Chicken Gonads Research Articles

Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination

IntroductionUnderstanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. ObjectivesWe aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. MethodsSingle-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. ResultsHere, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. ConclusionsOverall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.

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.

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.

Exploring right ovary degeneration in duck and goose embryos by histology and transcriptome dynamics analysis

BackgroundThe development of asymmetric chick gonads involves separate developmental programs in the left and right gonads. In contrast to the left ovary developing into a fully functional reproductive organ, the right ovary undergoes gradual degeneration. However, the molecular mechanisms underlying the the degeneration of the right ovary remain incompletely understood. In the present study, we investigated the histomorphological and transcriptomic changes in the right ovary of ducks and geese during the the embryonic stage up to post-hatching day 1.ResultHematoxylin–eosin stainings revealed that the right ovary developed until embryonic day 20 in ducks (DE20) or embryonic day 22 in geese (GE22), after which it started to regress. Further RNA-seq analyses revealed that both the differentially expressed genes (DEGs) in ducks and geese right ovary developmental stage were significantly enriched in cell adhesion-related pathway (ECM-receptor interaction, Focal adhesion pathway) and Cellular senescence pathway. Then during the degeneration stage, the DEGs were primarily enriched in pathways associated with inflammation, including Herpes simplex virus 1 infection, Influenza A, and Toll-like receptor signaling pathway. Moreover, duck-specific DEGs showed enrichment in Steroid hormone biosynthesis, Base excision repair, and the Wnt signaling pathway, while geese-specifically DEGs were found to be enriched in apoptosis and inflammation-related pathways, such as Ferroptosis, Necroptosis, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. These findings suggest that the degeneration process of the right ovary in ducks occurs at a slower pace compared to that in geese. Additionally, the observation of the left ovary of the geese varying degeneration rates in the right ovary after hatching indicated that the development of the left ovary may be influenced by the degeneration of the right ovary.ConclusionThe data presented in this study provide valuable insights into the dynamic changes in histological structure and transcriptome during the degeneration of the right ovary in ducks and geese. In addition, through the analysis of shared characteristics in the degeneration process of the right ovary in both ducks and geese, we have uncovered the patterns of degradation and elucidated the molecular mechanisms involved in the regression of the right ovary in poultry. Furthermore, we have also made initial discoveries regarding the relationship between the degeneration of the right ovary and the development of the left ovary.

The m6A methylation regulates gonadal sex differentiation in chicken embryo

BackgroundAs a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation.ResultsThe chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation.ConclusionsThis work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

Time-Course Transcriptional and Chromatin Accessibility Profiling Reveals Genes Associated With Asymmetrical Gonadal Development in Chicken Embryos.

In birds, male gonads form on both sides whereas most females develop asymmetric gonads. Multiple early lines of evidence suggested that the right gonad fails to develop into a functional ovary, mainly due to differential expression of PITX2 in the gonadal epithelium. Despite some advances in recent years, the molecular mechanisms underlying asymmetric gonadal development remain unclear. Here, using bulk analysis of whole gonads, we established a relatively detailed profile of four representative stages of chicken gonadal development at the transcriptional and chromatin levels. We revealed that many candidate genes were significantly enriched in morphogenesis, meiosis and subcellular structure formation, which may be responsible for asymmetric gonadal development. Further chromatin accessibility analysis suggested that the transcriptional activities of the candidate genes might be regulated by nearby open chromatin regions, which may act as transcription factor (TF) binding sites and potential cis-regulatory elements. We found that LHX9 was a promising TF that bound to the left-biased peaks of many cell cycle-related genes. In summary, this study provides distinctive insights into the potential molecular basis underlying the asymmetric development of chicken gonads.

