HSD17B1 Compensates for HSD17B3 Deficiency in Fetal Mouse Testis but Not in Adults.

Hydroxysteroid (17ß) dehydrogenase (HSD17B) enzymes convert 17-ketosteroids to 17beta-hydroxysteroids, an essential step in testosterone biosynthesis. Human XY individuals with inactivating HSD17B3 mutations are born with female-appearing external genitalia due to testosterone deficiency. However, at puberty their testosterone production reactivates, indicating HSD17B3-independent testosterone synthesis. We have recently shown that Hsd17b3 knockout (3-KO) male mice display a similar endocrine imbalance, with high serum androstenedione and testosterone in adulthood, but milder undermasculinization than humans. Here, we studied whether HSD17B1 is responsible for the remaining HSD17B activity in the 3-KO male mice by generating a Ser134Ala point mutation that disrupted the enzymatic activity of HSD17B1 (1-KO) followed by breeding Hsd17b1/Hsd17b3 double-KO (DKO) mice. In contrast to 3-KO, inactivation of both HSD17B3 and HSD17B1 in mice results in a dramatic drop in testosterone synthesis during the fetal period. This resulted in a female-like anogenital distance at birth, and adult DKO males displayed more severe undermasculinization than 3-KO, including more strongly reduced weight of seminal vesicles, levator ani, epididymis, and testis. However, qualitatively normal spermatogenesis was detected in adult DKO males. Furthermore, similar to 3-KO mice, high serum testosterone was still detected in adult DKO mice, accompanied by upregulation of various steroidogenic enzymes. The data show that HSD17B1 compensates for HSD17B3 deficiency in fetal mouse testis but is not the enzyme responsible for testosterone synthesis in adult mice with inactivated HSD17B3. Therefore, other enzymes are able to convert androstenedione to testosterone in the adult mouse testis and presumably also in the human testis.

Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric Müllerian duct development by eliminating asymmetric gene expression in Japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3ß-HSD) and estrogen receptor-ß, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Fatty acid compositions of immature and mature testis are differently responsive to dietary docosahexasenoic acid during development in rats exposed to prenatal ethanol.

BACKGROUND: Ethanol (EtOH) exposure impairs, but docosahexaenoic acid (DHA) supports testis functions. This study investigated whether dietary DHA and prenatal EtOH exposure affected fatty acid profiles equally in immature and mature testis during developmental stages. METHODS: Female rats were exposed to ± EtOH (3g/kg BW, twice a day via gavage) throughout pregnancy, while consuming a diet supplemented ± DHA (1.4%, w/w). Pups were continued on their mother's diet after weaning with testes collected for fatty acid analysis at different stages of reproductive development, at gestational day 20 (GD20) and postnatal day (PD) 4, 21, 49, and 90, to present fetal, neonatal, weaning, prepubertal and adult stages, respectively. RESULTS: Regardless of EtOH exposure, dietary DHA significantly increased in testis DHA at all ages, with testis at weaning and prepuberty being more responsive to the diet (p<0.0002). Immature testis at GD20 and PD4 contained more DHA than n-6 docosapentaenoic acid (n-6 DPA) compared to mature testis while being well responsive to the maternal DHA diet through gestation and lactation. The level of n-6 very long chain fatty acids and (VLCFA) and n-6 DPA, distinctively increased from weaning and prepuberty, respectively, and were not reduced by the DHA diet at prepuberty and adulthood. Prenatal EtOH minimally affected testis fatty acids during development. CONCLUSION: Immature and mature testis responds differently to dietary DHA. The age around sexual maturity might be a critical time for dietary intervention as testis was more responsive to diet at this time point. The increase in DPA and n-6 VLCFA in matured testis while not affected by dietary DHA, indicates their critical roles in male reproductive function in rodents.

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses.

The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHß-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Localization and regulatory function of Yin Yang 1 (YY1) in chicken testis.

