Morc1 reestablishes H3K9me3 heterochromatin on piRNA-targeted transposons in gonocytes.

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.

External genitalia phenotypes of a Mab21l1-null mouse model for cerebellar, ocular, craniofacial, and genital (COFG) syndrome.

The cerebellar, ocular, craniofacial, and genital (COFG) syndrome is a human genetic disease that is caused by MAB21L1 mutations. A COFG mouse model with Mab21l1-null mutation causes severe microphthalmia and fontanelle dysosteogenesis, similar to the symptoms in human patients. One of the typical symptoms is scrotal agenesis in male infants, while male Mab21l1-null mice show hypoplastic preputial glands, a rodent-specific derivative of the cranial scrotal fold. However, it is still unclear where and how MAB21Ll acts in the external genitalia in both mice and humans. Here we show that, at the neonatal stage, MAB21L1 expression in the external genitalia was restricted to two mesenchymal cell populations-underneath the scrotal and labial skin and around the preputial and clitoral glands (PG/CG). Morphometric analyses of the Mab21l1-/- pups revealed a significant reduction in the external size of the scrotum, vulva, and CG, as well as PG. In the periglandular region around PG and CG, the periglandular mesenchymal cells showed a drastic reduction in both cell density and immunoreactive signals for several extracellular matrix proteins (e.g., collagen I, fibronectin, and proteoglycans), together with their reduced Ki67-positive cell proliferation index. In the Mab21l1-/- PG/CG, together with reduced vascularization, the glandular epithelia displayed atrophy with discontinuous basal lamina along the basal surface and defective glycogen accumulation in their cytoplasm. Under a 5-day organ culture of the isolated PG, the Mab21l1-/- explants showed poor outgrowth and retention of the glandular structure in vitro. However, the addition of exogenous Matrigel could partially rescue such tissue-autonomous phenotypes, showing glandular morphology similar to that of the wild-type explants. These findings suggest that MAB21L1+ mesenchymal cells play a crucial role in providing nutrient ECM support for glandular outgrowth and morphogenesis in the peripheral external genitalia.

A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo.

BACKGROUND: During mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta's intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17 is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity. METHODS: RNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study, and firstly, the whole-mount immunostaining was done. We conducted luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells. RESULTS: The increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity. CONCLUSIONS: Rasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

Partial male-to-female reprogramming of mouse fetal testis by Sertoli cell ablation.

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions: near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3ßHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

A possible function of Nik-related kinase in the labyrinth layer of delayed delivery mouse placentas.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

SOX17-positive rete testis epithelium is required for Sertoli valve formation and normal spermiogenesis in the male mouse.

Seminiferous tubules (STs) in the mammalian testes are connected to the rete testis (RT) via a Sertoli valve (SV). Spermatozoa produced in the STs are released into the tubular luminal fluid and passively transported through the SV into the RT. However, the physiological functions of the RT and SV remain unclear. Here, we identified the expression of Sox17 in RT epithelia. The SV valve was disrupted before puberty in RT-specific Sox17 conditional knockout (Sox17-cKO) male mice. This induced a backflow of RT fluid into the STs, which caused aberrant detachment of immature spermatids. RT of Sox17-cKO mice had reduced expression levels of various growth factor genes, which presumably support SV formation. When transplanted next to the Sox17+ RT, Sertoli cells of Sox17-cKO mice reconstructed the SV and supported proper spermiogenesis in the STs. This study highlights the novel and unexpected modulatory roles of the RT in SV valve formation and spermatogenesis in mouse testes, as a downstream action of Sox17.

Gonadal Sex Differentiation and Ovarian Organogenesis along the Cortical-Medullary Axis in Mammals.

In most mammals, the sex of the gonads is based on the fate of the supporting cell lineages, which arises from the proliferation of coelomic epithelium (CE) that surfaces on the bipotential genital ridge in both XY and XX embryos. Recent genetic studies and single-cell transcriptome analyses in mice have revealed the cellular and molecular events in the two-wave proliferation of the CE that produce the supporting cells. This proliferation contributes to the formation of the primary sex cords in the medullary region of both the testis and the ovary at the early phase of gonadal sex differentiation, as well as to that of the secondary sex cords in the cortical region of the ovary at the perinatal stage. To support gametogenesis, the testis forms seminiferous tubules in the medullary region, whereas the ovary forms follicles mainly in the cortical region. The medullary region in the ovary exhibits morphological and functional diversity among mammalian species that ranges from ovary-like to testis-like characteristics. This review focuses on the mechanism of gonadal sex differentiation along the cortical-medullary axis and compares the features of the cortical and medullary regions of the ovary in mammalian species.