H3K27ac chromatin acetylation and gene expression analysis reveal sex- and situs-related differences in developing chicken gonads

BackgroundBirds exhibit a unique asymmetry in terms of gonad development. The female left gonad generates a functional ovary, whereas the right gonad regresses. In males, both left and right gonads would develop into testes. How is this left/right asymmetry established only in females but not in males remains unknown. The epigenetic regulation of gonadal developmental genes may contribute to this sex disparity. The modification of histone tails such as H3K27ac is tightly coupled to chromatin activation and gene expression. To explore whether H3K27ac marked chromatin activation is involved in the asymmetric development of avian gonads, we probed genome-wide H3K27ac occupancy in left and right gonads from both sexes and related chromatin activity profile to the expression of gonadal genes. Furthermore, we validated the effect of chromatin activity on asymmetric gonadal development by manipulating the chromatin histone acetylation levels.MethodsThe undifferentiated gonads from both sides of each sex were collected and subjected to RNA-Seq and H3K27ac ChIP-Seq experiments. Integrated analysis of gene expression and active chromatin regions were performed to identify the sex- and situs-specific regulation and expression of gonadal genes. The histone deacetylase inhibitor trichostatin A (TSA) was applied to the undifferentiated female right gonads to assess the effect of chromatin activation on gonadal gene expression and cell proliferation.ResultsEven before sex differentiation, the gonads already show divergent gene expression between different sexes and between left/right sides in females. The sex-specific H3K27ac chromatin distributions coincide with the higher expression of male/female specification genes in each sex. Unexpectedly, the H3K27ac marked chromatin activation show a dramatic difference between left and right gonads in both sexes, although the left/right asymmetric gonadal development was observed only in females but not in males. In females, the side-specific H3K27ac occupancy instructs the differential expression of developmental genes between the pair of gonads and contributes to the development of left but not right gonad. However, in males, the left/right discrepancy of H3K27ac chromatin distribution does not drive the side-biased gene expression or gonad development. The TSA-induced retention of chromatin acetylation causes up-regulation of ovarian developmental genes and increases cell proliferation in the female right gonad.ConclusionsWe revealed that left/right asymmetry in H3K27ac marked chromatin activation exists in both sexes, but this discrepancy gives rise to asymmetric gonadal development only in females. Other mechanisms overriding the chromatin activation would control the symmetric development of male gonads in chicken.

A low-tech, cost-effective and efficient method for safeguarding genetic diversity by direct cryopreservation of poultry embryonic reproductive cells.

Chickens are an important resource for smallholder farmers who raise locally adapted, genetically distinct breeds for eggs and meat. The development of efficient reproductive technologies to conserve and regenerate chicken breeds safeguards existing biodiversity and secures poultry genetic resources for climate resilience, biosecurity, and future food production. The majority of the over 1600 breeds of chicken are raised in low and lower to middle income countries under resource-limited, small-scale production systems, which necessitates a low-tech, cost-effective means of conserving diversity is needed. Here, we validate a simple biobanking technique using cryopreserved embryonic chicken gonads. The gonads are quickly isolated, visually sexed, pooled by sex, and cryopreserved. Subsequently, the stored material is thawed and dissociated before injection into sterile host chicken embryos. By using pooled GFP and RFP-labelled donor gonadal cells and Sire Dam Surrogate mating, we demonstrate that chicks deriving entirely from male and female donor germ cells are hatched. This technology will enable ongoing efforts to conserve chicken genetic diversity for both commercial and smallholder farmers, and to preserve existing genetic resources at poultry research facilities.

PAX2 + Mesenchymal Origin of Gonadal Supporting Cells Is Conserved in Birds.

During embryonic gonadal development, the supporting cell lineage is the first cell type to differentiate, giving rise to Sertoli cells in the testis and pre-granulosa cells in the ovary. These cells are thought to direct other gonadal cell lineages down the testis or ovarian pathways, including the germline. Recent research has shown that, in contrast to mouse, chicken gonadal supporting cells derive from a PAX2/OSR1/DMRT1/WNT4 positive mesenchymal cell population. These cells colonize the undifferentiated genital ridge during early gonadogenesis, around the time that germ cells migrate into the gonad. During the process of somatic gonadal sex differentiation, PAX2 expression is down-regulated in embryonic chicken gonads just prior to up-regulation of testis- and ovary-specific markers and prior to germ cell differentiation. Most research on avian gonadal development has focused on the chicken model, and related species from the Galloanserae clade. There is a lack of knowledge on gonadal sex differentiation in other avian lineages. Comparative analysis in birds is required to fully understand the mechanisms of avian sex determination and gonadal differentiation. Here we report the first comparative molecular characterization of gonadal supporting cell differentiation in birds from each of the three main clades, Galloanserae (chicken and quail), Neoaves (zebra finch) and Palaeognathe (emu). Our analysis reveals conservation of PAX2+ expression and a mesenchymal origin of supporting cells in each clade. Moreover, down-regulation of PAX2 expression precisely defines the onset of gonadal sex differentiation in each species. Altogether, these results indicate that gonadal morphogenesis is conserved among the major bird clades.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

Effect of Post-Hatch Heat-Treatment in Heat-Stressed Transylvanian Naked Neck Chicken.

Simple SummaryHeat stress due to high environmental temperature negatively influences animal productivity. Extensive studies have been carried out to evaluate the mechanisms of heat stress in chickens. It was shown that the expression level of heat-shock factors (HSFs) and heat-shock proteins (HSPs) were affected. Tissue-specific responses to the thermal challenge were also found in the heart, liver and muscle. Our study examined the changes in primary production parameters and four heat-shock factor and two heat-shock protein expression profiles in chicken gonads. In the first experiment, 24 h after hatching, 80 Transylvanian Naked Neck chickens were heat-treated at 38.5 °C ambient temperature with 60% humidity for 12 h. In this experiment, we studied the primary productivity parameters of matured chickens after the performed heat stress. In the second experiment, the heat treatment was the same, and we examined the expression pattern of heat-shock factors and heat-shock proteins in the control and treated gonads. We collected the samples immediately after the heat-treatment in case of half of the treated and control group. We found a significant difference in egg production, and increased expression level of HSP90 and HSF4 in heat-treated female gonads.Although numerous studies reported the effects of heat stress in chickens, it was not investigated in the Transylvanian Naked Neck breed. In our research, Transylvanian Naked Neck chickens, 24 h after hatching, were heat-treated at 38.5 °C for 12 h. We compared the control and heat-treated adult chickens’ productivity parameters following 12 weeks of heat-stress at 30 °C. We found that the heat-treated layers had significantly higher egg production in heat stress, but in cockerels, the sperm quality did not differ significantly between the two groups. To detect the effect of heat-treatment on a molecular level, the expression of two heat-shock proteins and four heat-shock factors were analysed in the gonads of control and heat-treated chickens. We found that the expression level of HSP90 and HSF4 increased significantly in heat-treated female chicken gonads. Still, in adult females, the expression of HSF2 and HSF3 were substantially lower compared to the control. In adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads compared to the control. We think that the presented significant differences in egg production might be related to the increased expression level of HSP90 and HSF4 in heat-treated female gonads.

Fadrozole-Mediated Sex Reversal Induces PAX2+Undifferentiated Supporting Cells in Female Chicken Gonads

