Early Sex Differentiation Research Articles

Association between maternal androgen levels and early fetal sex differentiation: Anogenital distance and genital tubercle length in the first trimester.

To evaluate the use of anogenital distance (AGD) and genital tubercle length (GTL) between 11 and 13+6weeks of gestation for fetal sex determination and to assess the impact of maternal androgen levels on these measurements. A cross-sectional study was conducted from February to June 2017 with patients undergoing first trimester Down syndrome screening. Inclusion criteria were: (1) female age 18-49, (2) gestational age between 11 and 13+6weeks, (3) optimal visualization of AGD and GTL, and (4) nonsmoking status. Maternal androgen levels (total testosterone, free testosterone, androstenedione, and dehydroepiandrostenedione) were measured simultaneously with ultrasound. AGD was significantly higher in male fetuses compared to females. With a cutoff of 5.0mm, fetal sex was identified in 82% of female fetuses and 70% of male fetuses after the 12th week. Linear regression analysis showed that only AGD was a significant predictor of fetal sex (β: 0.54, p<0.001). In the 12-12+6week group, a positive correlation between AGD and maternal androstenedione was found in female fetuses (r: 0.23, p=0.038). In contrast, negative correlations were observed between AGD and both androstenedione (r: -0.475, p<0.001) and total testosterone (r: -0.282, p=0.026) in male fetuses. AGD and GTL measurements show sexual dimorphism in the first trimester, with AGD correlating positively with maternal androstenedione in females and negatively in males. These findings suggest that AGD and GTL are reliable, non-invasive methods for early fetal sex determination.

Transcriptome analysis of four types of gonadal tissues in largemouth bass (Micropterus salmoides) to reveal its sex-related genes.

The sex determination system of largemouth bass (Micropterus salmoides, LMB) is XX/XY; however, the underlying molecular mechanisms involved in early sex differentiation, gonadal development, and exogenous hormone-induced sex reversal remain unknown. In this study, LMB at 15days post-hatching (dph) were fed diets containing 20mg/kg of 17α-methyltestosterone (17α-MT) or 30mg/kg of 17β-estradiol (17β-E2) for 60days, respectively. Serum steroid levels, histological observations of the gonads, and identification of sex-specific markers were employed to screen the gonads of 60-day-old normal female fish (XX-F), normal male fish (XY-M), 17β-E2 induced pseudo-female fish (XY-F), and 17α-MT-induced pseudo-male fish (XX-M) for transcriptome sequencing in order to uncover genes and pathway involved in the process of sexual reversal. The results from histology and serum sex steroid hormone analysis showed that both 17α-MT and 17β-E2 were capable of inducing sex reversal of LMB at 15dph. Transcriptome results revealed a total of 2,753 genes exhibiting differential expression, and the expression pattern of these genes in the gonads of XX-M or XY-F resembled that of normal females or males. The male sex-biased genes that are upregulated in XX-M and downregulated in XY-F are referred to as key genes for male reversal, while the female sex-biased genes that are upregulated in XY-F and downregulated in XX-M are referred to as key genes for female reversal. Finally, 12 differentially expressed genes (DEGs) related to male sex reversal were screened, including star2, cyp17a, cyp11b1, dmrt1, amh, sox9a, katnal1, spata4, spata6l, spata7, spata18 and foxl3. 2 DEGs (foxl2a and cyp19a1b) were found to be associated with female sex reversal. The changes in these genes collectively influence the direction of sex differentiation of LMB. Among them, star2, dmrt1 and cyp19a1b with significantly altered expression levels may play potentially crucial role in the process of gender reversal. The expression patterns of 21 randomly selected genes were verified using qRT-PCR which confirmed the reliability and accuracy of the RNA-seq results. These findings not only enhance our understanding of the molecular basis underlying sex reversal but also provide crucial data support for future breeding research on unisexual LMB.

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.

