Chicken Ovary Research Articles

Morphogenesis of the ovary of a chicken embryo in the germinal period of embryonic ontogenesis

The article is devoted to the morphogenesis of the ovary of chickens in embryonic ontogenesis, namely, in the embryonic period, which lasts from the beginning of incubation to the 8-day age of the chicken embryo. The aim of the study was to study the morphology of the development of the ovary of chickens in early embryonic ontogenesis. The objectives of the study included: determining the timing of the onset of ovarian development in chicken embryos; determining the age of the chicken embryo in which the reduction of one of the gonads begins, namely the right one; determining the size of epithelial cells; describing the morphology of gonocytes. It has been established that the development of gonads in chicken embryos, in particular the ovary, begins on the fourth day of embryogenesis in the form of two strips. By the sixth day, the right and left gonads are already roller-shaped bodies with a covering epithelium and a capillary system. At the seven day age of the embryos, the reduction of the right ovary begins. At the eight day age of embryos, the thickness of the integumentary epithelium of the left ovary is 1,3 times higher than that of the right ovary, since the thickness of the epithelium in the left ovary was 6,9 microns, whereas in the right one 5,4 microns. By the end of the embryonic period, namely by the age of 8 days, there is no clear division of the organ into cortical and cerebral matter in the developing ovaries, there is an increase in vascularization of the organ and a decrease in the density of gonocyte placement. It should also be noted the intensive growth of the chicken embryo itself in the embryonic period, since the weight of the embryo increased 11 times from 4 to 8 days of age.

Expression of Relaxin 3 in the Ovarian Follicle of Japanese Quail.

The relaxin (RLN) gene is expressed in the reproductive tracts, such as the ovary and uterus, of mammalian species. Although RLN expression is detected in the chicken ovary, detailed clarification of the physiological role of RLN has not yet been reported. To address this issue, in the present study we aimed to examine the spatiotemporal expression and hormonal control of RLN in Japanese quail. By performing semi-quantitative and quantitative reverse transcription-polymerase chain reaction analysis, we found that RLN mRNA was mainly expressed in the granulosa and theca layers of the ovary. The expression level in the granulosa layer increased with the stage of follicular development. Results from granulosa layer culture experiments revealed that RLN mRNA expression increased with the addition of estradiol-17β, whereas the addition of progesterone suppressed RLN transcription. More detailed analysis indicated that RLN expression was highest in the stigma region of the follicle but significantly decreased as the time of the expected luteinizing hormone (LH) surge approached. Together, our findings demonstrated that the granulosa cells in the mature preovulatory follicles constitute the main source of RLN in the Japanese quail. Because RLN expression was highest in the stigma region and the expression dramatically decreased following the LH surge, the results further suggest that RLN may be related to tissue remodeling for the ovulation process in birds.

Nucleolus Organizer Region Functioning in Chicken Growing Oocytes: A Revision of the Existing Views

It is believed that the nucleolus organizer region (NOR) in the ovary of adult birds is inactivated at all stages of oocyte growth. However, in the ovary of juvenile chicken (Gallus g. domesticus) one or two nucleoli were observed in 50% of early diplotene oocytes. These nucleoli were functioning up to the lampbrush stage. It was assumed that diplotene oocytes with inactivated NOR are stored as a pool for development in the ovary of adult hens. Such a contradiction in the NOR functional activity in the oocytes of juvenile and adult hens required special studies. Here we represent new results of a comparative study of the nucleoli in oocytes of juvenile and adult hens. We show for the first time that in the oocytes of laying hens the nucleolus appears at the early diplotene stage and the pre-rRNA synthesis continues up to the lampbrush stage. At the lampbrush stage, the NOR is getting inactivated. The nucleolus disintegrates into large fragments containing both pre-rRNA and fibrillarin. Then these larger fragments break down into small granules that do not contain pre-rRNA but contain fibrillarin. The results resolve the above contradiction and demonstrate a similar pattern of NOR functioning in oocytes of adult and juvenile birds.

Identification of oogonial stem cells in chicken ovary.

Oogonial stem cells (OSCs) are germ cells that can sustain neo-oogenesis to replenish the pool of primary follicles in adult ovaries. In lower vertebrates, fresh oocytes are produced by numerous OSCs through mitosis and meiosis during each reproduction cycle, but the OSCs in adult mammals are rare. The birds have retained many conserved features and developed unique features of ovarian physiology during evolution, and the presence of OSCs within avian species remain unknown. In this study, we investigated the existence and function of OSCs in adult chickens. The chicken OSCs were isolated and expanded in culture. We then used cell transplantation system to evaluate their potential for migration and differentiation in vivo. DDX4/SSEA1-positive OSCs were identified in both the cortex and medulla of the adult chicken ovary. These putative OSCs undergo meiosis in the reproductively active ovary. Furthermore, the isolated OSCs were expanded in vitro for months and found to express germline markers similar to those of primordial germ cells. When transplanted into the bloodstream of recipient embryos, these OSCs efficiently migrated into developing gonads, initiated meiosis, and then derived oocytes in postnatal ovaries. This study has confirmed the presence of functional OSCs in birds for the first time. The identification of chicken OSCs has great potential for improving egg laying and preserving endangered species.

Tracking the dynamics of female germ cell development during peri-hatch periods using a gene-edited chicken model

In hens, egg production depends on the development of germ cells in the ovary. Germ cells are established before birth, and their number gradually decreases during their lifespan. Therefore, it is essential to determine the time points of massive germ cell loss and the underlying mechanism. In this study, a gene-edited chicken with mCherry fluorescence specifically expressed in the germline was generated by the integration of the mCherry gene into the 3'-end of the DAZL locus, which facilitated the isolation of germ cells from the gonads of DAZL-mCherry embryos or chicks and quantification using flow cytometry based on the observation of red fluorescence. The results demonstrated the dynamics of germ cell development from embryos at 17 d of hatching (dh) to chickens at 7 d post-hatch (dph) and revealed a substantial loss of germ cells in the late embryonic stage (18 -19 dh) and post-hatch period (2 -3 dph). Additionally, the number of germ cells in DAZL×Guangxi Ma chicken was significantly higher than that in DAZL×Lohmann Pink chicken at 19 dh and 3 dph (P < 0.05). Furthermore, the numbers of germ cells positively correlated with the body weight in DAZL×Lohmann Pink chicken. In conclusion, our results showed the dynamics of germ cell development in chicken ovaries during peri-hatch periods and indicated the time point of substantial germ cell loss. The results provide evidence for further exploration of the underlying mechanism and serve as a reference for chicken breeding and management.

Ovarian mRNA Expression and Regulation of Matrix Metalloproteinase 16 in the Domestic Hen

In mammals, membrane-bound matrix metalloproteinases (MT-MMPs) are thought to play an important role in ovarian remodeling. However, the role and regulation of these proteases in the ovary of birds remain largely unknown. One of MT-MMPs, i. e., MMP-16, has been found in the hen ovary; therefore, this study was undertaken to examine whether the transcript level of MMP-16 changes during follicle development and whether gonadotropins and estrogen are involved in the regulation of this enzyme expression. The relative expression of MMP-16 mRNA in the ovarian follicles (white, yellowish, small yellow, and the granulosa and theca layers of three of the largest yellow preovulatory [F3-F1]) was examined 22 h and 3 h before F1 follicle ovulation as well as following equine chorionic gonadotropin (eCG) or tamoxifen (estrogen receptor modulator, TMX) treatments by quantitative real-time polymerase chain reaction (qRT-PCR). MMP-16 transcripts were detected in all examined ovarian tissues of control and treated hens. The relative expression of MMP-16 depended on follicular size/maturation and the layer of the follicular wall. A relatively higher expression of MMP-16 mRNA in the granulosa layer at 3 h compared to 22 h before ovulation of F1 was found. The injections of eCG decreased transcript abundance of MMP-16 in white and small yellow follicles, as well as in the theca layer of F3-F2 and the granulosa layer of the F1 follicle. In turn, TMX caused an increase in mRNA expression of MMP-16 in the theca layer of the largest preovulatory follicles and a decrease in the granulosa layer of the F1 follicle. Our results provide the first mRNA expression analysis of MMP-16 in the hen ovary under different physiological states. In addition, results indicate a possible role of gonadotropins and estrogen in regulating the transcription of MMP-16 in the chicken ovary.

Effects of fumonisin and Salmonella infection in the expression of Toll-like receptors in chicken ovary

Mycotoxins are secondary metabolites produced mainly by fungi that contaminate animal feed and basic food products throughout the world. Currently, more than 500 mycotoxins are reported and one of the most important concern to public health and agriculture is Fumonisin B1 (FB1). FB1, a mycotoxin produced by Fusarium moniliforme, is a contaminant of animal feed with various and complex cellular effects. Although FB1 has been associated with various diseases in animals, to date few studies have been performed to evaluate the endocrine disrupting effects of FB1 and more particularly the effects in the innate immune mechanisms of farm animals and more specifically in poultry species. As the family of Toll-like receptors (TLRs) is one of the key molecules of the innate immune system, the aim of the present study was to evaluate the transcriptional changes of TLRs in the chicken ovary in vivo, in chicks fed with FB1 and in response to Salmonella Enteritidis (SE) infection. RNA was extracted from the ovaries of sexually mature and aged birds, treated with FB1 and SE. Quantitative real-time PCR analysis revealed that FB1 and SE infection resulted in a significant down regulation of 5 TLR genes in the ovary of sexual mature and aged birds. These findings suggest that the mycotoxin FB1 suppresses the innate immune system of the chicken reproductive organs, through alteration in the expression of certain TLR genes.

Characterization of relaxin 3 and its receptors in chicken: Evidence for relaxin 3 acting as a novel pituitary hormone.

Mammalian relaxin (RLN) family peptides binding their receptors (RXFPs) play a variety of roles in many physiological processes, such as reproduction, stress, appetite regulation, and energy balance. In birds, although two relaxin family peptides (RLN3 and INSL5) and four receptors (RXFP1, RXFP2, RXFP2-like, and RXFP3) were predicated, their sequence features, signal properties, tissue distribution, and physiological functions remain largely unknown. In this study, using chickens as the experimental model, we cloned the cDNA of the cRLN3 gene and two receptor (cRXFP1 and cRXFP3) genes. Using cell-based luciferase reporter assays, we demonstrate that cRLN3 is able to activate both cRXFP1 and cRXFP3 for downstream signaling. cRXFP1, rather than cRXFP3, is a cognate receptor for cRLN3, which is different from the mammals. Tissue distribution analyses reveal that cRLN3 is highly expressed in the pituitary with lower abundance in the hypothalamus and ovary of female chicken, together with the detection that cRLN3 co-localizes with pituitary hormone genes LHB/FSHB/GRP/CART and its expression is tightly regulated by hypothalamic factors (GnRH and CRH) and sex steroid hormone (E2). The present study supports that cRLN3 may function as a novel pituitary hormone involving female reproduction.

Transcriptomics and Metabolomics Analysis of the Ovaries of High and Low Egg Production Chickens.

Simple SummaryThe ovarian tissues of different breeds of hens during egg production were investigated through transcriptomics and metabolomics to provide a more comprehensive understanding of the molecular mechanisms of the ovary during egg production. Four genes involved in egg production were predicted by the transcriptome, including P2RX1, INHBB, VIPR2, and FABP3, and several close metabolites associated with reproduction were identified in the metabolome, including 17α-hydroxyprogesterone, iloprost, spermidine and adenosine. Correlation analysis of specific differential genes and differential metabolites identified important gene-metabolite pairs VIPR2–Spermidine and P2RX1–Spermidine in the reproductive process.Egg production is a pivotal indicator for evaluating the fertility of poultry, and the ovary is an essential organ for egg production and plays an indispensable role in poultry production and reproduction. In order to investigate different aspects of egg production mechanisms in different poultry, in this study we performed a metabolomic analysis of the transcriptomic combination of the ovaries of two chicken breeds, the high-production Ninghai indigenous chickens and the low-production Wuliangshan black-boned chickens, to analyze the biosynthesis and potential key genes and metabolic pathways in the ovaries during egg production. We predicted four genes in the transcriptomic that are associated with egg production, namely P2RX1, INHBB, VIPR2, and FABP3, and identified three important pathways during egg production, “Calcium signaling pathway”, “Neuroactive ligand–receptor interaction” and “Cytokine–cytokine receptor interaction”, respectively. In the metabolomic 149 significantly differential metabolites were identified, 99 in the negative model and 50 in the positive model, of which 17α-hydroxyprogesterone, iloprost, spermidine, and adenosine are important metabolites involved in reproduction. By integrating transcriptomics and metabolomics, the correlation between specific differential genes and differential metabolites identified important gene-metabolite pairs “VIPR2-Spermidine” and “P2RX1-Spermidine” in egg production. In conclusion, these data provide a better understanding of the molecular differences between the ovaries of low- and high-production hens and provide a theoretical basis for further studies on the mechanics of poultry egg production.