During early embryogenesis, the undifferentiated gonad is bipotential and subsequently commits to an ovarian or testicular fate. In birds, double dose of the Z-linked gene DMRT1 is required for testicular differentiation in male embryos (genetically ZZ). In female birds, estrogen plays a key role in ovarian differentiation. 17β-estradiol (E2) induces gonadal feminization when applied to male embryos (ZZ). Conversely, inhibition of estrogen synthesis with the drug fadrozole (FAD) results in testicular development in genetically female embryos (ZW). However, activation of male markers in sex-reversed ZW embryos is typically delayed, raising the possibility that FAD-treated embryos may transition through an undifferentiated state before masculinization. Recently, PAX2 was identified as a marker of undifferentiated supporting cells in the chicken embryo, being downregulated in both sexes at the onset of gonadal sex determination. To investigate the supporting cell differentiation process in estrogen-mediated sex reversal, we injected 1 mg of fadrozole in 100µl of PBS or vehicle into embryonic day 3.5 (E3.5) chicken eggs. Eggs were incubated until E9.5, genotypically sexed (ZZ or ZW) and processed for qRT-PCR and immunofluorescence. Quantitative RT-PCR confirmed that sex reversal had occurred in FAD-treated females, showing a reduction of pre-granulosa cell markers aromatase (P<0.005) and FOXL2 (P<0.05), compared to the control. Interestingly, PAX2 mRNA expression was up-regulated (P<0.05) in sex-reversed females, suggesting an increase in undifferentiated supporting cells (n=6). To confirm this observation, immunofluorescence was used to detect aromatase, SOX9 (male marker) and PAX2. In FAD-treated females, both SOX9+ (male) and aromatase+ (female) cells co-existed in the same gonad, but in separated defined regions. Aromatase positive cells were located in the most apical region of the gonad whereas SOX9 positive cells were detected in the basal region. We detected an increase in PAX2 positive cells in the gonadal medulla between the SOX9 and aromatase positive supporting cells. No SOX9 or PAX2 positive cells were detected in control female gonads (n=3). For feminization experiments 100µl of a 1mg/ml solution of E2 or vehicle (Oil) was injected into E3.5 chicken eggs. No significant increase in PAX2 was detected by qRT-PCR (p>0.05, n=6) and no PAX2 positive cells were detected in E2 treated gonads at E9.5. These results suggest that in fadrozole-mediated masculinization (but not in estrogen-induced feminization) there is an increase in undifferentiated supporting cells. The absence of both estrogens (feminizing) and elevated DMRT1 (masculinizing) could explain why the supporting cells remain in an undifferentiated state in ZW (genetically female) embryos. Further research is required to evaluate the fate of these undifferentiated cells in gonadal sex differentiation.

HMGCS1 Promotes male differentiation of chicken embryos by regulating the generate of cholesterol

Gender research shows excellent significance in the breeding and production of poultry, achieving the actual needs of poultry through targeted gender regulation could increase economic benefits. However, the mechanism of sex determination in poultry remains unclear. We respectively constructed HMGCS1 overexpression and interference vectors for lentivirus packaging. Vascular microinjection was performed when fertilized eggs developed to 2.5 days. qRT-PCR and ELISA results indicated that HMGCS1 could promote the expression of male germ-cell development-related genes (SOX9) and inhibit the expression of female germ-cell development-related genes (CYP19A1 and DMRT1). Furthermore, the synthesis of cholesterol and testosterone in the gonads were increased. In addition, the injection of lovastatin into chicken embryos, an inhibitor of cholesterol synthesis, showed a significant decrease in gonadal testosterone. This experiment revealed that HMGCS1 could regulate the synthesis of testosterone by affecting the content of cholesterol in gonads, and promote the differentiation of chicken gonads into males.

Comprehensive analysis of miRNAs, lncRNAs, and mRNAs reveals potential players of sexually dimorphic and left-right asymmetry in chicken gonad during gonadal differentiation

Despite thousands of sex-biased genes being found in chickens, the genetic control of sexually dimorphic and left-right asymmetry during gonadal differentiation is not yet completely understood. This study aimed to identify microRNAs (miRNAs), long noncoding RNAs (lncRNAs), messenger RNAs (mRNAs), and signaling pathways during gonadal differentiation in chick embryos (day 6/stage 29). The left and right gonads were collected for RNA sequencing. Sex-biased, side-biased miRNAs, lncRNAs, mRNAs, and shared differentially expressed miRNAs (DEmiRNA)–differentially expressed mRNAs (DEmRNA)–differentially expressed lncRNAs (DElncRNA) interaction networks were performed. A total of 8 DEmiRNAs, 183 DElncRNAs, and 123 DEmRNAs were identified for the sex-biased genes, and 7 DEmiRNAs, 189 DElncRNAs, and 183 DEmRNAs for the side-biased genes. The results of quantitative real-time PCR were generally consistent with the RNA-sequencing results. The study suggested that miRNAs and lncRNAs regulation were novel gene-specific dosage compensation mechanism and they could contribute to left-right asymmetry of chicken, but sex-biased and side-biased miRNAs, lncRNAs, and mRNAs were independent of each other. The competing endogenous RNA (ceRNA) networks showed that 17 target pairs including miR-7b (CYP19A1, FSHR, GREB1, STK31, CORIN, and TDRD9), miR-211 (FSHR, GREB1, STK31, CORIN, and TDRD9), miR-204 (FSHR, GREB1, CORIN, and TDRD9), and miR-302b-5p (CYP19A1 and TDRD9) may play crucial roles in ovarian development. These analyses provide new clues to uncover molecular mechanisms and signaling networks of ovarian development.