DNMT1 Expression and DNA Methylation in Intraductal Papillary Neoplasms of the Bile Duct.

BACKGROUND/AIM: Intraductal papillary neoplasms of the bile duct (IPNB) are histologically and clinically classified as type 1 and 2. This study aimed to identify the differences between these two types. MATERIALS AND METHODS: Based on multiple gene expression analysis (MGEA) using type 1, type 2, and pancreatic intraductal papillary mucinous neoplasms (n=4, 6, and 5, respectively), immunohistochemistry of DNMT1 and methylation-specific PCR for p16, APC, BRCA1, hMLH1, TIMP3, and SOX17 were performed on type 1 and 2 IPNBs (n=14, each). RESULTS: The DNMT1 protein was highly expressed (p<0.001) in 28.6% of type 1 cases and all type 2 cases. The DNA methylation ratio for the six genes in total as well as for SOX17 was lower in type 1 than in type 2 (p<0.05 each). CONCLUSION: Type 2 IPNB showed increased DNMT1 protein expression and increased DNA methylation frequency of the examined tumor suppressor genes compared to type 1. DNMT1 IHC may be helpful in discriminating between these two types.

MAB21L1 modulates gene expression and DNA metabolic processes in the lens placode.

Mutations in human MAB21L1 cause aberrations in lens ectoderm morphogenesis and lead to congenital cerebellar, ocular, craniofacial and genital (COFG) syndrome. Murine Mab21l1-null mutations cause severe cell-autonomous defects in lens formation, leading to microphthalmia; therefore, Mab21l1-null mice are used as a mouse model for COFG syndrome. In this study, we investigated the early-onset single-cell-level phenotypes of murine Mab21l1-null lens ectoderms using electron microscopy and single-cell RNA sequencing (scRNA-seq). Electron microscopy and immunohistochemical analyses indicated endoplasmic reticulum stress at the 24- to 26-somite stage in Mab21l1-null lens placodes. scRNA-seq analysis revealed that 131 genes were downregulated and 148 were upregulated in Mab21l1-null lens ectoderms relative to the wild type. We successfully identified 21 lens-specific genes that were downregulated in Mab21l1-null cells, including three key genes involved in lens formation: Pitx3, Maf and Sfrp2. Moreover, gene ontology analysis of the 279 differentially expressed genes indicated enrichment in housekeeping genes associated with DNA/nucleotide metabolism prior to cell death. These findings suggest that MAB21L1 acts as a nuclear factor that modulates not only lens-specific gene expression but also DNA/nucleotide metabolic processes during lens placode formation.

Sertoli cell replacement in explanted mouse testis tissue supporting host spermatogenesis†.

Spermatogenesis takes place in the seminiferous tubules, starting from the spermatogonial stem cell and maturing into sperm through multiple stages of cell differentiation. Sertoli cells, the main somatic cell constituting the seminiferous tubule, are in close contact with every germ cell and play pivotal roles in the progression of spermatogenesis. In this study, we developed an in vitro Sertoli cell replacement method by combining an organ culture technique and a toxin receptor-mediated cell knockout system. We used Amh-diphtheria toxin receptor transgenic mice, whose Sertoli cells specifically express human diphtheria toxin receptor, which renders them sensitive to diphtheria toxin. An immature Amh-diphtheria toxin receptor testis was transplanted with the donor testis cells followed by culturing in a medium containing diphtheria toxin. This procedure successfully replaced the original Sertoli cells with the transplanted Sertoli cells, and spermatogenesis originating from resident germ cells was confirmed. In addition, Sertoli cells in the mouse testis tissues were replaced by transplanted rat Sertoli cells within culture conditions without requiring immunosuppressive treatments. This method works as a functional assay system, making it possible to evaluate any cells that might function as Sertoli cells. It would also be possible to investigate interactions between Sertoli and germ cells more closely, providing a new platform for the study of spermatogenesis and its impairments.

Comparative anatomy of the hepatobiliary systems in quail and pigeon, with a perspective for the gallbladder-loss.

