Female Embryonic Gonads Research Articles

Direct in vitro propagation of avian germ cells from an embryonic gonad biorepository

Direct introduction of cryopreserved embryonic gonadal germ cells (GGC) into a sterile chicken surrogate host to reconstitute a chicken breed has been demonstrated as a feasible approach for preserving and utilizing chicken genetic resources. This method is highly efficient using male gonads; however, a large number of frozen female embryonic gonads is needed to provide sufficient purified GGC for the generation of fertile surrogate female hosts. Applying this method to indigenous chicken breeds and other bird species is difficult due to small flock numbers and poor egg production in each egg laying cycle. Propagating germ cells from the frozen gonadal tissues may be a solution for the biobanking of these birds. Here, we describe a simplified method for culture of GGC from frozen embryonic 9.5 d gonads. At this developmental stage, the germ cells are autonomously shed into medium, yielding hundreds to thousands of mitosis-competent germ cells. The resulting cultures of GGC have over 90% purity, uniformly express SSEA-1 and DAZL antigens and can re-colonize recipient's gonads. The GGC recovery rate from frozen gonads are 42% to 100%, depending on length of cryopreservation and the breed or line of chickens. Entire chicken embryos can also be directly cryopreserved for later gonadal isolation and culture. This storage method is a supplementary approach to safeguard local indigenous chicken breeds bearing valuable genetic traits and should be applicable to the biobanking of many bird species.

Differential expression and genetic polymorphism of DMRT1 and FOXL2 genes in the gonads of Kadaknath chicken

In chicken, the left ovary is functional and the right ovary becomes rudimentary in the later stage of the embryonic development. The expression and genetic polymorphism of the DMRT1 and FOXL2 genes are assessed in order to understand the distinct developing process of gonads in male and female embryonic gonads. In the current study, 19th day Kadaknath chicken gonadal tissue samples were collected from the embryos and RT-PCR analysis and DNA sequencing was done. According to the expression studies, the left ovaries relative expression of FOXL2 was 2.14 and 12.65 factor greater than that of the right ovaries and testes, while the right testis relative expression of DMRT1 was 3.78 and 3.83 fold higher than that of the left and right ovaries. DNA sequence analysis in the left ovary identified a nonsynonymous alteration (c.289C>G) involving histidine to aspartic acid at the 289th position in the FOXL2 coding region. The 51st position of exon 6 of DMRT1 has a G→A transition SNP that is not part of the coding sequence (CDS). Males showed higher levels of the DMRT1 gene than females, while females expressed higher levels of FOXL2. It was found that the DMRT1 and FOXL2 genes are polymorphic. The genetic variability in the gonads and the differences in DMRT1 and FOXL2 gene expression allow learning more about the critical mechanisms underlying chicken gonadal development.

WGBS of embryonic gonads revealed that long non-coding RNAs in the MHM region might be involved in cell autonomous sex identity and female gonadal development in chickens

ABSTRACT DNA methylation plays a key role in sex determination and differentiation in vertebrates. However, there are few studies on DNA methylation involved in chicken gonad development, and most focused on male hypermethylated regions (MHM). It is unclear whether there are specific differentially methylated regions (DMRs) in chicken embryonic gonads regulating sex determination and differentiation. Here, the DNA methylation maps showed that the difference of DNA methylation level between sexes was much higher at embryonic day 10 (E10) than that at embryonic day 6 (E6), and the significant differentially methylated regions at both stages were mainly distributed on the Z chromosome, including MHM1 and MHM2. The results of bisulphite sequencing PCR (BSP) and qRT-PCR showed hypomethylation of female MHM and upregulation of long non-coding RNAs (lncRNAs) whose promoter in the MHM region was consistent with the sequencing results, and similar results were in brain and muscle. In female sex-reversed gonads, the methylation pattern of MHM remained unchanged, and the expression levels of the three candidate lncRNAs were significantly decreased compared with those in females, but were significantly increased compared to males. The fluorescence in situ hybridization (FISH) results also showed that these lncRNAs were highly expressed in female embryonic gonads. The results of methyltransferase inhibitor and dual-luciferase reporter assay suggest that lncRNA expression may be regulated by DNA methylation within their promoters. Therefore, we speculated that MHM may be involved in cell-autonomous sex identity in chickens, and that lncRNAs regulated by MHM may be involved in female sexual differentiation.

