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Yes-associated protein (YAP) and WW-containing transcription regulator 1 (WWTR1) are two functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway, and that act as major regulators of cell growth and differentiation. To elucidate their role in Sertoli cells, primary Sertoli cell culture from Yapflox/flox; Wwtr1flox/flox animals were infected with a Cre recombinase-expressing adenovirus. Concomitant inactivation of Yap and Wwtr1 resulted in a decrease in the mRNA levels of the male sex differentiation genes Dhh, Dmrt1, Sox9, and Wt1, whereas those of genes involved in female differentiation (Wnt4, Rspo1, and Foxl2) were induced. SOX9, FOXL2, and WNT4 proteins were regulated in the same manner as their mRNAs in response to loss of YAP and WWTR1. To further characterize the role of YAP and WWTR1 in Sertoli cells, we generated a mouse model (Yapflox/flox; Wwtr1flox/flox; Amhcre/+) in which Yap and Wwtr1 were conditionally deleted in Sertoli cells. An increase in the number of apoptotic cells was observed in the seminiferous tubules of 4 dpp mutant mice, leading to a reduction in testis weights and a decrease in the number of Sertoli cells in adult animals. Gene expression analyses of testes from 4 dpp Yapflox/flox; Wwtr1flox/flox; Amhcre/+ mice showed that Sertoli cell differentiation is initially altered, as Dhh, Dmrt1, and Sox9 mRNA levels were downregulated, whereas Wnt4 mRNA levels were increased. However, expression of these genes was not changed in older animals. Together, these results suggest a novel role of the Hippo signaling pathway in the mechanisms of sex differentiation.

Background Over the past decades, an increasing trend in male external genital malformations such as cryptorchidism and hypospadias has led to the suspicion that environmental chemicals are detrimental to male fetal sexual development. Several environmental pollutants, including organochlorine pesticides, polychlorinated biphenyls, bisphenol A, phthalates, dioxins and furans have estrogenic and anti-androgenic activity and are thus considered as endocrine-disrupting chemicals (EDCs). Since male sex differentiation is critically dependent on the normal production and action of androgens during fetal life, EDCs may be able to alter normal male sex differentiation. Objective The objective of this study was to determine the incidence of external genital malformations in a population of full-term newborn males in southern France. We also performed a case-control study to identify the risk factors for male external genital malformations, with a focus on parental occupational exposure to EDCs. Methods Over a 16-month period, 1615 full-term newborn males with a birth weight above 2500 g were registered on a level-1 maternity ward, and the same pediatrician systematically examined 1442 of them (89%) for cryptorchidism, hypospadias and micropenis. For every male newborn with genital malformation, we enrolled nearly two males matched for age, parity and term. All parents of the case and control newborns were interviewed about pregnancy aspects, personal characteristics, lifestyle and their occupational exposure to EDCs using a detailed questionnaire. RESULTS We report 39 cases of genital malformation (2.70%), with 18 cases of cryptorchidism (1.25%), 14 of hypospadias (0.97%), 5 of micropenis (0.35%) and 2 of 46,XY disorders of sexual differentiation (DSD; 0.14%). We observed a significant relationship between newborn cryptorchidism, hypospadias or micropenis and parental occupational exposure to pesticides [odds ratio (OR) = 4.41; 95% confidence interval (95% CI), 1.21-16.00]. Familial clustering for male external genital malformations (OR = 7.25; 95% CI, 0.70-74.30) and medications taken by mothers during pregnancy (OR = 5.87; 95% CI, 0.93-37.00) were associated with the risk of cryptorchidism, hypospadias and micropenis, although the association was not statistically significant. Conclusions Although the causes of male genital malformation are multifactorial, our data support the hypothesis that prenatal contamination by pesticides may be a potential risk factor for newborn male external genital malformation and it should thus be routinely investigated in all undervirilized newborn males.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with male sex differentiation at the juvenile stage, a bisexual gonad during first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. The present study investigated the role of aromatase (cyp19a1a/Cyp19a1a) in gonadal development in this species, especially in relation to sexual differentiation and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment has an impact on these processes. We report an integrative immunohistochemical, cellular, and molecular data set describing these interesting phenomena. During male sex differentiation, high levels of cyp19a1a/Cyp19a1a expression were observed in the undifferentiated gonad (4 mo of age), in marked contrast to the low cyp19a1a/Cyp19a1a levels detected in the differentiated testis at the age of 5–6 mo. A low dose of E2 (0.25 mg/kg ...

