Distinct Transcriptional Profiles of the Female, Male, and Finasteride-Induced Feminized Male Anogenital Region in Rat Fetuses.

A short anogenital distance (AGD) in males is a marker for incomplete masculinization and a predictor of adverse effects on male reproductive health. For this reason, AGD is used to assess the endocrine disrupting potential of chemicals for risk assessment purposes. The molecular mechanisms underpinning this chemically induced shortening of the AGD, however, remains unclear. Although it is clear that androgen receptor-mediated signaling is essential, evidence also suggest the involvement of other signaling pathways. This study presents the first global transcriptional profile of the anogenital tissue in male rat fetuses with chemically induced short AGD, also including comparison to normal male and female control animals. The antiandrogenic drug finasteride (10 mg/kg bw/day) was used to induce short AGD by exposing time-mated Sprague Dawley rats at gestation days 7-21. The AGD was 37% shorter in exposed male fetuses compared with control males at gestation day 21. Transcriptomics analysis on anogenital tissues revealed a sexually dimorphic transcriptional profile. More than 350 genes were found to be differentially expressed between the 3 groups. The expression pattern of 4 genes of particular interest (Esr1, Padi2, Wnt2, and Sfrp4) was also tested by RT-qPCR analyses, indicating that estrogen and Wnt2 signaling play a role in the sexually dimorphic development of the anogenital region. Our transcriptomics profiles provide a stepping-stone for future studies aimed at characterizing the molecular events governing development of the anogenital tissues, as well as describing the detailed Adverse Outcome Pathways for short AGD; an accepted biomarker of endocrine effects for chemical risk assessment.

Sex Reversal and Comparative Data Undermine the W Chromosome and Support Z-linked DMRT1 as the Regulator of Gonadal Sex Differentiation in Birds.

The exact genetic mechanism regulating avian gonadal sex differentiation has not been completely resolved. The most likely scenario involves a dosage mechanism, whereby the Z-linked DMRT1 gene triggers testis development. However, the possibility still exists that the female-specific W chromosome may harbor an ovarian determining factor. In this study, we provide evidence that the universal gene regulating gonadal sex differentiation in birds is Z-linked DMRT1 and not a W-linked (ovarian) factor. Three candidate W-linked ovarian determinants are HINTW, female-expressed transcript 1 (FET1), and female-associated factor (FAF). To test the association of these genes with ovarian differentiation in the chicken, we examined their expression following experimentally induced female-to-male sex reversal using the aromatase inhibitor fadrozole (FAD). Administration of FAD on day 3 of embryogenesis induced a significant loss of aromatase enzyme activity in female gonads and masculinization. However, expression levels of HINTW, FAF, and FET1 were unaltered after experimental masculinization. Furthermore, comparative analysis showed that FAF and FET1 expression could not be detected in zebra finch gonads. Additionally, an antibody raised against the predicted HINTW protein failed to detect it endogenously. These data do not support a universal role for these genes or for the W sex chromosome in ovarian development in birds. We found that DMRT1 (but not the recently identified Z-linked HEMGN gene) is male upregulated in embryonic zebra finch and emu gonads, as in the chicken. As chicken, zebra finch, and emu exemplify the major evolutionary clades of birds, we propose that Z-linked DMRT1, and not the W sex chromosome, regulates gonadal sex differentiation in birds.

Intersex gonad differentiation in cultured Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon.

Among sturgeons, the occurrence of individuals with gonads containing both testis and ovary components is considered pathological, and such fish are described as intersex individuals or intersexes. Intersexes are observed in both wild and cultured populations of sturgeon, usually at low frequencies. In the present study, intersex Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons constituted 30% of the studied populations. Macroscopically, intersex gonads were recognizable from 500 days posthatching (dph). Initially, gonads with predominantly male characteristics (testis-ova) were observed, but in older fish gonads with predominantly female traits (ova-testis) were more frequent. Using microscopic analysis, intersex gonads were discernible by 130-200 dph. Observations of intersex germinal epithelium development and analysis of sex distribution in the study populations indicated that feminization was occurring. Histological analysis revealed that differentiation of the germinal epithelium in such gonads was accompanied by various morphological alterations (transformations) that were described using quantitative and localization criteria. The most common type of transformations, massive subepithelial transformations, was manifested by the presence of abundant female germinal tissue located under the gonad surface epithelium in the developing testis. These transformations were identified in the early development stage (100-200 dph). In this type of transformation, differentiation of female germinal tissue at the gonad surface and male tissue at the mesorchium/mesovarium resulted in complete formation of both male and female germinal epithelia within the same gonad.