Early Sex Differentiation of Climbing Perch (Anabas testudineus Bloch.): A Pathway to Feminization

The phenomenon of sexual dimorphism in climbing perch, which shows that female fish grow faster than males, underlies the development of mono-sex culture. Female mono-sex culture is more applicable for farmers by crossing neo-male fish with normal females. The timing of sexual differentiation in climbing perch is still unknown. It is very useful in sex reversal procedures to produce neo-male climbing perch. This study revealed the time and status of climbing perch sexual differentiation. Ten samples of climbing perch from the spawning of five pairs of parents were taken from the nursery pond at 10–29 days post-hatching (dph). Samples were prepared through a histology preparation procedure. Observations of the structure and characteristics of the gonads were carried out using a light microscope and analyzed histologically. The results indicated that gonad samples aged 10, 11, 12, 13, 15, and 16 dph showed primordial germ cells surrounded by somatic tissue forming genital ridges and mitotic division. Meanwhile, the gonads begin to differentiate as ovaries found at 18 dph with the presence of oogonia and ovarian cavities. Gonads aged 20–21 dph increasingly showed single oogonia cells (size 20–37.5 µm), germ cell cysts, genital ridges, oocytes undergoing the vitellogenesis process, perinucleolar oocytes, and the formation of the ovarian cavity. Sex differentiation of climbing perch was predicted from 18–21 dph. This conclusion underlies that the sex reversal procedure in climbing perch must be carried out before 18 dph.

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.

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.

Sex biased expression of Fem-1 in larval stages suggests its function in early sex differentiation of Litopeneaus vannamei

The Pacific white shrimp Litopenaeus vannamei (L. vannamei) is an economically important species worldwide. As females exhibit a faster growth rate than males, it is crucial to understand the mechanism of sex determination and differentiation for the purpose of mono-sex breeding in shrimp. The feminization-1 gene (Fem-1) serves as a sex-determining gene in Caenorhabditis elegans, and the Fem-1 family is implicated in sex development in several crustaceans. In this study, three Fem-1 family members, namely LvFem-1a, LvFem-1b, and LvFem-1c, were identified and characterized in L. vannamei. Based on genetic sex marker, individuals were separated by sex during the early developmental stages. The expression profiles of these genes were analyzed in female or male larvae. Significant differences between females and males were observed during the early stages of development. In Zoea III or earlier stages, the expressions levels in males were significantly higher than those in females. Intriguingly, there was a shift towards higher expression in females during the mysis and post-larval stages. These findings indicate that the Zoea III stage may be crucial for sex differentiation in shrimp. In addition, the analysis of gene expression across various ovarian stages and tissues was conducted. Results showed that all three genes were highly expressed in the ovary, with particularly elevated levels in ovarian development stage II. These findings suggest that the Fem-1 genes play a critical role in early ovary development. This study is the first research focusing on the sex-specific expression of sex differentiation genes in shrimp. The obtained results provide new insights into to the role of the Fem-1 gene family in sex differentiation and gonadal development.

Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model

BackgroundSex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems.ResultsWe conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory.Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation.ConclusionOur data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

A testis-specific gene doublesex is involved in spermatogenesis and early sex differentiation of the Chinese mitten crab Eriocheir sinensis

The doublesex (Dsx) is a DM domain gene involved in sex determination and/or differentiation in insects. In this study, a novel sex-specific Dsx homolog, designated as EsDsx-like, was identified from the Chinese mitten crab Eriocheir sinensis. The full-length cDNA of EsDsx-like was 921 bp, encoding a protein of 215 amino acids. The EsDsx-like mRNA and protein were detected exclusively in the testis. During spermatogenesis, the EsDsx-like mRNA were continuously expressed in spermatogonium, spermatocyte, spermatid and spermatozoon. In situ hybridization analysis showed that the EsDsx-like mRNA was mainly detected in the nucleus of spermatogonium and spermatocyte, and then concentrated in a dot on the edge of the nucleus in spermatid. In spermatozoa, the hybridization signal was localized to the acrosomal tubule. Immunofluorescence analysis revealed that the EsDsx-like protein was detected mainly in the cytoplasm around the nucleus in spermatocyte and spermatid, and then appeared as a dot immuno-signal in the acrosomal tubule and distributed in the cup-shaped nucleus of the spermatozoa. Together, these results suggest a potential role for EsDsx-like in the formation of acrosome complex and nuclear morphogenesis during sperm maturation. In the early development, similar with the male sex regulator, insulin-like androgenic gland hormone (EsIAG), the EsDsx-like mRNA was specifically detected in all male individuals tested at juvenile III stage just before the first appearance of the petasma in morphological sex differentiation at juvenile IV. Further, RNAi knockdown of EsDsx-like significantly downregulated the expression of EsIAG in adult and juvenile-III males. The direct interaction between the EsDsx-like protein and EsIAG promoter was confirmed by dual-luciferase reporter assays, indicating that EsDsx-like could act as an upstream regulatory gene of EsIAG in sex differentiation. This study provided valuable insights into the role of sex-specific gene associated with morphological sex differentiation and might contribute to development of sex control techniques in the Chinese mitten crab.