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with a mono-male sex differentiation at the juvenile stage and male-to-female sex change at 3 yr of age. We report for the first time integrative molecular data on these interesting phenomena. Sex differentiation occurred between 4 and 5 mo of age. Testicular nr5a4 transcripts increased to high levels during sex differentiation (5 mo old), whereas nr0b1 (Dax1) did not increase until the age of 8 mo. High nr5a4 and nr0b1 expression in testicular tissue, in contrast to low nr5a4 and high nr0b1 expression in ovarian tissue, were found in the male phase of 0 + - to 2-yr-old fish (before sex change). Increased nr5a4, decreased nr0b1, and increased cyp19a1a were found in the ovarian tissues undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in 2 + -yrold fish. Removal of testicular tissue in 1 + -yr-old fish resulted in both increased ovarian nr5a4 and genes in the steroidogenic pathway and decreased nr0b1 together with the appearance of vitellogenic oocytes. Ovary developed into the active stage with the increased expression of star and steroidogenic enzymes, including aromatase, in concordance with the decreased expression of nr0b1 in the testis-excised fish. Long-term estradiol (E2) administration resulted in early sex change, but the ovaries were mainly with primary oocytes. Low nr5a4, high nr0b1, and low steroidogenic enzymes, including cyp19a1a expression, were also observed in these E2-fed ovarian tissues. Thus, nr5a4 but not nr0b1 was associated with male sex differentiation. Testicular development required cooperative functions of both nr5a4 and nr0b1. The present study suggests that nr5a4 and nr0b1 have an antagonistic interaction for the oocyte development. Testicular tissue exerted inhibitory effects on ovarian development. It is probable that nr0b1 regulates the timing of vitellogenic development and sex change in black porgy. early development, gene regulation, oocyte development, ovary, testis

Insulin-like 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the testis and by theca and luteal cells of the adult ovary. In males, INSL3 regulates testicular descent during fetal life, whereas in adults, it acts as a germ cell survival factor. In the ovary, INSL3 regulates oocyte maturation. Despite its importance for male sex differentiation and reproductive function in both sexes, very little is known regarding the molecular mechanisms that regulate Insl3 expression. So far, the nuclear receptor NR5A1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. NR5A1 by itself, however, cannot explain the spatiotemporal expression pattern of the Insl3 gene. In the present study, we have identified the orphan nuclear receptor NR4A1 as a novel regulator of INSL3 transcription in Leydig cells. Using RT-PCR, we found that Nr4a1 is coexpressed with Insl3 in purified Leydig cells and in several Leydig cell lines. Through detailed analyses of the mouse and human INSL3 promoter in Leydig cells, we have mapped a novel regulatory element located at -100 bp that is essential and sufficient to confer NR4A1 responsiveness. Consistent with a role for NR4A1 in Insl3 transcription, chromatin immunoprecipitation assays revealed that endogenous NR4A1 binds to the proximal Insl3 promoter in vivo. Finally, we found that NR4A1 is also implicated in cAMP-induced Insl3 transcription in Leydig cells. Taken together, our identification of NR4A1 as an important regulator of mouse and human INSL3 promoter activity helps us to better define the tissue-specific regulation of the INSL3 gene in gonadal cells.

Aetiology of mixed gonadal dysgenesis (MGD) has not been completely elucidated. Molecular analyses have failed to demonstrate the presence of mutations in sex-determining region on Y chromosome (SRY); it has been suggested that these individuals may bear mutations in other genes involved in the testis-determining pathway. Desert hedgehog's (DHH) importance regarding male sex differentiation has been demonstrated in various studies we describe here, for the first time, two cases of MGD in which a monoallelic single base deletion in DHH is associated with the disorder. Genomic DNA was isolated from paraffin-embedded gonad tissue from 10 unrelated patients with MGD and three controls; in addition to, DNA from peripheral blood leukocytes in 100 controls. Coding sequence abnormalities in DHH were assessed by exon-specific PCR, single-stranded conformation polymorphism (SSCP) and direct sequencing. In two patients, a heterozygous 1086delG in exon 3 was found. Comparing previously described mutations in DHH to the one observed in this study, we can affirm that the phenotypic spectrum of patients with gonadal dysgenesis due to mutations in DHH is variable. This study continues to demonstrate the importance that DHH has in mammalian male sexual differentiation, providing extended evidence that DHH constitutes a key gene in gonadal differentiation.

SYNOPSIS. Over the past thirty years, a global occurrence of sexual aberration has occurred whereby females among populations of prosobranch snails exhibit male sex characteristics. This condition, called imposex, has been causally associated with exposure to the biocide tributyltin. Tributyltin-exposed, imposex snails typically have elevated levels of testosterone which have led to the postulate that this endocrine dysfunction is responsible for imposex. This overview describes recent evidence that supports this postulate. Gastropods maintain circulating testosterone levels and administration of testosterone to females or castrates stimulates male sex differentiation in several snail species. Studies in the mud snail ( Ilyanassa obsoleta) have shown that gastropods utilize a unique strategy for regulating free testosterone levels. Excess testosterone is converted to fatty acid esters by the action of a testosterone-inducible, high capacity/low affinity enzyme, acyl-CoA:testosterone acyl transferase, and stored within the organisms. Free testosterone levels are regulated during the reproductive cycle apparently due to changes in esterification/desterification suggesting that testosterone functions in the reproductive cycle of the organisms. Testosterone esterification provides a unique target in the testosterone regulatory machinery of snails that is altered by tributyltin. Indeed, imposex and free testosterone levels were elevated in field collected snails containing high tin levels, while testosteronefatty acid ester pools were reduced in these organisms. These observations indicate that tributyltin elevates free testosterone by reducing the retention of testosterone as fatty acid-esters. This endocrine effect of tributyltin may be responsible for imposex.

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation.