8-day-old male or female chick embryos were cultured in the presence of human recombinant MIS at concentrations between 2.5 and 12.5 micrograms/ml. None of 20 ducts regressed at any concentration. In contrast, at concentrations of 2.5-5 micrograms/ml, all 12 Müllerian ducts from 13-day-old male mouse embryos and 13 out of 14 female ducts were inhibited to varying degrees. It is concluded that avian Müllerian ducts are unresponsive to mammalian MIS. There may be a difference in structure between the MIS of birds and mammals, or the signal-transduction system may be different.

A series of 13470 hysterosalpingographic investigations demonstrated congenital uterine malformations in 1160 cases (8.6%). Uterus arcuatus and uterine hypoplasia, the latter ranking as a malformation on embryological grounds, together accounted for two-thirds of the cases, uterus unicornis unicollis and uterus bicornis unicollis together for over a quarter and the others, uterine didelphys, uterus bicornis bicollis and uterus septus/subseptus, for much smaller percentages. Each malformation is described and illustrated by typical hysterosalpingograms. In addition to its decisive importance in diagnosis, hysterosalpingography it also valuable in planning surgical correction in many cases.