Although the gallbladder is one of the characteristic component of the vertebrate body, it has been independently lost in several lineages of mammals and birds. Gallbladder loss is a widely reported phenomenon; however, there have been few descriptive comparisons of entire hepatobiliary structures between birds with and without a gallbladder. Here, we discuss the evolution of avian hepatobiliary morphology by describing the gross anatomy of the hepatobiliary system in the quail and pigeon. Quails have two major extrahepatic bile ducts: the right cystic-enteric duct, which has a gallbladder, and the left hepatic-enteric duct, which does not. Together with two pancreatic ducts, they share one opening to the ascending part of duodenum. Pigeons lack a gallbladder, but also have two extrahepatic ducts similar to those of quails. However, the hepatic-enteric duct opens solely to the descending part of the duodenum close to the stomach. The pancreatic duct opens to the very posterior part of the duodenum independent from the biliary tracts, giving rise to three separate openings in the duodenum. The hepatobiliary anatomy of the pigeon represents a highly derived condition not only because of gallbladder loss. Avian gallbladder loss may be related to remodeling of the entire hepatobiliary system, and may have occurred via a different mechanism from that of mammals, which can be explained simply by the disappearance of the gallbladder primordium.

Single-cell transcriptional analysis reveals developmental stage-dependent changes in retinal progenitors in the murine early optic vesicle.

The optic vesicle in the developing embryonic eye contains a multitude of neuroepithelial progenitors that subsequently differentiate into functionally distinct domains of the optic cup, such as the neural retina, pigment epithelium, and optic stalk. To investigate cell-type diversity across early optic vesicles before regionalization of the optic cup, we performed single-cell RNA-sequencing (scRNA-seq) using 7989 cells from the presumptive eye area in mouse embryos at the 12-26-somite stages at five developmental time points. We demonstrated the presence of seven optic vesicle populations. Moreover, the four populations of retinal progenitor cells could be classified according to their stage-dependent time point, and these cells exhibited altered expression of several structural and metabolic key genes, such as Col9a1 and Ckb, just before regionalization of the optic cup. From these data, we provide the first report on stage-dependent transcriptional profiles during initial retinal specification at single-cell resolution and highlight the unexpected developmental heterogeneity of the murine optic vesicle structure.

Gene expression and functional abnormalities in XX/Sry Leydig cells.

The SRY gene induces testis development even in XX individuals. However, XX/Sry testes fail to produce mature sperm, due to the absence of Y chromosome carrying genes essential for spermatogenesis. XX/Sry Sertoli cells show abnormalities in the production of lactate and cholesterol required for germ cell development. Leydig cells are essential for male functions through testosterone production. However, whether XX/Sry adult Leydig cells (XX/Sry ALCs) function normally remains unclear. In this study, the transcriptomes from XY and XX/Sry ALCs demonstrated that immediate early and cholesterogenic gene expressions differed between these cells. Interestingly, cholesterogenic genes were upregulated in XX/Sry ALCs, although downregulated in XX/Sry Sertoli cells. Among the steroidogenic enzymes, CYP17A1 mediates steroid 17α-hydroxylation and 17,20-lyase reaction, necessary for testosterone production. In XX/Sry ALCs, the latter reaction was selectively decreased. The defects in XX/Sry ALCs, together with those in the germ and Sertoli cells, might explain the infertility of XX/Sry testes.

Low retinoic acid levels mediate regionalization of the Sertoli valve in the terminal segment of mouse seminiferous tubules.

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.

Anatomical and histological characteristics of the hepatobiliary system in adult Sox17 heterozygote mice.

Biliary atresia (BA) is a rare neonatal disease characterized by inflammation and obstruction of the extrahepatic bile ducts (EHBDs). The Sox17-haploinsufficient (Sox17+/- ) mouse is an animal model of BA that encompasses bile duct injury and subsequent BA-like inflammation by the neonatal stage. Most Sox17+/- neonates die soon after birth, but some Sox17+/- pups reach adulthood and have a normal life span, unlike human BA. However, the phenotype and BA-derived scars in the hepatobiliary organs of surviving Sox17+/- mice are unknown. Here, we examined the phenotypes of the hepatobiliary organs in post-weaning and young adult Sox17+/- mice. The results confirmed the significant reduction in liver weight, together with peripheral calcinosis and aberrant vasculature in the hepatic lobule, in surviving Sox17+/- mice as compared with their wild-type (WT) littermates. Such hepatic phenotypes may be sequelae of hepatobiliary damage at the fetal and neonatal stages, a notion supported by the slight, but significant, increases in the levels of serum markers of liver damage in adult Sox17+/- mice. The surviving Sox17+/- mice had a shorter gallbladder in which ectopic hepatic ducts were more frequent compared to WT mice. Also, the surviving Sox17+/- mice showed neither obstruction of the EHBDs nor atrophy or inflammation of hepatocytes or the intrahepatic ducts. These data suggest that some Sox17+/- pups with BA naturally escape lethality and recover from fetal hepatobiliary damages during the perinatal period, highlighting the usefulness of the in vivo model in understanding the hepatobiliary healing processes after surgical restoration of bile flow in human BA.