Insights into Gonadal Sex Differentiation Provided by Single-Cell Transcriptomics in the Chicken Embryo

Although the genetic triggers for gonadal sex differentiation vary across species, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using single-cell sequencing in the embryonic chicken gonad during sexual differentiation. The data show that chicken embryonic-supporting cells do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, they derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes and derivation of embryonic steroidogenic cells from a differentiated supporting-cell lineage. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.

FOXL2 antagonises the male developmental pathway in embryonic chicken gonads.

FOXL2 is a conserved transcription factor with a central role in ovarian development and function. Studies in humans and mice indicate that the main role of FOXL2 is in the postnatal ovary, namely folliculogenesis. To shed light on the function and evolution of FOXL2 in the female gonad, we examined its role in embryonic avian gonads, using in ovo over-expression and knockdown. FOXL2 mRNA and protein are expressed female-specifically in the embryonic chicken gonad, just prior to the onset of sexual differentiation. FOXL2 is expressed in the medullary cord cells, in the same cell type as aromatase (CYP19A1). In addition, later in development, expression also becomes localised in a subset of cortical cells, distinct from those expressing estrogen receptor alpha. Mis-expression of FOXL2 in the male chicken embryonic gonad suppresses the testis developmental pathway, abolishing local expression of the male pathway genes, SOX9, DMRT1 and AMH, and repressing Sertoli cell development. Conversely, knockdown of FOXL2 expression allows ectopic activation of SOX9 in female gonads. However, mis-expression of FOXL2 alone was insufficient to activate aromatase expression in male gonads, while FOXL2 knockdown did not affect aromatase expression in females. These results indicate that FOXL2 plays an important role in embryonic differentiation of the avian ovary via antagonism of SOX9, but may be dispensable for aromatase activation at embryonic stages. The data suggest that FOXL2 has different roles in different species, more central for embryonic ovarian differentiation in egg-laying vertebrates.

Determination of testicular estrogen receptor alpha expression of male chickens (Gallus domesticus) with age.

Background and Aim:Estrogen activity, a central component of reproductive growth, is regulated by the receptor proteins, estrogen receptor alpha (ERα), and ER beta (ERβ) in chickens as in many other species. ERα expresses predominantly in gonads. Although the expression of ERα in embryonic gonads has been studied in detail, the expression of ERα in post-hatching male gonads has not been studied adequately. Therefore, the current research was conducted to determine the post-hatching changes in the expression of ERα in the left gonads of male chickens with age.Materials and Methods:Shaver Brown male chickens were raised and cared for according to the management guide and sacrificed at the intervals of 1, 4, and 8 weeks of age. The total RNA was extracted from the left gonads using the Trizol method and reverse transcribed using a pair of gene-specific primers. Following polymerase chain reaction amplification, the expression of ERα was quantified relative to the expression of the reference gene GAPDH.Results:The results showed that ERα expression significantly increases with age at p=0.0032. However, the increment of ERα expression from week 1 to week 4 was 2.04-fold and from week 4 to week 8 was 1.39-fold, with the later age reflecting a diminishing pattern in the increment.Conclusion:These results differentiate the post-hatching ERα expression of the left gonads of male chickens increase with age but with a diminishing gradient that may support their reproductive functions in later stages of life.