Identification and Expression Analysis of Wnt2 Gene in the Sex Differentiation of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture animal in China. The Wnt gene family plays important regulatory roles in the development and growth of mammals. However, the precise function of these family genes has not been well understood in the sex differentiation of Chinese soft-shelled turtles. Here, we cloned a member of the Wnt family, Wnt2, which obtained a 1077 bp open reading frame that encoded a 358-aa protein. The putative amino acid sequences of proteins are exceeded 80% identical to other turtles. The expression level of Wnt2 peaked at the 14th stage both in female and male embryos during the early gonadal differentiation period of Chinese soft-shelled turtles, which occurred before gonadal differentiation. Wnt2 mRNA was expressed at higher levels in the brains and gonads of mature P. sinensis females compared with those in mature males. Wnt agonists significantly affected the expression level of Wnt2 during the gonadal differentiation period. After Wnt agonists (1.0 μg/μL, 2.5 μg/μL, 5.0 μg/μL) treatment, the expression level of the Wnt2 generally appeared to have an inverted-V trend over time in female embryonic gonads. The results suggested that Wnt2 may participate in the regulation of gonad development in P. sinensis during the early embryonic stages. These results could provide a theoretical basis for the reproduction process of the Chinese soft-shelled turtle.

Transcriptome analysis reveals key genes and pathways related to sex differentiation in the Chinese soft-shelled turtle (Pelodiscus sinensis)

Most vertebrates exhibit sexual dimorphisms in size, colour, behaviour, physiology and many others. The Chinese soft-shelled turtle (Pelodiscus sinensis) male individuals reach a larger size than females which produce significant economic implications in aquaculture. However, the mechanisms of sex determination and plastic patterns of sex differentiation in P. sinensis remain unclear. Here, comparative transcriptome analysis on male and female embryonic gonads prior to gonad formation and stages mediated gonadal differentiation of P. sinensis were performed to characterize the potential sex-related genes and their molecular pathways in P. sinensis. A total of 6369 differentially expressed genes (DEGs) were identified from day 9 and day 16 and assigned to 626 GO pathways and 161 KEGG signalling pathways, including ovarian steroidogenesis pathway, steroid hormone biosynthesis pathways, and the GnRH signalling pathway (P < 0.05). Moreover, protein interaction network analyses revealed that Akr1c3, Sult2b1, Sts, Cyp3a, Cyp1b1, Sox30 and Lhx9 might be key candidate genes for sex differentiation in P. sinensis. These data provide a genomic rationale for the sex differentiation of P. sinensis and enrich the candidate gene pool for sex differentiation.

Characterization and expression pattern of Wnt5b gene in Pelodiscus sinensis

Wingless-type MMTV integration site family member 5B (Wnt5b) regulates life activities such as cell proliferation, tissue morphogenesis and embryo development. We investigated the role of Wnt5b in sex differentiation in Pelodiscus sinensis by analyzing its expression in diverse tissues and embryonic gonads using quantitative reverse transcription PCR (qRT-PCR). Moreover, Wnt5b expression in gonads of P. sinensis embryos treated with three concentrations of a Wnt agonist was also measured. Sequence alignment indicated that P. sinensis Wnt5b (psWnt5b) protein shared a high sequence similarity with that of Trachemys scripta elegans. Wnt5b gene was highly expressed in embryonic gonads at stages 14–16, but the expression level decreased significantly at stage 17, and Wnt5b expression at stage 14 was significantly higher in female gonads than that in male gonads. Wnt5b had significantly higher expression level in the ovaries than in other tissues. Wnt agonists (5 μg/μl) significantly affected the expression of Wnt5b in male and female embryonic gonads. After Wnt agonists was injected, the expression level of the Wnt5b increased firstly and then decreased overtime. The results suggested that Wnt5b may play a potential important role in gonad development of P. sinensis during the early embryonic stages.

Sexually dimorphic expression of a chicken sex chromosome gene (VCP) reflects differences in gonadal development between males and females

The chicken has a Z-W sex chromosome system, in which the males are the homogametic sex (ZZ) and the females the heterogametic sex (ZW). The smaller W chromosome is generally considered to be a highly degraded copy of the Z chromosome that retains around 28–30 homologous protein-coding genes’ These Z-W homologues are thought to have important, but undefined, roles in development, and here we explore the role of one of these genes, VCP (Valosin Containing Protein) in gonadogenesis. We established RNA expression levels of both Z and W VCP homologues, the levels of VCP protein, and the cellular localization of VCP protein in male and female embryonic gonads during development. We also assessed the effects of female-to-male sex-reversal on VCP expression in developing gonads. The results showed that both VCP RNA and protein are expressed at higher levels in female than male gonads, and the expression levels of VCP protein and VCP-Z transcript, but not VCP-W transcript, are decreased in female-to-male sex reversed gonads. In addition, the spatial expression of VCP protein differs between male and female embryonic gonads: in testes, VCP protein is mainly confined to the medullary sex cords, while in ovaries, VCP protein is expressed throughout the medulla and at higher levels in the cortex. The results suggest that sexually dimorphic expression of chicken VCP reflects differences in gonadal morphology between sexes.