Masculine epigenetic sex marks of the CYP19A1/aromatase promoter in genetically male chicken embryonic gonads are resistant to estrogen-induced phenotypic sex conversion.

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

[Endocrine disruptor compounds and their role in the developmental programming of the reproductive axis]. / Compuestos disruptores endocrinos y su participación en la programación del eje reproductivo.

Different perturbations during fetal and postnatal development unleash endocrine adaptations that permanently alter metabolism, increasing the susceptibility to develop later disease, process known as "developmental programming." Endocrine disruptor compounds (EDC) are widely spread in the environment and display estrogenic, anti-estrogenic or anti-androgenic activity; they are lipophilic and stored for long periods in the adipose tissue. Maternal exposure to EDC during pregnancy and lactation produces the exposure of the fetus and neonate through placenta and breast milk. Epidemiological and experimental studies have demonstrated reproductive alterations as a consequence of intrauterine and/or neonatal exposure to EDC. Diethystilbestrol (DES) is the best documented compound, this synthetic estrogen was administered to pregnant women in the 1950s and 1960s to prevent miscarriage. It was implicated in urogenital abnormalities in children exposed in utero and was withdrawn from the market. The "DES daughters" are women with high incidence of vaginal hypoplasia, spontaneous abortion, premature delivery, uterine malformation, menstrual abnormalities and low fertility. The "DES sons" show testicular dysgenesis syndrome, which is characterized by hypospadias, cryptorchidism and low semen quality. This entity is also associated wtih fetal exposure to anti-androgens as flutamide. The effects on the reproductive axis depend on the stage of development and the window of exposure, as well as the dose and the compound. The wide distribution of EDC into the environment affects both human health and ecosystems in general, the study of their mechanisms of action is extremely important currently.

Compuestos disruptores endocrinos y su participación en la programación del eje reproductivo / Endocrine disruptor compounds and their role on the developmental programming of the reproductive axis

Different perturbations during fetal and post natal development unleash endocrine adaptations that permanently alter metabolism, increasing the susceptibility to develop later disease, process known as "developmental programming"'. Endocrine disruptor compounds (EDC) are widely spread on the environment and display estrogenic, anti-estrogenic or anti-androgenic activity; they are lypophilyc and stored for long periods on the adipose tissue. Maternal exposure to EDC during pregnancy and lactation produces the exposure of the fetus and neonate through placenta and breast milk. Epidemiological and experimental studies have demonstrated reproductive alterations as a consequence of intrauterine and/or neonatal exposure to EDC. Diethystilbestrol (DES) is the best documented compound, this synthetic estrogen was administered to pregnant women at the BO and 60 to prevent miscarriage. It was implicated in urogenital abnormalities in children exposed in utero and withdrawn from the market. The "DES daughters" are women with high incidence of vaginal hypoplasia, spontaneous abortion, premature delivery, uterine malformation, menstrual abnormalities and low fertility. The "DES sons" show testicular dysgenesis syndrome, which is characterized by hypospadias, cryptorchidism and low semen quality. This entity is also associated to the fetal exposure to anti-androgens as flutamide. The effects on the reproductive axis depend on the stage of development and the window of exposure, as well as the dose and the compound. The wide distribution of EDC into the environment affects both human health and ecosystems in general, the study of their mechanisms of action is extremely important currently.

Diversas perturbaciones durante el desarrollo fetal y posnatal desencadenan adaptaciones endocrinas que modifican permanentemente el metabolismo, incrementando la susceptibilidad para el desarrollo de enfermedades, proceso conocido como "programación durante el desarrollo". Los compuestos disruptores endocrinos (CDE) se encuentran en el medio ambiente y presentan actividad estrogénica, antiestrogénica o antiandrogénica; son altamente lipofílicos y se almacenan por periodos prolongados en el tejido adiposo. La exposición materna a CDE durante el embarazo y la lactancia permite su paso al producto a través de la placenta y la leche materna. Estudios epidemiológicos y experimentales han demostrado alteraciones en el eje reproductivo como consecuencia de la exposición intrauterina y/o neonatal a CDE. El compuesto mejor documentado es el dietilestilbestrol (DES), este estrógeno sintético fue administrado a mujeres embarazadas durante los 50s y 60s y retirado del mercado por su implicación en anormalidades urogenitales de los bebés expuestos in útero. Las denominadas "hijas del DES" son mujeres con alta incidencia de hipoplasia vaginal, malformaciones uterinas, irregularidades menstruales, baja fertilidad y alta prevalencia de aborto espontáneo y parto prematuro. Por su parte, "los hijos del DES" presentan una entidad clínica conocida como síndrome de disgenesia testicular caracterizado por hipospadias, criptorquidia y baja calidad del semen. Este síndrome también se asocia a la exposición fetal a compuestos antiandrogénicos como la ñutamida. Los efectos en el eje reproductivo dependen del estadio de desarrollo y del tiempo de exposición, así como de la dosis y el compuesto del que se trate. La extensa presencia de CDE en el ambiente afecta la salud humana e impacta al ecosistema en general por lo cual es de suma importancia el estudio de los mecanismos involucrados en su acción.

Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz.

The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17alpha-hydroxylase/17,20-lyase (P450c17) and a reduction in 17beta-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level.

Ovarian hormones can organize the rat corpus callosum in adulthood.

The rat corpus callosum (CC) is larger in males than females, and is responsive to hormone manipulations during development. Previous data suggest that CC sensitivity to testosterone ends by postnatal day 8 (P8). In contrast, responsivity to ovarian hormones extends as late as P25. The current series of experiments investigates whether ovarian hormone effects on the callosum are permanent and whether CC sensitivity to ovarian hormones extends beyond P25. We found that P70 ovariectomy (Ovx) did not affect callosal size, suggesting that ovarian hormone exposure sometime prior to P70 is sufficient to feminize the CC, and that once the callosum is feminized, the effects can not be reversed. We also found that P25 ovariectomy enlarged, or defeminized, adult female CC, whereas ovary transfer starting on P55 or P70 counteracted this enlarging effect, resulting in feminized adult CC. Thus, although a previously feminized callosum is not affected by P70 ovarian hormone removal, a not-yet feminized callosum can still be feminized after P70. These findings indicate that there is flexibility in the developmental window within which the female brain is responsive to the active feminization process initiated by ovarian hormones.

A dose-finding study of in utero and lactational exposure to diethylstilboestrol and flutamide in 129/Sv mice.

The purpose of the study was to test the experimental conditions and find the appropriate dose range for further investigations of the disruptive effects of oestrogens and antiandrogens on the development of testis. Groups of four 129/Sv mice were exposed from day 6 after mating until weaning to 1, 10 or 100 microg/kg/day of the non-steroidal oestrogen diethylstilboestrol and to 0.1, 1 or 10 mg/kg/day of the non-steroidal antiandrogen flutamide. The number of implantation sites, the litter size and reproductive parameters were examined and nipple development, anogenital distance and testicular morphology were investigated in the offspring. Dose-related post-implantation loss was seen in both diethylstilboestrol- and flutamide-treated dams and the mean litter size was smaller in the groups given the high dosages of diethylstilboestrol and flutamide. Disturbance of testicular development was seen in males exposed to diethylstilboestrol. Because of the small data material, no statistical analyses were performed. Our findings indicate that very high doses of both diethylstilboestrol and flutamide given at early stages of gestation result in a high post-implantation loss and should be avoided in further experiments using this strain which is known to have a poor reproductive performance. Exposure of the dams before mating may better reflect human exposure, but will presumably require even lower dose levels.

Action of estradiol and tamoxifen on the Müllero-regressive activity of the chick embryonic testis assayed in vivo by organotypic grafting.

In the chick, the implantation of a testis graft from a 13-day-old male donor embryo into the extra-embryonic coelom of 3-day-old female embryos induces the total regression of their Müllerian ducts because of the anti-Müllerian hormone (AMH or MIS) secreted by the implant. Pre-treatment of the donors with estradiol (E2), between day 12 and day 13, counteracts in a significant way the Müllero-regressive activity of the implant. Co-treatment of donors at the same stage with both Tamoxifen (TAM) and E2 restores the initially observed activity, thus demonstrating the presence of Tamoxifen-sensitive estrogen receptors at the late stage of treatment in the Sertoli cells responsible for AMH secretion. The treatment of 3-day-old male donor embryos with E2 causes the differentiation of their left gonad into an ovotestis which provides implants totally devoid of Müllero-regressive activity. The additional treatment with TAM of the grafted host embryos, does not modify the results obtained when E2-treated male gonads are grafted to host embryos not treated with TAM. This shows that the lack of Müllero-regressive activity exhibited by the E2-treated male gonads does not depend on the estrogens they may secrete during the time of the assay, i.e., it cannot be attributed to a protecting action of estrogens on the MDs of the host. Our results therefore favor the idea that E2 down-regulates AMH. The relevance of such a regulation to the phenomenon of Müllerian duct maintenance, either in the E2-feminized male or in the female chick embryo, is discussed.