In mammals, Yin Yang 1 (YY1), a pervasively expressed transcription factor related to many biological processes as an activator or inhibitor of the transcription of various genes, plays a critical role in the development of male gonads and spermatogenesis. Although the role of YY1 on the development of male gonads and spermatogenesis in mammals has been reported, its function on chicken testis are yet to be clarified. In this study, we used immunofluorescence analysis to investigate the location of YY1 in chicken testis. In embryo testis, YY1 was detected in spermatogonia and Sertoli cells, while in adult testis, YY1 was shown to be expressed in spermatogenic cells and Sertoli cells, but not in spermatozoa. Furthermore, we investigated the regulatory functions of YY1 in chicken testicular Sertoli cells by combining overexpression with RNA-sequencing. Overexpression of YY1 in Sertoli cells revealed a total of 2955 differentially expressed genes involved in various biological processes, such as male gonad development and seminiferous tubule development. Overexpression of YY1 also caused significant differences in the expression of the androgen receptor gene and the inhibin ßA gene, two major genes involved in the regulation of spermatogonia in Sertoli cells. These observations indicate that YY1 may regulate the development and function of the gonads by affecting the secretion of cytokines and hormones in Sertoli cells to mediate the production and differentiation of spermatogonia.

Insights into the Evolution of Spermatogenesis-Related Ubiquitin-Proteasome System Genes in Abdominal Testicular Laurasiatherians.

During embryonic development in mammals, the testicles generally descend into the scrotum, making the testicular temperature 2-4 °C lower than the core temperature via heat exchange and clearance, and thus more beneficial for normal spermatogenesis. Failure to descend, known as cryptorchidism, carries a series of risks such as infertility and testicular cancer. However, some mammals have evolved abdominal testes while maintaining healthy reproduction. To explore the underlying molecular mechanism, we conducted comparative genomic analyses and functional assays on the spermatogenesis-related ubiquitin-proteasome system (UPS) genes essential to sperm formation in representative laurasiatherians. Here, positive selection and rapid evolution of spermatogenesis-related UPS genes were identified in the abdominal testicular laurasiatherians. Moreover, potential convergent amino acids were found between distantly related species with similar abdominal testicles and functional analyses showed RNF8 (V437I) in abdominal testicular species (437I) has a stronger ubiquitination ability, which suggests that the mammals with abdominal testes might exhibit enhanced sperm cell histone clearance to maintain sperm formation. This evidence implies that, in response to "cryptorchidism injury", spermatogenesis-related UPS genes in the abdominal testicular species might have undergone adaptive evolution to stabilize sperm formation. Thus, our study could provide some novel insights into the reproductive adaptation in abdominal testicular mammals.

DYRK1A Overexpression in Mice Downregulates the Gonadotropic Axis and Disturbs Early Stages of Spermatogenesis.

Down syndrome (DS) is the most common chromosomal disorder. It is responsible for intellectual disability (ID) and several medical conditions. Although men with DS are thought to be infertile, some spontaneous paternities have been reported. The few studies of the mechanism of infertility in men with DS are now dated. Recent research in zebrafish has indicated that overexpression of DYRK1A (the protein primarily responsible for ID in DS) impairs gonadogenesis at the embryonic stage. To better ascertain DYRK1A's role in infertility in DS, we investigated the effect of DYRK1A overexpression in a transgenic mouse model. We found that overexpression of DYRK1A impairs fertility in transgenic male mice. Interestingly, the mechanism in mice differs slightly from that observed in zebrafish but, with disruption of the early stages of spermatogenesis, is similar to that seen in humans. Unexpectedly, we observed hypogonadotropic hypogonadism in the transgenic mice.

hnRNPU in Sertoli cells cooperates with WT1 and is essential for testicular development by modulating transcriptional factors Sox8/9.

Background: Sertoli cells are essential regulators of testicular fate in the differentiating gonad; however, its role and underlying molecular mechanism of regulating testicular development in prepubertal testes are poorly understood. Although several critical regulatory factors of Sertoli cell development and function have been identified, identifying extrinsic factors that regulate gonocyte proliferation and migration processes during neonatal testis development remains largely unknown. Methods: We used the Sertoli cell-specific conditional knockout strategy (Cre/Loxp) in mice and molecular biological analyses (Luciferase assay, ChIP-qPCR, RNA-Seq, etc.) in vitro and in vivo to study the physiological roles of hnRNPU in Sertoli cells on regulating testicular development in prepubertal testes. Results: We identified a co-transcription factor, hnRNPU, which is highly expressed in mouse and human Sertoli cells and required for neonatal Sertoli cell and pre-pubertal testicular development. Conditional knockout of hnRNPU in murine Sertoli cells leads to severe testicular atrophy and male sterility, characterized by rapid depletion of both Sertoli cells and germ cells and failure of spermatogonia proliferation and migration during pre-pubertal testicular development. At molecular levels, we found that hnRNPU interacts with two Sertoli cell markers WT1 and SOX9, and enhances the expression of two transcriptional factors, Sox8 and Sox9, in Sertoli cells by directly binding to their promoter regions. Further RNA-Seq and bioinformatics analyses revealed the transcriptome-wide of key genes essential for Sertoli cell and germ cell fate control, such as biological adhesion, proliferation and migration, were deregulated in Sertoli cell-specific hnRNPU mutant testes. Conclusion: Our findings demonstrate an essential role of hnRNPU in Sertoli cells for prepubertal testicular development and testis microenvironment maintenance and define a new insight for our understanding of male infertility therapy.

Genetic Regulation of Avian Testis Development.

As in other vertebrates, avian testes are the site of spermatogenesis and androgen production. The paired testes of birds differentiate during embryogenesis, first marked by the development of pre-Sertoli cells in the gonadal primordium and their condensation into seminiferous cords. Germ cells become enclosed in these cords and enter mitotic arrest, while steroidogenic Leydig cells subsequently differentiate around the cords. This review describes our current understanding of avian testis development at the cell biology and genetic levels. Most of this knowledge has come from studies on the chicken embryo, though other species are increasingly being examined. In chicken, testis development is governed by the Z-chromosome-linked DMRT1 gene, which directly or indirectly activates the male factors, HEMGN, SOX9 and AMH. Recent single cell RNA-seq has defined cell lineage specification during chicken testis development, while comparative studies point to deep conservation of avian testis formation. Lastly, we identify areas of future research on the genetics of avian testis development.

Improving In Vitro Culture of Human Male Fetal Germ Cells.

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.

The role of StAR2 gene in testicular differentiation and spermatogenesis in Nile tilapia (Oreochromis niloticus).

Sex steroids play critical roles in sex differentiation and gonadal development in teleosts. Steroidogenic acute regulatory protein (StAR), transporting cholesterol (the substrate for steroidogenesis) from the outer mitochondrial membrane to the inner membrane, is the first rate-limiting factor of steroidogenesis. Interestingly, two StAR genes (named as StAR1 and StAR2) have been isolated from non-mammalian vertebrates. To characterize the functions of the novel StAR2 gene in the gonadal differentiation and fertility, we generated a StAR2 homozygous mutant line in Nile tilapia (Oreochromis niloticus). StAR2 gene knockout in male tilapia impeded meiotic initiation, associate with the down-regulation of meiosis related gene expressions of vasa, sycp3 and dazl at 90 days after hatching (dah). Meanwhile, cyp11b2 expression and serum 11-KT production significantly declined in StAR2-/- XY fish at 90 dah. From 120-300 dah, spermatogenesis gradually recovered, and so did the expressions of vasa, sycp3 and dazl in StAR2-/- XY fish testes. However, seminiferous lobules arranged disorderly in StAR2-/- XY fish testes at 300 dah. The number of Leydig cells and expressions of downstream steroidogenesis enzymes including cyp11a1, 3ß-HSD-I, 3ß-HSD-II, cyp17a1 and cyp17a2 decreased in StAR2-/- XY fish testes at 300 dah. Serum testosterone and 11-KT levels were significantly lower in StAR2-/- XY fish than that of their control counterparts. Furthermore, significantly elevated ar, fsh and lh expressions in StAR2-deficient XY fish testes and pituitaries were found when compared with the control XY fish. Testes degeneration and spermatogenic cell apoptosis were observed, while no sperm were squeezed out in StAR2-/- XY fish testes at 540 dah. Taken together, our results suggest that StAR2 has a role in testicular development, spermatogenesis and spermiation by regulating androgen production in tilapia, but may not be essential and could be compensated.

Three-dimensional structure of testis cords in mice and rats.