Androgens and androgen receptor signaling contribute to ovarian development in the chicken embryo

Androgens and androgen receptor (AR) signaling play important roles throughout development. In the chicken, AR signaling is involved in reproduction; however, its specific role is unclear. We show that AR signaling is involved in the normal development of the female embryonic gonads. The AR mRNA level was detected in male and female embryonic gonads by quantitative RT-PCR, and its expression was higher in females than in males at all developmental stages examined. In female embryos, the AR localized to nuclei of cells in the left gonad. Although AR expression was low in the majority of the medulla, high expression was detected in cells of lacunae within the medulla. In addition, AR expression increased in cells of cortical cords within the cortex with the progression of development. AR expression in the right gonad was lower than that in left gonad throughout development. In the male gonad, the AR localized to the cytoplasm of cells in seminiferous tubules at all stages. Female AR knockdown (ARKD) embryos infected with a retrovirus expressing micro RNAs targeting the AR showed normal asymmetric gonads (development of the left and depression of the right gonads), whereas the number of lacunae decreased. Furthermore, there was a disruption in the structure of cortical cords. By contrast, the gonads of ARKD males developed normally during embryogenesis. These results indicate that androgens and AR signaling are essential for the development of lacunae and cortical cords in gonads of female embryos.

Molecular cloning and expression pattern of &amp;lt;italic&amp;gt;Amh &amp;lt;/italic&amp;gt;and its preliminary functional analysis in male sexual differentiation in &amp;lt;italic&amp;gt;Pelodiscus sinensis&amp;lt;/italic&amp;gt;

To investigate the regulatory role of Amh in male sexual differentiation in Pelodiscus sinensis ( P. sinensis ), we performed the full-length cDNA cloning of Amh and its differential expression analysis, and subsequently conducted the loss-of-function of Amh through the lentivirus-mediated RNA interference approach in this study. RACE results showed that the complete cDNA sequence of P. sinensis Amh is 3233 bp, including 997 bp of 5′ un-translated region, 834 bp of 3′ un-translated region and 1401 bp of open reading frame, which encodes 466 amino acids. Real-time PCR analysis showed the testis-specific expression of Amh in P. sinensis . The male-specific expression of Amh in embryonic gonads initiated early at stage 16, preceding the onset of gonadal sex differentiation. However, the female embryonic gonads exhibited extremely low expression level of Amh throughout the embryogenesis. In addition, the expression of Amh was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment, whereas, Amh expression was dramatically decreased in feminized ZZ embryonic gonads induced by estrogen. Most importantly, RNA interference analysis showed that ZZ embryos with Amh knockdown exhibited male to female sex reversal, characterized by obviously feminized gonads with developed cortical regions and degenerated medulla, a decline in the testicular marker Sox9 , and up-regulation of ovarian marker Cyp19a1 . These data indicate that Amh is essential for primary male sexual differentiation and might functions as an upstream regulator in male pathway cascade in P. sinensis . This study provides a foundation for understanding the mechanism of sex determination in P. sinensis .

Sex-specific expression of CTNNB1 in the gonadal morphogenesis of the chicken

BackgroundBeta-catenin (CTNNB1), as a key transcriptional regulator in the WNT signal transduction cascade, plays a pivotal role in multiple biological functions such as embryonic development and homeostasis in adults. Although it has been suggested that CTNNB1 is required for gonad development and maintenance of ovarian function in mice, little is known about the expression and functional role of CTNNB1 in gonadal development and differentiation in the chicken reproductive system.MethodsTo examine sex-specific, cell-specific and temporal expression of CTNNB1 mRNA and protein during gonadal development to maturation of reproductive organs, we collected left and right gonads apart from mesonephric kidney of chicken embryos on embryonic day (E) 6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens and performed quantitative PCR, in situ hybridization, and immunohistochemical analyses. In addition, localization of Sertoli cell markers such as anti-Müllerian hormone (AMH), estrogen receptor alpha (ESR1), cyclin D1 (CCND1) and N-cadherin (CDH2) during testicular development was evaluated.ResultsResults of the present study showed that CTNNB1 mRNA and protein are expressed predominantly in the seminiferous cords on E6 to E14 in the male embryonic gonad, and are mainly localized to the medullary region of female embryonic gonads from E6 to E9. In addition, CTNNB1 mRNA and protein are abundant in the Sertoli cells in the testes and expressed predominantly in luminal epithelial cells of the oviduct, but not in the ovaries from 12-week-old and adult chickens. Concomitant with CTNNB1, AMH, ESR1, CCND1 and CDH2 were detected predominantly in the seminiferous cord of the medullary region of male gonads at E9 (after sex determination) and then maintained or decreased until hatching. Interestingly, AMH, ESR1, CCND1 and CDH2 were located in seminiferous tubules of the testes from 12-weeks-old chickens and ESR1, CCND1 and CDH2 were expressed predominantly in the Sertoli cells within seminiferous tubules of adult testes.ConclusionsCollectively, these results revealed that CTNNB1 is present in gonads of both sexes during embryonic development and it may play essential roles in differentiation of Sertoli cells during formation of seminiferous tubules during development of the testes.