The transcriptional landscape of the giant freshwater prawn: Embryonic development and early sexual differentiationmechanisms.

The giant freshwater prawn pjMacrobrachium rosenbergii is one of the best studied species in aquaculture. However, the transcriptional changes associated with embryonic development and the sexual differentiation mechanism of M. rosenbergii remain to be elucidated. To characterize the embryonic development of this prawn and to determine whether differential expression and differential splicing play roles in the early sexual differentiation of M. rosenbergii, we profiled five developmental days of male and female embryos by RNA sequencing. We identified modules of co-expressed genes representing waves of transcription that correspond to physiological processes in early embryonic development (such as the maternal-to-zygotic transition) up to preparation for life outside the egg (development of muscles, cuticle etc.). Additionally, we found that hundreds of genes are differentially expressed between sexes, most of them uncharacterized, suggesting that the sex differentiation mechanism of M. rosenbergii might contain clade-specific elements. The resulting first-of-a-kind transcriptional map of embryonic development of male and female M. rosenbergii will guide future studies to reveal the roles of specific genes and splicing isoforms in the embryonic development and sexual differentiation process of M. rosenbergii.

Comparative transcriptome analysis of early sexual differentiation in the male and female gonads of common carp (Cyprinus carpio)

The growth rate of allotetraploid common carp (Cyprinus carpio) is sex dimorphic. Thus, intervention for controlling sex is crucial for farming this fish. However, few studies have investigated the spatiotemporal interaction of interaction of various genes related to sex determination/early gonadal differentiation in carp. In this study, comparative gonadal transcriptomic analyses were conducted during 4 key sex differentiaton stages for common carp of both sexes including sex determination stage, pre-gonadal, gonadal differentiation, and maturation stages. Altogether, 305.66 Gb raw reads were obtained form 48 libraries. Differentially expressed genes (DEGs) associated with sex differentiation and gonadal development were identified at each time point. Male-specific DEGs including dmrt1, amh, hmga2, sox3, dmrt2, wnt2b, and wnt9b, and female-biased DEGs including foxl2, cyp19a1a, zp3, zp4, and gper1 were identified. The trend analysis of the key genes of sex determination and gonadal differentiation indicated that 35 dph or earlier is the male sex determination period, 90–100 dph is the key window period of male sex differentiation, and 90–100 dph may be the key window period of female sex differentiation. Functional enrichment analysis showed that DEGs at the testicular undifferentiated stages were mainly enriched in androgen receptor signaling, while DEGs from the onset of testicular differentiation to maturity were enriched in male sex differentiation, spermatogenesis, and male gonadal development. In genetic females, DEGs at the undifferentiated ovarian stage are mainly enriched in insulin-like growth factor (IGF) transport, whereas DEGs from the onset of ovarian differentiation to maturation are mainly enriched in ovarian steroidogenesis and oocyte meiosis. Protein-protein interaction (PPI) analysis revealed that 8 DEGs, including Foxl2, Src, Ncor1, and Smad4, were identified as hub proteins. Additionally, plasma 17β-estradiol and 11-Ketotestosterone levels were sexually dimorphic in C. carpo. The study provide insights into the genetic network of the underlying mechanisms of early sex determination and gonadal differentiation in C. carpo for intervening all-female production in better way.

MiR-34b/c play a role in early sex differentiation of Amur sturgeon, Acipenser schrenckii.