BACKGROUND: Testis cord elongation and coiling, which occur in the final stage of testis formation, have been attributed to Sertoli cell proliferation; however, the underlying mechanisms remain unclear. OBJECTIVE: The aim of the present study was to clarify the precise three-dimensional structure of testis cords in the final stage of testis formation in mice and rats. MATERIALS AND METHODS: We reconstructed whole testis cords in the final stage of testis formation in mice (on embryonic days 15.5 and 18.5) and rats (on embryonic days 16.5 and 19.5) using serial paraffin sections and high-performance three-dimensional reconstruction software. RESULTS: Detailed morphometric parameters were calculated for three-dimensionally reconstructed testis cords in six mouse and rat testes each. The mean numbers of testis cords in mice and rats were 12.7 and 27.8, respectively. The mean number of branching points per testis cord was 1.52 in mice, whereas it was only 0.30 in rats. In contrast, the mean ratio of the inner cords, that is, cords not in contact with the tunica albuginea, was 23.0% in rats, whereas it was only 6.5% in mice. In both species, the cords on the cranial side coiled more strongly than those on the caudal side, consistent with the greater expansion of the testis volume on the caudal side. All cords formed right-handed helices from the rete testis side. DISCUSSION AND CONCLUSIONS: The present results suggest that testis cords undergo anastomosis at a higher frequency in mice than in rats and that the coiling of testis cords proceeds from the cranial to caudal side of the testis in both species.

Merlin and expanded integrate cell signaling that regulates cyst stem cell proliferation in the Drosophila testis niche.

The Drosophila testis is a model organism stem cell niche in which two stem cell populations coordinate together to produce sperm; thus, these stem cells must be balanced in the niche. Merlin, a tumor-suppressor and human disease gene required for contact inhibition of proliferation, is known to limit the proliferation of the somatic cyst stem cells in the testis niche. Expanded encodes a protein that is structurally similar to Merlin in Drosophila, and is semi-redundant with Merlin in multiple tissues. We found that expanded depletion caused similar cyst lineage cell over-proliferation as observed with Merlin, and double mutants showed more severe phenotypes than either gene individually. Thus, these genes have partially redundant functions in the cyst lineage cells of this niche. We also expressed non-phosphorylatable constitutively "tumor suppressing" alleles of Merlin in cyst lineage cells, and surprisingly, we observed a similar cyst lineage over-proliferation phenotype. Merlin is known to impact multiple different signaling pathways to exert its effect on proliferation. We found that the Merlin loss of function phenotype was associated with an increase in MAPK/ERK signaling, consistent with Merlin's established role in transmembrane receptor inhibition. Constitutive Merlin displayed a reduction in both MAPK/ERK signaling and PI3K/Tor signaling. PI3K/Tor signaling is required for cyst cell differentiation, and inhibition of this pathway by Merlin activation phenocopied the Tor cyst lineage loss of function phenotype. Thus, Merlin impacts and integrates the activity of multiple signaling pathways in the testis niche. The ability of Merlin to dynamically change its activity via phosphorylation in response to local contact cues provides an intriguing mechanism whereby the signaling pathways that control these stem cells might be dynamically regulated in response to the division of a neighboring germ cell.

Loss of Mafb and Maf distorts myeloid cell ratios and disrupts fetal mouse testis vascularization and organogenesis†.

Testis differentiation is initiated when Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. Sertoli cells are essential for testis development, but cell types within the interstitial compartment, such as immune and endothelial cells, are also critical for organ formation. Our previous work implicated macrophages in fetal testis morphogenesis, but little is known about genes underlying immune cell development during organogenesis. Here, we examine the role of the immune-associated genes Mafb and Maf in mouse fetal gonad development, and we demonstrate that deletion of these genes leads to aberrant hematopoiesis manifested by supernumerary gonadal monocytes. Mafb; Maf double knockout embryos underwent initial gonadal sex determination normally, but exhibited testicular hypervascularization, testis cord formation defects, Leydig cell deficit, and a reduced number of germ cells. In general, Mafb and Maf alone were dispensable for gonad development; however, when both genes were deleted, we observed significant defects in testicular morphogenesis, indicating that Mafb and Maf work redundantly during testis differentiation. These results demonstrate previously unappreciated roles for Mafb and Maf in immune and vascular development and highlight the importance of interstitial cells in gonadal differentiation.