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

GADD45G Functions in Male Sex Determination by Promoting p38 Signaling and Sry Expression

Male sex determination in mammals is induced by Sry, a gene whose regulation is poorly understood. Here we show that mice mutant for the stress-response gene Gadd45g display complete male-to-female sex reversal. Gadd45g and Sry havea strikingly similar expression pattern in the genital ridge, and they are coexpressed in gonadal somatic cells. In Gadd45g mutants, Sry expression is delayed and reduced, and yet Sry seemed to remain poised for expression, because its promoter is demethylated on schedule and is occupied by active histone marks. Instead, p38 MAPK signaling is impaired in Gadd45g mutants. Moreover, the transcription factor GATA4, which is required for Sry expression, binds tothe Sry promoter invivo in a MAPK-dependent manner. The results suggest that a signaling cascade, involving GADD45G → p38 MAPK → GATA4 → SRY, regulates male sex determination.

Expression of Wsb2 in the developing and adult mouse testis

We present a detailed study of the expression pattern of WD repeat and SOCS box-containing 2 (Wsb2) in mouse embryonic and adult gonads. Wsb2 was previously identified in a differential screen aimed at identifying the genes involved in male- and female-specific gonadal development. Wsb2 expression was analysed during mouse gonadogenesis by real-time PCR, whole-mount and section in situ hybridisation and immunofluorescence. Wsb2 mRNA expression was initially detected in gonads of both sexes from 11.5 days post coitum (dpc) until 12.0 dpc. By 12.5 dpc and thereafter, Wsb2 expression rapidly decreased in the female, while persisting in the male gonads. In foetal, newborn and juvenile testes, Wsb2 mRNA and protein were readily detected in the seminiferous cords within both Sertoli and germ cells. Wsb2 mRNA was present in spermatogonia, spermatocytes and in Sertoli cells of the adult mouse testis. The differential expression of Wsb2 in male versus female embryonic gonads suggests some male-specific role in gonad development, and its expression in the first wave of spermatogenesis indicates a role in germ cells. Real-time analysis of adult mouse testis tubules cultured in the presence of the Hedgehog signalling inhibitor, cyclopamine, showed a downregulation of Wsb2 mRNA after treatment which suggests that Wsb2 may be a target of Hedgehog signalling.

218. Expression of Wsb2 in the mouse testis

We present a detailed study on the expression pattern of Wsb2 in the mouse foetal and adult gonad. Wsb2 expression was analysed during mouse embryogenesis by whole-mount, section in situ hybridisation and immunohistochemistry. Wsb2 was found to be expressed in the developing mouse gonads from 11.5 dpc to 16.5 dpc. Expression is initially equal in both sexes from 10.5 dpc until 12.0 dpc, then it persists in the male gonad. Wsb2 expression was confined to the cords in both Sertoli cell and germ cells. Other sites of Wsb2 embryonic expression were the somites, dorsal root ganglia and the lateral mantle layer of the neural tube. mRNA encoding Wsb2 and Wsb2 protein has been detected in the newborn testis in both gonocytes and Sertoli cells. Wsb2 mRNA in the adult mouse testis was observed in Sertoli cells, spermatogonia, spermatocytes and the corresponding Wsb2 protein expression was in pachytene spermatocytes, round and elongated spermatids, Sertoli cells and Leydig cells. The differential expression of Wsb2 in male versus female embryonic gonads suggests it may play a role in mammalian sex determination during embryonic development and its expression in the first wave of spermatogenesis and in the adult suggests a later role in spermatogenesis.