BackgroundSex differentiation can be viewed as a controlled regulatory balance between sex differentiation-related mRNAs and post-transcriptional mechanisms mediated by non-coding RNAs. In mammals, increasing evidence has been reported regarding the importance of gonad-specific microRNAs (miRNAs) in sex differentiation. Although many fishes express a large number of gonadal miRNAs, the effects of these sex-biased miRNAs on sex differentiation in teleost fish remain unknown. Previous studies have shown the exclusive and sexually dimorphic expression of miR-34b/c in the gonads of the Amur sturgeon (Acipenser schrenckii), suggesting its potential role in the sex differentiation process.ResultsUsing quantitative real-time PCR (qPCR), we observed that miR-34b/c showed consistent spatiotemporal expression patterns; the expression levels significantly increased during early sex differentiation. Using in situ hybridization, miR-34c was found to be located in the germ cells. In primary germ cells in vitro, the group subjected to overexpression and inhibition of miR-34c showed significantly higher proliferation ability and lower apoptosis, respectively, compared to the corresponding control group. Luciferase reporter assays using the ar-3′UTR-psiCHECK-2 luciferase vector suggested a targeted regulatory interaction between miR-34b/c and the 3′UTR of the androgen receptor (ar) mRNA. Furthermore, miR-34b/c and ar showed negative expression patterns during early sex differentiation. Additionally, a negative feedback regulation pattern was observed between foxl2 expression in the ovaries and amh and sox9 expression in the testes during early sex differentiation.ConclusionsThis study sheds new light on the roles of miR-34b/c in gonad development of Amur sturgeon, and provides the first comprehensive evidence that the gonad-predominant microRNAs may have a major role in sex differentiation in teleost fish.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12983-022-00469-6.

2,4-Dichlorophenol Increases Primordial Germ Cell Numbers via ESR2a-Dependent Pathway in Zebrafish Larvae.

Previous studies have reported the feminizing effects of 2,4-dichlorophenol (2,4-DCP) on zebrafish (Danio rerio). However, the effect of 2,4-DCP on the number of primordial germ cells (PGCs), an indicator for early sex differentiation, remains elusive. In the present study, Tg (piwil1:egfp-UTR nanos3) zebrafish (GFP-labeled PGCs) were treated with 2,4-DCP (10, 20, and 40 μg/L) from 5 to 15 days postfertilization to explore the effect on PGC numbers and to elucidate associated molecular mechanisms. The results showed that 2,4-DCP exposure increased PGC numbers, as evidenced by larger GFP fluorescent areas, upregulated expressions of PGC marker genes (vasa and dnd), and raised the female ratio. Notably, the mRNA level of estrogen receptor 2a (esr2a) was also increased subsequently. Moreover, docking studies revealed stable 2,4-DCP interactions with ESR2a, speculating a role of ESR2a signaling pathway in 2,4-DCP toxicity. Furthermore, in esr2a knockout (esr2a-/-) zebrafish, the effects of 2,4-DCP were considerably minimized, proving the involvement of the ESR2a signaling pathway in the 2,4-DCP-mediated increase in PGC numbers. Dual-luciferase reporter gene assay and point mutation studies demonstrated that 2,4-DCP-stimulated promoter activity was mediated by estrogen response element (ERE) located in -686/-674 of the vasa promoter and -731/-719 of the dnd promoter. Overall, 2,4-DCP can potentially enhance the expression of vasa and dnd by binding to zebrafish ESR2a, thus leading to increased PGC numbers and subsequent female-biased sex differentiation.

ACL Research Retreat IX Summary Statement: The Pediatric Athlete, March 17-19, 2022; High Point, North Carolina.

ACL Research Retreat IX Summary Statement: The Pediatric Athlete, March 17-19, 2022; High Point, North Carolina.

Feminization of channel catfish with 17β-oestradiol involves methylation and expression of a specific set of genes independent of the sex determination region

ABSTRACT Exogenous oestrogen 17β-oestradiol (E2) has been shown to effectively induce feminization in teleosts. However, the molecular mechanisms underlying the process remain unclear. Here, we determined global DNA methylation and gene expression profiles of channel catfish (Ictalurus punctatus) during early sex differentiation after E2 treatment. Overall, the levels of global DNA methylation after E2 treatment were not significantly different from those of controls. However, a specific set of genes were differentially methylated, which included many sex differentiation-related pathways, such as MARK signalling, adrenergic signalling, Wnt signalling, GnRH signalling, ErbB signalling, and ECM–receptor interactions. Many genes involved in these pathways were also differentially expressed after E2 treatment. Specifically, E2 treatments resulted in upregulation of female-related genes and downregulation of male-related genes in genetic males during sex reversal. However, E2-induced sex reversal did not cause sex-specific changes in methylation profiles or gene expression within the sex determination region (SDR) on chromosome 4, suggesting that E2-induced sex reversal was a downstream process independent of the sex determination process that was regulated by sex-specific methylation within the SDR.

The transcriptional regulator HDP1 controls expansion of the inner membrane complex during early sexual differentiation of malaria parasites.