The Role of miRNA in Differentiation, Cell Proliferation, and Pathogenesis of Poultry Diseases

To date, several noncoding RNAs are known that play a prominent role in the processes of transcription, translation, and structural conformation of RNA. By binding miRNA to 3'-untranslated regions, mRNA regulates gene expression in animals through inhibition of translation initiation, elongation, and other mechanisms. There is evidence for the differential expression of miRNA that regulates transcription with the inclusion of several stages of myoblast proliferation, differentiation, and apoptosis. These molecules play a significant role in cell differentiation, proliferation, apoptosis, and tumorigenesis. Some miRNAs are known to target 100–200 genes. Polymorphisms of miRNA genes, especially in the competent regions of the genome, can be biomarkers for phenotypic traits important for breeding birds. The findings confirm the key role of miRNA in controlling the metabolic switch that occurs between the embryo development and the chick hatching. Differentially expressed miRNAs and their possible target genes are involved in egg production functions. The available miRNA data complement our understanding of the molecular genetic control underlying abdominal fat accumulation and myogenesis in chickens. Hundreds of differentially expressed miRNAs from individuals differing in body weight were found. A new understanding into the functions of miRNA during chicken gonads’ rapid development is being formed. Hundreds of miRNAs expressed by hypothalamic genes and involved in the initial phase of rapid gonad growth have been identified. There is increasingly clear evidence that miRNA play a major role in regulating the innate immune response and are important effectors in complex host–pathogen interaction networks for salmonellosis, Marek’s disease, carcinogenic, and other diseases. Many of the miRNAs are associated with cell proliferation, apoptosis, and tumorigenesis. Bacterial pathogens are able to modulate the expression of the host miRNAs and affect the regulation of miRNAs and the outcome of infection. Several miRNAs are induced by TLR activation in innate immune cells and target the 3′-untranslated regions of the mRNA encoding the components of the TLR signaling system. Modern genome-editing tools suggest an artificial increase in the diversity of miRNAs and an increased use of miRNAs for directional action. Structure determination of RNA, RNA–RNA, RNA–DNA, and ribonucleoprotein complexes is becoming a rapidly developing area requiring the development of new technologies.

Derivation of chicken primordial germ cells using an indirect Co-culture system

Primordial germ cells (PGCs) are promising genetic resources for avian studies including modified animals. However, chicken PGCs are slow to proliferate and gradually lose germline competency after long-term culture, which hinders their application in avian biotechnology. Thus, we developed a robust method for the isolation and rapid propagation of PGCs using an indirect co-culture system. PGCs derived from a pair of embryonic chicken gonads were expanded to 1 × 106 within 2 weeks, and no sex bias was observed in. These PGCs presented high capacity of germline transmission and produced donor-derived offspring after injection into the chicken embryos. This system allows the efficient gene-banking of chicken species and can facilitate the production of chickens bearing a desired phenotype via genomic editing.

Logical modelling uncovers developmental constraints for primary sex determination of chicken gonads.

In the chicken, sex determination relies on a ZZ (male)/ZW (female) chromosomal system, but underlying mechanisms are still not fully understood. The Z-dosage and the dominant W-chromosome hypotheses have been proposed to underlie primary sex determination. We present a modelling approach, which assembles the current knowledge and permits exploration of the regulation of this process in chickens. Relying on published experimental data, we assembled a gene network, which led to a logical model that integrates both the Z-dosage and dominant W hypotheses. This model showed that the sexual fate of chicken gonads results from the resolution of the mutual inhibition between DMRT1 and FOXL2, where the initial amount of DMRT1 product determines the development of the gonads. In this respect, at the initiation step, a W-factor would function as a secondary device, by reducing the amount of DMRT1 in ZW gonads when the sexual fate of the gonad is settled, that is when the SOX9 functional level is established. Developmental constraints that are instrumental in this resolution were identified. These constraints establish qualitative restrictions regarding the relative transcription rates of the genes DMRT1, FOXL2 and HEMGN. Our model further clarified the role of OESTROGEN in maintaining FOXL2 function during ovary development.