TheDazhGene Is Expressed in Male and Female Embryonic Gonads before Germ Cell Sex Differentiation

The autosomal homologs of the human Y-chromosomal DAZ gene (DAZH and Dazh in human and mouse, respectively) are strong candidate for Azoospermia factor and encode a testis specific RNA-binding proteins. We studied the expression pattern of the mouse Dazh during embryonic development by using Northern-blotting of developing gonads. In the mouse, we have detected 3.5kb and 4.5kb transcripts in male and female embryonic gonads at 12.5 dpc (days post coitum). During this period, the only germ cells present in the gonad are primordial germ cells. Dazh transcripts were not detected in embryonic gonads of mice that lack germ cells because of mutation in W gene, suggesting that expression is limited to germ-cells. In females, oogonia enter meiosis at 13.5-14.5 dpc: at this time Dazh transcription levels are similar to those of the male (when prospermatogonia are in the male gonad). Transcription levels decrease steadily after birth as the number of oocytes is depleted and is hardly detectable by puberty. A human DAZH transcript was also detected by Northern-blotting in the human ovary in levels which are of about 100 fold lower than those observed in the human testis. The expression of the Dazh in male and female gonad before germ cell sex differentiation suggests that these genes may act at the first phase of male and female gametogenesis.

Molecular cloning of an avian anti-Müllerian hormone homologue

Anti-Müllerian hormone (AMH) is responsible for regression of the Müllerian ducts in males during embryonic development. This peptide hormone of the transforming growth factor-β family is also believed to play a broader role in sex determination, affecting differentiation and morphogenesis of the testes. Accordingly, in mammals, AMH is produced at much higher levels in male fetuses than in female fetuses. In contrast, in birds, both male and female embryonic gonads produce AMH at high levels, although in males it is still responsible for regression of the Müllerian ducts. Its persistent expression by the embryonic ovaries and its role in female sex determination in birds is not understood. We have cloned an avian homologue to AMH. Avian AMH cDNA encodes a 644 amino acid protein that is 42% identical to human AMH overall with increased identity at the carboxyl terminus. Similarities to human AMH include motifs of sequence identity, a conserved putative plasmin cleavage site and cysteine alignments, and similar genomic intron/exon structure. Antibodies to recombinant avian AMH cross-react with recombinant human AMH and were used to show that avian AMH is glycosylated as has been shown for the human form. The avian AMH gene is transcribed in both male and female gonads but not in liver, heart, kidney or muscle.

Normal structure and expression of Zfy genes in XY female mice mutant in Tdy

Zfy-1 and Zfy-2 are candidate genes for Tdy, the testis-determining gene in mice. We have analysed these genes in a line of XY female mice that have been shown to be mutated in Tdy. We have used Southern blot analysis to show that the Zfy genes have not undergone any major structural alterations, and have also demonstrated that both genes are transcribed normally from the mutant Y chromosome (Y) in both adult XYY testis and XY female embryonic gonads. The fact that these genes show a normal structure and expression pattern in mice with a Y chromosome known to carry a mutation in Tdy and that mutant embryos develop into females despite Zfy-1 expression, strongly supports other recent evidence that Zfy genes are not directly involved in primary testis determination.

Functional differentiation of chick gonads following depletion of primordial germ cells

ABSTRACT The endocrine capacity of embryonic chick gonads depleted in germ cells was compared to that of controls to determine whether the somatic elements of germ-cell-depleted gonads will undergo normal functional sex differentiation. Primordial germ cells were removed from early embryos, Hamburger and Hamilton stages 7 – 11, by excision of the anterior germinal crescent. Embryos were sacrificed at 14 days of incubation, and their gonads were analysed for functional differentiation by: (1) electron microscopy to detect ultrastructural cellular morphology characteristic of steroid-secreting cells; (2) growth in cell culture to detect development of characteristic cell morphologies ; (3) radioimmunoassay of cell-culture media to detect androgens and oestrogens (androstenedione and oestradiol 17 β) secreted by gonadal cells; and (4) measurement of steroid levels produced by cultures treated with human chorionic gonadotropin (hCG) to detect the ability of gonadal cells to increase steroid production in response to gonadotropin stimulation. As a bioassay of gonadal endocrine activity, a gross morphological analysis was performed on the genital ducts, the development of which is ovary-dependent in females and testis-dependent in males. This study demonstrated that both male and female embryonic gonads exhibit normal functional differentiation following a significant reduction in the number of primordial germ cells. These results confirm and extend our previous finding that morphological differentiation of sterile embryonic gonads is normal (McCarrey &amp; Abbott, 1978). It is concluded frofn the present study that a normal complement of germ cells is not essential to either morphological or functional sex differentiation of the somatic elements of the embryonic ovary or testis, thus arguing against any inductive role for the germ cells in this process.