Gonadal Sex Differentiation Research Articles

Two phases of gonadal sex differentiation in zebrafish with ZZ/ZW sex determination system

Zebrafish sex chromosomes have been identified in the wild Nadia (NA) strain, and its sex determination belongs to the female-heterogametic ZZ/ZW system. Here, we investigate the correlation between ZZ/ZW sex chromosomes in the NA strain with sex-related factors, and sort out the complicated process of sex determination in zebrafish. Two phases exist during zebrafish sex differentiation. In the first phase, ZW gonads differentiate into juvenile ovary while ZZ gonads remain indifferent. In the second phase, ZW gonads either continue ovary development or undergo female-to-male transition, while ZZ gonads undergo direct male development. The W chromosome may contribute to the first phase while the abundance of germ cells and other factors may be involved in the second phase of sex differentiation in zebrafish.

MiR-34 negatively regulates the expression of Dmrt and related genes in the testis of mud crab Scylla paramamosain

The mud crab (Scylla paramamosain) is a commercially significant marine decapod crustacean. Due to its obvious sexual dimorphism, the mechanism of sex differentiation and gonadal development has attracted significant research interest. The Dmrt (double-sex and mab-3 related transcription factor) genes are vital in animal gonadal development and sex differentiation. In the present study, miR-34 was predicted to target the 3′ end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like genes by prediction software, and the interactions between miR-34 and these Dmrt genes were validated by in vivo and in vitro experiments. Dual luciferase assay results indicated that miR-34 mimics/inhibitors co-transfected with plasmid vectors with 3′ end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like, respectively, led to a significant decrease/increase of fluorescence activity in HEK293T cells. In vivo experiments showed that injection of agomir-34 significantly inhibited Dmrt-1, idmrt-2, Dsx and Dmrt-like expression, while injection of antagomir-34 caused the opposite result. However, Dmrt-3 expression was not affected by injection of miR-34 reagents. Meanwhile, the expression of spermatogenesis and testicular development-related molecular marker genes (IAG, foxl2 and vasa) in mud crabs was significantly changed after injecting the miR-34 reagent in vivo. Furthermore, the result of immunoblotting proved that the expression level of Dmrt-like protein can be regulated by miR-34. These results imply that miR-34 is indirectly involved in sex differentiation and testicular development of S. paramamosain by regulating Dmrt-1, idmrt-2, Dsx and Dmrt-like genes.

A New Investigation to Discriminate Sexes in Alive Nile Tilapia (Oreochromis niloticus) Using Cyp19a1a and Dmrt1 Gene Expression in Tail Fin Tissues

The Nile Tilapia (Oreochromis niloticus), a gonochoristic teleost fish with a XX/XY sex-determination system, is an ideal model for investigating gonadal sex differentiation. During gonadal differentiation, the expression of cyp19a1a in XX gonads and dmrt1 in XY gonads are required for undifferentiated tissues to develop into ovary or testis. In this study, quantitative real-time RT-PCR assessed the expression of cyp19a1a and dmrt1 genes in gonads and tail fin tissues. Differences in gene expression mean among sexually differentiated fish were analyzed using two-way analysis of variance (ANOVA) and validation of mixed model using discriminant analysis (DA) for morphometric traits and the gene expression in gonads and tail fin tissues used to validate and utilize them in discriminating sexes in sex-differentiated Nile Tilapia fish. The results revealed that, cyp19a1a gene expression in female ovaries was more significant than dmrt1 in male testis. In the other hand, the dmrt1 gene expression in the tail fin was higher in males than females. Both, cyp19a1a and dmrt1 genes, can discriminate fish sexes by 100% by using their expression in tail fin tissues. In conclusion, the cyp19a1a and dmrt1 genes could be used as a genetic marker to discriminate between the Nile Tilapia sexes, whereas used as an indicator for ovarian or testis differentiation in sexually differentiated Nile Tilapia using tail fin tissues. It is worth mentioning that this is the first investigation for using cyp19a1a and dmrt1 genes from Nile Tilapia tail fin tissues in sex determination.

Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch

In this study, we first sequenced the whole genome of the yellow perch (YP, Perca favencens) and screened transcripts of brain, liver, and gonadal tissues from fish of different sexes. By comparing known female sex-related genes in fish such as cyp19a1 and dmrt1 with YP genome, we obtained full-length sequences through genomic homology analysis. Then an experiment was conducted to evaluate the effects of cyp19a1-siRNA interference on gonadal development and sex differentiation and determination in YP. Transcriptome analysis revealed that the gonadal tissue of female YP exhibited predominantly and differentially up-regulated expression of genes than males, and cyp19a1 was predominantly expressed in the gonads while cyp19a2 showed predominant expression in the brain. KEGG pathway enrichment analysis illustrated the metabolic pathways of the top 25 enriched genes and confirmed CYP19 (P450) pathway in YP. For the cyp19a1-siRNA interference experiment, four intermittent in vivo injections of siRNA were administered to interfere with cyp19a1 gene expression in an all-female group of YP. The results collected at 180 dph revealed that the siRNA interference resulted in an incomplete masculinization and hermaphroditism of all-female monosex YP, displaying the presence of male spermatocytes along with female oocytes. In contrast, tissues from the blank control and negative control groups only contained oocytes. The appearance of the gonads in the experimental group appeared more irregular, lacking the smooth surface and typical histoarchitecture observed in the blank control and negative control groups. In addition, the cyp19a1-siRNA treatment group had significantly (P < 0.05) lower body weight than the control groups, indicating that the interference with cyp19a1 might have affected the expression of estrogen, leading to a trend similar to the growth of males; similarly, the gonad weight and GSIs of the cyp19a1 group were significantly (P < 0.05) lower than those in the control groups, suggesting that the cyp19a1-siRNA interference might have led to a trend of male type of gonadal development. Meanwhile, the treatment group had significantly (P < 0.05) higher HSI and VSI levels compared to control groups, indicating that stimulation of external siRNA might have resulted in more vigorous liver metabolism processes. Furthermore, the expression level of the dmrt1 in the treatment group was significantly (P < 0.05) upregulated compared to the control groups, suggesting the correlation roles between these two genes in the regulation of sex development. The findings provide valuable insights for future research into the underlying mechanisms involved and hold significant promise for sex control in aquaculture.

Completely blocking the feminization by replacing Limnodilus spp. with Artemia salina to feeding XY Zig-zag eel (Mastacembelus armatus)

Zig-zag eel (Mastacembelus armatus), with male growing faster and larger than female, is a popular farmed fish in China due to its delicious taste and rich nutritional value. The culture of mono-sex M. armatus has great value and is the best choice for aquaculture industry. However, the high feminization rate in aquaculture severely limits the application of sex control in the breeding process of M. armatus. In this study, by analyzing the early gonadal development and sex differentiation process, as well as the expression profiles of male and female pathway genes in the early XX and XY gonads, we determined that the critical period for sex differentiation and gonadal differentiation in M. armatus was 10–30 dpf. In addition, we confirmed for the first time that the feminization of M. armatus is related to feeding Limnodilus spp., as only Limnodilus spp. are fed during 10–30 dpf. Replacing Limnodilus spp. with Artemia salina to feed M. armatus fry can completely block the feminization. These results lay a solid foundation for understanding the sex determination mechanism of M. armatus, and the method of blocking the feminization of M. armatus can be used for the development of all-male breeding.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17β (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Potential Implications of Acid-Sensing Ion Channels ASIC2 and ASIC4 in Gonadal Differentiation of Dicentrarchus labrax Subjected to Water Temperature Increase during Gonadal Development.

In this study, the expression and implication of acid-sensing ion channels 2 and 4 (ASIC2 and ASIC4) in the gonadal sex differentiation of Dicentrarchus labrax (D. labrax), subjected to increasing water temperatures during gonadal development, were evaluated. Two groups were selected: a control group (CG), in which the average water temperature was maintained at 15 °C and increased to 20 °C in 20 days until weaning; and an experimental group (EG), in which the water temperature was retained at 15 °C for 60 days; thereafter, the temperature was increased daily by 0.5 °C until it reached 20 °C up to the weaning time. Ten fish from the CG and 13 fish from the EG were sampled randomly on the 335th day after hatching (dph). A higher percentage of gonad differentiation in ovaries rather than in testes was observed in the EG compared to the CG (p = 0.01). ASIC2 and ASIC4 were detected for the first time in D. labrax ovaries by indirect immunofluorescence. Both ASIC2 and ASIC4 were expressed in previtellogenic oocytes of ovaries and in scattered cells within some testes, and were most likely intratesticular previtellogenic oocytes in both the CG and EG groups. The CG group showed a higher expression of ASIC4 than the EG cohort (p < 0.05). The results gathered in this study revealed the capacity of water temperature to influence both gonadal differentiation and growth in this gonochoristic fish species, and suggests the possible role of ASIC2 and ASIC4 in gonad differentiation and gamete development in D. labrax.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and β (ERα and Erβ), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences.