Genetic Basis of Sexual Maturation Heterosis: Insights From Ovary lncRNA and mRNA Repertoire in Chicken.

Sexual maturation is fundamental to the reproduction and production performance, heterosis of which has been widely used in animal crossbreeding. However, the underlying mechanism have long remained elusive, despite its profound biological and agricultural significance. In the current study, the reciprocal crossing between White Leghorns and Beijing You chickens were performed to measure the sexual maturation heterosis, and the ovary lncRNAs and mRNAs of purebreds and crossbreeds were profiled to illustrate molecular mechanism of heterosis. Heterosis larger than 20% was found for pubic space and oviduct length, whereas age at first egg showed negative heterosis in both crossbreeds. We identified 1170 known lncRNAs and 1994 putative lncRNAs in chicken ovary using a stringent pipeline. Gene expression pattern showed that nonadditivity was predominant, and the proportion of nonadditive lncRNAs and genes was similar between two crossbreeds, ranging from 44.24% to 49.15%. A total of 200 lncRNAs and 682 genes were shared by two crossbreeds, respectively. GO and KEGG analysis showed that the common genes were significantly enriched in the cell cycle, animal organ development, gonad development, ECM-receptor interaction, calcium signaling pathway and GnRH signaling pathway. Weighted gene co-expression network analysis (WGCNA) identified that 7 out of 20 co-expressed lncRNA-mRNA modules significantly correlated with oviduct length and pubic space. Interestingly, genes harbored in seven modules were also enriched in the similar biological process and pathways, in which nonadditive lncRNAs, such as MSTRG.17017.1 and MSTRG.6475.20, were strongly associated with nonadditive genes, such as CACNA1C and TGFB1 to affect gonad development and GnRH signaling pathway, respectively. Moreover, the results of real-time quantitative PCR (RT-qPCR) correlated well with the transcriptome data. Integrated with positive heterosis of serum GnRH and melatonin content detected in crossbreeds, we speculated that nonadditive genes involved in the GnRH signaling pathway elevated the gonad development, leading to the sexual maturation heterosis. We characterized a systematic landscape of ovary lncRNAs and mRNAs related to sexual maturation heterosis in chicken. The quantitative exploration of hybrid transcriptome changes lays foundation for genetic improvement of sexual maturation traits and provides insights into endocrine control of sexual maturation.

Autophagy participates in germline cyst breakdown and follicular formation by modulating glycolysis switch via Akt signaling in newly-hatched chicken ovaries

In females, the establishment of the primordial follicle pool is accompanied by a remarkable programmed oocyte loss for unclear reasons. In this study, the role of autophagy was investigated to serve as a protective mechanism for oocyte survival during chicken folliculogenesis. Inhibition of autophagy by 3-methyladenine (3-MA) led to a remarkable delay in germ cell cyst breakdown that resulted in fewer primordial follicles and retarded sequent follicular development either in vivo or in the ovarian organ culture. Furthermore, the glycolysis level was downregulated in ovaries treated with 3-MA, while Recilisib (a specific activator of Akt) reversed this inhibiting effect of 3-MA on primordial folliculogenesis. Collectively, these data indicate that autophagy functions to maintain germ cell cyst breakdown and primordial follicle assembly by regulating ovarian glycolysis involving Akt signaling in the ovaries of newly-hatched chickens.

Integrated Proteomic and Metabolomic Analyses of Chicken Ovary Revealed the Crucial Role of Lipoprotein Lipase on Lipid Metabolism and Steroidogenesis During Sexual Maturity.