In utero exposure to dipentyl phthalate disrupts fetal and adult Leydig cell development.

Phthalates as plasticizers are widely used in many consumer products. Dipentyl phthalate (DPeP) is one of phthalates. However, there are currently few data on whether DPeP exposure affects rat Leydig cell development. In this study, we investigated the effects of in utero DPeP exposure on Leydig cell development in the testes of male newborn and adult rats. From gestational days 14 to 21, Sprague-Dawley pregnant rats were gavaged vehicle (corn oil, control) or DPeP (10, 50, 100, and 500 mg/kg body weight/day). Testosterone and the expression of Leydig cell genes and proteins in the testis at birth and at postnatal day 56 were examined. DPeP dose-dependently reduced serum testosterone levels of male offspring at birth and at postnatal day 56 at 100 and 500 mg/kg and lowered serum luteinizing hormone levels at adult males at ≥10 mg/kg when compared with the control. In addition, DPeP increased number of fetal Leydig cells by inducing their proliferation but down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Insl3 in fetal Leydig cells per se. DPeP reduced number of adult Leydig cells by inducing cell apoptosis and down-regulated the expression of Lhcgr and Star in adult Leydig cells at postnatal day 56. DPeP lowered SIRT1 and BCL2 levels in the testis of adult rats. In conclusion, DPeP adversely affects both fetal and adult Leydig cell development after in utero exposure.

RNA kinase CLP1/Cbc regulates meiosis initiation in spermatogenesis.

CLP1, TSEN complex, and VCP are evolutionarily conserved proteins whose mutations are associated with neurodegenerative diseases. In this study, we have found that they are also involved in germline differentiation. To optimize both quantity and quality in gametes production, germ cells expand themselves through limited mitotic cycles prior to meiosis. Stemming from our previous findings on the correlation between mRNA 3'-processing and meiosis entry, here we identify that the RNA kinase Cbc, the Drosophila member of the highly conserved CLP1 family, is a component of the program regulating the transition from mitosis to meiosis. Using genetic manipulations in Drosophila testis, we demonstrate that nuclear Cbc is required to promote meiosis entry. Combining biochemical and genetic methods, we reveal that Cbc physically and/or genetically intersects with Tsen54 and TER94 (VCP ortholog) in this process. The C-terminal half of Tsen54 is both necessary and sufficient for its binding with Cbc. Further, we illustrate the functional conservation between Cbc and mammalian CLP1 in the assays of subcellular localization and Drosophila fertility. As CLP1, TSEN complex, and VCP have also been identified in neurodegenerations of animal models, a mechanism involving these factors seems to be shared in gametogenesis and neurogenesis.

MAP3K4 kinase activity dependent control of mouse gonadal sex determination†.

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein-protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination.

Cholesterol Contributes to Male Sex Differentiation Through Its Developmental Role in Androgen Synthesis and Hedgehog Signaling.

Two specialized functions of cholesterol during fetal development include serving as a precursor to androgen synthesis and supporting hedgehog (HH) signaling activity. Androgens are produced by the testes to facilitate masculinization of the fetus. Recent evidence shows that intricate interactions between the HH and androgen signaling pathways are required for optimal male sex differentiation and defects of either can cause birth anomalies indicative of 46,XY male variations of sex development (VSD). Further, perturbations in cholesterol synthesis can cause developmental defects, including VSD, that phenocopy those caused by disrupted androgen or HH signaling, highlighting the functional role of cholesterol in promoting male sex differentiation. In this review, we focus on the role of cholesterol in systemic androgen and local HH signaling events during fetal masculinization and their collective contributions to pediatric VSD.

Steroidogenesis during prenatal testicular development in Spix's cavy Galea spixii.

Spix's cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17ß-hydroxysteroid dehydrogenase (17ß-HSDI) and mitochondrial microsomal 3ß-hydroxysteroid dehydrogenase (3ß-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17ß-HSDI and 3ß-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P<0.05) and a decrease at Day 50 of gestation (P<0.05). It is concluded that the increase in androgenic enzymes in Leydig cells coincides with the functional differentiation of the testes, and with the stabilisation and differentiation of mesonephric ducts forming the epididymis.