Extraordinary variability in gene activation and repression programs during gonadal sex differentiation across vertebrates.

Genes involved in gonadal sex differentiation have been traditionally thought to be fairly conserved across vertebrates, but this has been lately questioned. Here, we performed the first comparative analysis of gonadal transcriptomes across vertebrates, from fish to mammals. Our results unambiguously show an extraordinary overall variability in gene activation and repression programs without a phylogenetic pattern. During sex differentiation, genes such as dmrt1, sox9, amh, cyp19a and foxl2 were consistently either male- or female-enriched across species while many genes with the greatest expression change within each sex were not. We also found that downregulation in the opposite sex, which had only been quantified in the mouse model, was also prominent in the rest of vertebrates. Finally, we report 16 novel conserved markers (e.g., fshr and dazl) and 11 signaling pathways. We propose viewing vertebrate gonadal sex differentiation as a hierarchical network, with conserved hub genes such as sox9 and amh alongside less connected and less conserved nodes. This proposed framework implies that evolutionary pressures may impact genes based on their level of connectivity.

Effect of diisobutyl adipate on the expression of biomarker genes that respond to endocrine disruption and on gonadal sexual differentiation in Japanese medaka (Oryzias latipes)

Phthalate and non-phthalate plasticizers are used in polymer materials, such as plastic and rubber. It has recently been found that diisobutyl adipate (DIBA), which is considered an environmentally safe non-phthalate plasticizer, potentially acts as a thyroid disruptor in fish. Here, we investigated the sexual hormone effects of DIBA based on the expression levels of genes that respond to endocrine disruption and sexual hormone activity in the livers and gonads, and on gonadal sexual differentiation in Japanese medaka. Compared with the control group, the mRNA expression of chgH, vtg1, vtg2, and esr1 was significantly suppressed in the livers of DIBA exposed XX individuals. Furthermore, the mRNA expression of gsdf was significantly upregulated and downregulated in the gonads of XX and XY individuals, respectively. The mRNA expressions of esr1 and esr2b were significantly suppressed by DIBA exposure in the gonads of both XX and XY individuals. These observations suggest that DIBA has potential androgenic activity in Japanese medaka. However, normal testes and ovaries were observed in respective XY and XX medaka after DIBA exposure; therefore, these results suggest that DIBA may have weak androgenic activity.

Histological Analysis of Gonadal Ridge Development and Sex Differentiation of Gonads in Three Gecko Species.

Reptiles constitute a highly diverse group of vertebrates, with their evolutionary lineages having diverged relatively early. The types of sex determination exemplify the diversity of reptiles; however, there are limited data regarding the gonadal development in squamate reptiles. Geckos constitute a group that is increasingly used in research and that serves as a potential reptilian model organism. The aim of this study was to trace the changes in the structure of developing gonads in the embryos of three gecko species: the crested gecko, leopard gecko, and mourning gecko. These species represent different families of the Gekkota infraorder and exhibit different types of sex determination. Gonadal development was examined from the formation of the earliest gonadal ridges through the development of undifferentiated gonadal structures, sex differentiation of gonads, and the formation of testicular and ovarian structures. The study showed that the gonadal primordia of these three gecko species formed on the most dorsally located surface of the dorsal mesentery, and both the coelomic epithelium and the nephric mesenchyme contributed to their development. As in other reptile species, primordial germ cells settled in the gonadal ridges, and the undifferentiated gonad was composed of a cortex and a medulla. Ovarian differentiation started with the thickening of the gonadal cortex and proliferation of germ cells in this region. A characteristic feature of the developing gecko ovaries was the thickened crescent-shaped cortex on the medial and ventral surfaces of the ovaries. The ovarian medulla also grew and exhibited diverse tendencies to form cords. In the leopard gecko, advanced cord-like structures with lumens were observed in the ovaries, which were not seen in the crested gecko. Testicular differentiation was characterized by cortical thinning and the disappearance of germ cells in this region. In the medulla, the development of distinct cords with early lumen formation was noted. A characteristic feature of embryonic gonads was their growth in a horizontal plane. In this study, gonadal development was characterized by several features that are shared by geckos and other reptiles, along with features that are specific only to geckos.