During sexual maturation and ovulatory cycle in chickens, ovaries undergo dynamic morphological and functional changes. The aim of this study was to evaluate the integrated proteome and metabolome analyses of chicken ovaries to characterize the changes in protein and metabolite profiles during sexual maturity. The ovary of Rohman layers before (125 days of age) and after (139 days of age) sexual maturation were collected for proteome and metabolome sequencing. The results showed that a total of 680 differentially expressed proteins (DEPs) and 1,046 differential metabolites (DMs) were identified in the chicken ovary during sexual maturity. Among the DEPs, 595 proteins were up-regulated and 85 were down-regulated, whereas 519 metabolites were up-regulated and 527 were down-regulated. KEGG pathway enrichment analysis showed that DEPs were significantly enriched in glycerolipid metabolism, calcium signaling pathway, folate biosynthesis, fat digestion and absorption, NF-kB signaling pathway, and PPAR signaling pathway. However, DMs were significantly enriched in the metabolism pathways, PPAR signalling pathway, glycerolipid metabolism, ferroptosis, biosynthesis of amino acids, and biosynthesis of unsaturated fatty acids. The results of the integrated analyses of DEPs and DMs revealed that the PPAR signaling pathway and glycerolipid metabolism were the most significantly enriched pathways. Among the identified DEPs, lipoprotein lipase (LPL) was upregulated in sexually mature chicken ovaries and was significantly enriched in the glycerolipid metabolism pathway, which may partially explain the possible reasons for steroidogenesis and lipid reserves responsible for oocyte maturation and ovarian follicle development during sexual maturity in chickens. The results further revealed that LPL silencing decreased the content of lipid droplets (LDs), as well as the mRNA expression of lipid metabolism-related genes including; sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase (FASN); and steroidogenesis-related genes such as; cytochrome P450 11A1 (CYP11A1) and steroidogenic acute regulatory (StAR). The present study revealed that upregulation of LPL in the chicken ovary during sexual maturity promotes granulosa cell (GC) lipid metabolism and steroidogenesis. These findings provide a theoretical support for further studies to elucidate the mechanism of lipid metabolism to regulate the function of avian GCs during sexual maturity in chickens.

Ovarian Transcriptomic Analysis of Ninghai Indigenous Chickens at Different Egg-Laying Periods.

Egg production is an essential indicator of poultry fertility. The ovary is a crucial organ involved in egg production; however, little is known about the key genes and signaling pathways involved in the whole egg-laying cycle of hens. In order to explore the mechanism of egg production at different stages of the egg-laying process, ovarian tissues from four chickens were randomly selected for transcriptome analysis at each of the three ages (145 d, 204 d, and 300 d in the early, peak, and late stages of egg laying). A total of 12 gene libraries were constructed, and a total of 8433 differential genes were identified from NH145d vs. NH204d, NH145d vs. NH300d and NH300d vs. NH204d (Ninghai 145-day-old, Ninghai 204-day-old, and Ninghai 300-day-old), with 1176, 1653 and 1868 up-regulated genes, and 621, 1955 and 1160 down-regulated genes, respectively. In each of the two comparison groups, 73, 1004, and 1030 differentially expressed genes were found to be co-expressed. We analyzed the differentially expressed genes and predicted nine genes involved in egg production regulation, including LRP8, BMP6, ZP4, COL4A1, VCAN, INHBA, LOX, PTX3, and IHH, as well as several essential egg production pathways, such as regulation adhesion molecules (CAMs), calcium signaling pathways, neuroactive ligand–receptor interaction, and cytokine–cytokine receptor interaction. Transcriptional analysis of the chicken ovary during different phases of egg-lay will provide a useful molecular basis for study of the development of the egg-laying ovary.

Endoplasmic reticulum stress is involved in small white follicular atresia in chicken ovaries