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their limited number of transcriptional and chromatin regulators and the molecular mediators of these changes remain largely unknown. We recently identified HomeoDomain Protein 1 (HDP1) as a DNA-binding protein first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation. The discovery of HDP1 marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. In this study, using molecular biology, biochemistry, and microscopy techniques we showed that HDP1 is essential for gametocyte maturation, facilitating the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes their characteristic shape.

Food composition triggers early sex differentiation of a shrimp: a transcriptomic analysis

Food composition triggers early sex differentiation of a shrimp: a transcriptomic analysis

Molecular cloning and characterization of estrogen and androgen receptors in Mandarin fish, Siniperca chuatsi

Steroid hormones are essential for the normal reproductive activity in females and males, such as early sex differentiation, gonad development and sex reversal, which works by binding with the steroid receptors. In the present study, the full-length of estrogen receptors (ERα, ERβ1 and ERβ2) and androgen receptors (ARα and ARβ) cDNAs were isolated from the mandarin fish (Siniperca chuatsi). Real time quantitative PCR (RT-qPCR) revealed that ERα and ERβ2 expressed mainly in the liver of females and males, and ERβ1 mostly distributed in ovary and testis. Additionally, the high expressions of ARα and ARβ in both females and males were detected in gonads and livers respectively. During the early sex differentiation stage, the expressions of ERs and ARs in female were higher than in male during 5–30 day after hatching (dah), and reached the peak level at 30 dah. In males, the expressions of ERβ1, ERβ2, and ARβ increased largely during 30 dah to 60 dah, and revealed significantly higher levels than in females during 60–180 dah. In female, the expression of ERβ2 in ovary increased sharply at 180 dah. Moreover, the result of gonadal histology revealed that the spermatocytes were observed at 45 dah and the spermatozoon could be found largely at 60 dah in males and oil droplets appeared for the first time in the oocytes at 180 dah. The expression levels of three ERs and ARβ were up-regulated and the ARα was down-regulated during female to male sex reversal induced by 17α-methyltestosterone (MT). Furthermore, after MT treatment for 30 and 35 days, the expressions of ERβ1, ERβ2 and ARβ were significantly up-regulated. These results suggest that ERβ1, ERβ2 and ARβ may play critical roles in spermatogenesis and masculinization, while ERβ2 and ERβ1 may be essential for vitellogenesis and ovary development in female.

Cloning, pattern of gonadal soma-derived factor mRNA in the orange-spotted grouper, Epinephelus coioides

The orange-spotted grouper (Epinephelus coioides), as a protogynous hermaphrodite, provides a novel model for understanding the mechanisms of sex determination and differentiation in teleosts. Gonadal soma-derived factor (gsdf), involved in early germ cell development, is a new member of the transforming growth factor-beta (TGF-β) superfamily. In this study, a 2364-bp long gsdf gene was cloned from E. coioides and there was further analysis of its tissue distribution and patterns of gene expression during the female phase, bisexual gonads stage and testis stage. The cellular localization of Gsdf during the early sexual differentiation stage was also analyzed. In addition, the expression levels of gsdf, dmrt1, foxl2 and cyp19a1a in gonads during the early sexual differentiation stage were analyzed by real-time PCR. Tissue distribution analysis showed that gsdf was only expressed in the testis and ovary. The gene expression showed that gsdf was expressed significantly higher in testes and bisexual gonads compared to ovaries. The early-stage ovary showed a significant fluorescence signal in the supporting cells surrounding gonium and oogonium, but not pre-vitellogenic oocytes, indicating that gsdf expression was restricted to morphologically undifferentiated supporting cells in the early stages of oogenesis. During the early sexual differentiation stage, Real-time PCR showed that both the expression levels of gsdf and dmrt1 were progressively increased and the expression levels of foxl2 and cyp19a1a were growing slowly. These results suggest that gsdf could have roles in the early sexual differentiation in hermaphroditic, E. coioides.

Role and function of the Hintw in early sex differentiation in chicken (Gallus gallus) embryo

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, Hintw was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress Hintw in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of Hintw on gonadal development of chicken embryos. Results showed that Hintw exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that Foxl2, Cyp19a1 in females were upregulated under the overexpression of Hintw, while Sox9 and Dmrt1 were downregulated Hintw. Overexpression of Hintw can promote the development of gonadal cortex, while interference with Hintw show the opposite result. Additionally, we found that overexpression of the Hintw in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with Hintw in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.