Gastrulation-stage alcohol exposure induces similar rates of craniofacial malformations in male and female C57BL/6J mice.

Prenatal alcohol exposure during gastrulation (embryonic day [E] 7 in mice, ~3rd week of human pregnancy) impairs eye, facial, and cortical development, recapitulating birth defects characteristic of Fetal Alcohol Syndrome (FAS). However, it is not known whether the prevalence or severity of craniofacial features associated with FAS is affected by biological sex. The current study administered either alcohol (2.9 g/kg, two i.p. doses, 4 hr apart) or vehicle to pregnant C57BL/6J females on E7, prior to gonadal sex differentiation, and assessed fetal morphology at E17. Whereas sex did not affect fetal size in controls, alcohol-exposed females were smaller than both control females and alcohol-treated males. Alcohol exposure increased the incidence of eye defects to a similar degree in males and females. Together, these data suggest that females might be more sensitive to the general developmental effects of alcohol, but not effects specific to the craniofacies. Whole transcriptomic analysis of untreated E7 embryos found 214 differentially expressed genes in females vs. males, including those in pathways related to cilia and mitochondria, histone demethylase activity, and pluripotency. Gastrulation-stage alcohol induces craniofacial malformations in male and female mouse fetuses at similar rates and severity, though growth deficits are more prevalent females. These findings support the investigation of biological sex as a contributing factor in prenatal alcohol studies.

Chronological Changes in Gonadotropin-Releasing Hormone 1, Gonadotropins, and Sex Steroid Hormones along the Brain-Pituitary-Gonadal Axis during Gonadal Sex Differentiation and Development in the Longtooth Grouper, Epinephelus bruneus.

(1) Fshβ and Lhβ showed stronger signals and higher transcript levels from 590 to 1050 dph than at earlier stages, implying their active involvement during primary oocyte development. (2) Fshβ and Lhβ at lower levels were detected during the phases of ovarian differentiation and oogonial proliferation. (3) E2 concentrations increased significantly at 174, 333, and 1435 dph, while T concentrations exhibited significant increases at 174 and 333 dph. These findings suggest potential correlations between serum E2 concentrations and the phases of oogonial proliferation and pre-vitellogenesis.

Transcriptome Analyses Reveal Expression Profiles of Morphologically Undifferentiated and Differentiated Gonads of Yangtze Sturgeon Acipenser dabryanus.

Sturgeon is known as a primitive fish with the ZZ/ZW sex determination system and is highly prized for its valuable caviar. Exploring the molecular mechanisms underlying gonadal differentiation would contribute to broadening our knowledge on the genetic regulation of sex differentiation of fish, enabling improved artificial breeding and management of sturgeons. However, the mechanisms are still poorly understood in sturgeons. This study aimed to profile expression patterns between female and male gonads at morphologically undifferentiated and early differentiated stages and identify vital genes involved in gonadal sex differentiation of sturgeons. The sexes of Yangtze sturgeon (Acipenser dabryanus) juveniles were identified via the sex-specific DNA marker and histological observation. Transcriptome analyses were carried out on female and male gonads at 30, 80 and 180 days post-hatching. The results showed that there was a total of 17 overlapped DEGs in the comparison groups of between female and male gonads at the three developmental stages, in which there were three DEGs related to ovarian steroidogenesis, including hsd17b1, foxl2 and cyp19a1. The three DEGs were highly expressed in the female gonads, of which the expression levels were gradually increased with the number of days after hatching. No well-known testis-related genes were found in the overlapped DEGs. Additionally, the expression levels of hsd17b1 and cyp19a1 mRNA were decreased with the knockdown of foxl2 mRNA via siRNA. The results further suggested that foxl2 should play a crucial role in the ovarian differentiation of sturgeons. In conclusion, this study showed that more genes involved in ovarian development than testis development emerged with sexually dimorphic expression during early gonadal sex differentiation, and it provided a preliminary understanding of the molecular regulation on gonadal differentiation of sturgeons.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

No effects of the antiandrogens cyproterone acetate (CPA), flutamide and p,p'-DDE on early sexual differentiation but CPA-induced retardation of embryonic development in the domestic fowl (Gallus gallus domesticus).