Gallbladder wall abnormality in biliary atresia of mouse Sox17+/- neonates and human infants.

Biliary atresia (BA) is characterized by the inflammation and obstruction of the extrahepatic bile ducts (EHBDs) in newborn infants. SOX17 is a master regulator of fetal EHBD formation. In mouse Sox17+/- BA models, SOX17 reduction causes cell-autonomous epithelial shedding together with the ectopic appearance of SOX9-positive cystic duct-like epithelia in the gallbladder walls, resulting in BA-like symptoms during the perinatal period. However, the similarities with human BA gallbladders are still unclear. In the present study, we conducted phenotypic analysis of Sox17+/- BA neonate mice, in order to compare with the gallbladder wall phenotype of human BA infants. The most characteristic phenotype of the Sox17+/- BA gallbladders is the ectopic appearance of SOX9-positive peribiliary glands (PBGs), so-called pseudopyloric glands (PPGs). Next, we examined SOX17/SOX9 expression profiles of human gallbladders in 13 BA infants. Among them, five BA cases showed a loss or drastic reduction of SOX17-positive signals throughout the whole region of gallbladder epithelia (SOX17-low group). Even in the remaining eight gallbladders (SOX17-high group), the epithelial cells near the decidual sites were frequently reduced in the SOX17-positive signal intensity. Most interestingly, the most characteristic phenotype of human BA gallbladders is the increased density of PBG/PPG-like glands in the gallbladder body, especially near the epithelial decidual site, indicating that PBG/PPG formation is a common phenotype between human BA and mouse Sox17+/- BA gallbladders. These findings provide the first evidence of the potential contribution of SOX17 reduction and PBG/PPG formation to the early pathogenesis of human BA gallbladders.This article has an associated First Person interview with the joint first authors of the paper.

Early Crypt Formation Defects in the Uterine Epithelia of Sox17 Heterozygous Mice.

SOX17 activity in the uterine epithelium is essential for the implantation of mouse embryos. Previously, we demonstrated that female Sox17 heterozygous mutant mice are subfertile, and 2 active copies of Sox17 are required for the proper implantation of mouse embryos. To understand which implantation step is most sensitive to the Sox17 gene dosage, we comprehensively investigated the phenotypes and RNA transcriptomes of Sox17 heterozygous mutant mice. Uterine Sox17 expression drastically changed according to estrous cycle and during early pregnancy. The highest Sox17 expression was observed during the receptive period for blastocyst implantation. Sox17 heterozygous uterine epithelia showed ectopic high-level expression of SOX9, another SOX factor that is normally expressed in the uterine gland. Three-dimensional analysis of the uterus on day 5 of pregnancy revealed no crypt formation near the healthy blastocysts in the Sox17 heterozygous uterine epithelium, suggesting that early defects in embryo homing had occurred. Global transcriptional analysis revealed that the expression of Amphiregulin (Areg), a gene encoding a heparin-binding epidermal growth factor receptor ligand, was decreased drastically in Sox17+/- uterine epithelia. These data imply that full Sox17 activity is required to promote early crypt formation through proper regulation of SOX9 and AREG expression at the implantation site.

Endocardium differentiation through Sox17 expression in endocardium precursor cells regulates heart development in mice.

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.

Nr5a1 suppression during the murine fetal period optimizes ovarian development by fine-tuning Notch signaling.

The nuclear receptor NR5A1 is equally expressed and required for development of the gonadal primordia of both sexes, but, after sex determination, it is upregulated in XY testes and downregulated in XX ovaries. We have recently demonstrated, in mice, that this downregulation is mediated by forkhead box L2 (FOXL2) and hypothesized that adequate suppression of Nr5a1 is essential for normal ovarian development. Further, analysis of human patients with disorders/differences of sex development suggests that overexpression of NR5A1 can result in XX (ovo)testicular development. Here, we tested the role of Nr5a1 by overexpression in fetal gonads using a Wt1-BAC (bacterial artificial chromosome) transgene system. Enforced Nr5a1 expression compromised ovarian development in 46,XX mice, resulting in late-onset infertility, but did not induce (ovo)testis differentiation. The phenotype was similar to that of XX mice lacking Notch signaling. The expression level of Notch2 was significantly reduced in Nr5a1 transgenic mice, and the ovarian phenotype was almost completely rescued by in utero treatment with a NOTCH2 agonist. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine-tuning Notch signaling.

Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice.

In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7-10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.