Because a wide range of environmental contaminants are known to cause endocrine disorders in humans and animals, in vivo tests are needed to identify such endocrine disrupting chemicals (EDCs) and to assess their biological effects. Despite the lack of a standardized guideline, the avian embryo has been shown to be a promising model system which responds sensitively to EDCs. After previous studies on the effects of estrogenic, antiestrogenic and androgenic substances, the present work focuses on the effects of in ovo exposure to p,p'-DDE, flutamide and cyproterone acetate (CPA) as antiandrogenic model compounds regarding gonadal sex differentiation and embryonic development of the domestic fowl (Gallus gallus domesticus). The substances were injected into the yolk of fertilized eggs on embryonic day one. On embryonic day 19 sex genotype and phenotype were determined, followed by gross morphological and histological examination of the gonads. Treatment with flutamide (0.5, 5, 50 µg/g egg), p,p'-DDE (0.5, 5, 50 µg/g egg) or CPA (0.2, 2, 20 µg/g egg) did not affect male or female gonad development, assessed by gonad surface area and cortex thickness in both sexes and by the percentage of seminiferous tubules in males as endpoints. This leads to the conclusion that antiandrogens do not affect sexual differentiation during embryonic development of G. gallus domesticus, reflecting that gonads are not target organs for androgens in birds. In ovo exposure to 2 and 20 µg CPA/g egg, however, resulted in significantly smaller embryos as displayed by shortened lengths of skull, ulna and tarsometatarsus. Although gonadal endpoints were not affected by antiandrogens, the embryo of G. gallus domesticus is shown to be a suitable test system for the identification of substance-related mortality and developmental delays.

Wnt4a Is Indispensable for Genital Duct Elongation but Not for Gonadal Sex Differentiation in the Medaka, Oryzias latipes.

In most vertebrates, the oviducts and sperm ducts are derived from the Müllerian ducts and Wolffian ducts, respectively. However, in teleosts, the genital ducts are formed by the posterior extension of gonads in both sexes. Whether the genital ducts of teleosts are newly evolved organs or variants of Müllerian ducts is an important question for understanding evolutionary mechanisms of morphogenesis. One of the genes essential for Müllerian duct formation in mice is Wnt4, which is expressed in the mesenchyme and induces invagination of the coelomic epithelium and its posterior elongation. Here, we addressed the above question by examining genital duct development in mutants of two Wnt4 genes in the medaka (wnt4a is orthologous to mouse Wnt4, and wnt4b is paralogous). The wnt4b mutants had a short body but were fertile with normal genital ducts. In contrast, both male and female wnt4a mutants had their posterior elongation of the gonads stopped within or just outside the coelom. The mutants retained the posterior parts of ovarian cavities or sperm duct primordia, which are potential target tissues of Wnt4a. The gonads of female scl mutants (unable to synthesize sex steroids) lacked these tissues and did not develop genital ducts. Medaka wnt4a was expressed in the mesenchyme ventral to the genital ducts in both sexes. Taken together, the data strongly suggest that the mouse Müllerian ducts and the medaka genital ducts share homologous developmental processes. Additionally, the wnt4a or wnt4b single mutants and the double mutants did not show sex-reversal, implying that both genes are dispensable for gonadal sex differentiation in the medaka.

Gene Expression Profiling Reveals Potential Players of Sex Determination and Asymmetrical Development in Chicken Embryo Gonads.

Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with ovary and testis development via an analysis of the results of PacBio and Illumina transcriptome sequencing of embryonic chicken gonads at the initiation of sexual differentiation (E4.5, E5.5, and E6.5). PacBio sequencing detected 328 and 233 significantly up-regulated transcript isoforms in females and males at E4.5, respectively. Illumina sequencing detected 95, 296 and 445 DEGs at E4.5, E5.5, and E6.5, respectively. Moreover, both sexes showed asymmetrical expression in gonads, and more DEGs were detected on the left side. There were 12 DEGs involved in cell proliferation shared between males and females in the left gonads. GO analysis suggested that coagulation pathways may be involved in the degradation of the right gonad in females and that blood oxygen transport pathways may be involved in preventing the degradation of the right gonad in males. These results provide a comprehensive expression profile of chicken embryo gonads at the initiation of sexual differentiation, which can serve as a theoretical basis for further understanding the mechanism of bird sex determination and its evolutionary process.