During the hierarchical development of follicles in laying hens, a large number of small white follicles (SWFs) undergo atresia caused by apoptosis rather than growing into large white follicles (LWFs), which reduces the number of follicles developing into hierarchical preovulatory follicles and results in declines in egg production. Therefore, figuring out factors responsible for SWFs atresia is necessary to improve egg production of laying hens. Endoplasmic reticulum stress (ERS) is one of the possible causes of cell apoptosis. However, the association between SWFs atresia and ERS in hens is yet to be answered. In this study, cell proliferation and apoptosis, expression levels of proteins and genes related to ERS, and cell ultrastructure were compared to elucidate whether ERS occurred in atretic small white follicles (ASWFs). Subsequently, a tunicamycin-induced ERS model and a serum withdrawal-induced atresia model of SWFs in vitro were established to investigate whether ERS could lead to follicular atresia. The results showed that the cell apoptosis increased significantly while the cell proliferation decreased evidently in ASWFs compared with SWFs. Meanwhile, swollen and vacuolization deformation of ERs was observed in the GCs but not TCs of ASWFs. Results of experiments in vitro demonstrated that treatment with serum withdrawal or tunicamycin inhibited cell proliferation and promoted cell apoptosis in SWFs, and the adverse effects of tunicamycin on SWFs manifested a dose-dependent increase. In comparison with the controls, treatment with serum withdrawal or tunicamycin upregulated expression levels of proteins and genes in ERS pathway. Moreover, the dose of 5 μg/mL tunicamycin seemed to be the optimal concentration for establishing the tunicamycin-induced ERS model. Meanwhile, the results of cell ultrastructure observation showed that ERs in GCs of SWFs of serum withdrawal group and treatment with 5 μg/mL tunicamycin group became swollen, dilatation and vacuolization. In conclusion, the data of this study indicated that ERS is involved in SWF atresia in laying hens, and ERS in GCs might be more severe than that in TCs.

Transcriptome identification and characterization of long non-coding RNAs in the ovary of hens at four stages

Long non-coding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation. LncRNAs, which are defined as non-coding RNAs more than 200 bp in length, are involved in key biological processes, such as cell proliferation and differentiation, epigenetic regulation, and gene transcriptional translation. Recent studies have shown that lncRNAs also play major regulatory roles in the reproduction of mammals. However, knowledge of the roles of lncRNAs in the chicken ovary lacking. In this study, we performed RNA-seq analyses of ovarian tissue from Hy-Line brown laying hens at four physiological stages [15, 20, 30, and 68 weeks of age (W)]. We identified 657 lncRNA transcripts that were differentially expressed during ovarian development, the number of down-regulated lncRNAs was higher than the number of up-regulated lncRNAs during development. We predicted the cis and trans target genes of the DE lncRNAs and constructed a lncRNA-mRNA interaction network, which indicated that the DE genes (DEGs) and the target genes of the DE lncRNAs are mainly involved in signaling pathways associated with ovarian development, including oocyte meiosis, calcium signaling pathways, ECM-receptor interactions, and ribosome and focal adhesion. Overall, we found that twelve lncRNAs were strongly involved in ovarian development: LNC_013443, LNC_001029, LNC_005713, LNC_016762, ENSGALT00000101857, LNC_003913, LNC_013692, LNC_012219, LNC_004140, ENSGALT00000096941, LNC_009356, and ENSGALT00000098716. In summary, our study utilized RNA-seq analysis of hen ovaries to explore key lncRNAs involved in ovarian development and function. Furthermore, the comprehensive analysis identified the target genes of these lncRNAs providing a better understanding of the mechanisms underlying ovarian development in hens and a theoretical basis for further research.

WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells.

Multiple Wilms tumor gene 1 (WT1) splicing variants are expressed in mammals, and these variants regulate tumorigenesis and mediate the development of multiple tissues and organs, including gonads. However, WT1 splicing variants (+KTS or -KTS) are expressed in only two nonmammalian vertebrates, and unexpectedly, their functions in chicken ovaries remain elusive. Progesterone (P4) secreted by chicken granulosa cells (GCs) participates in various physiological processes and plays an important role in maintaining reproductive performance. The purpose of this study was to investigate the effect of WT1(+KTS) and WT1(-KTS) on chicken P4 secretion in preovulatory GCs. First, we detected WT1 mRNA expression in GCs from follicles of different developmental stages by Quantitative real-time PCR (RT-qPCR) and found that WT1 mRNA expression was considerably increased in preovulatory GCs compared with prehierarchical GCs. Primary cells collected from preovulatory follicles were treated with WT1(+KTS) or WT1(-KTS) overexpression vectors and subsequently cultured in the absence or presence of follicle-stimulating hormone (FSH). The mRNA levels of FSH-receptor (FSHR) and steroidogenesis genes were determined by RT-qPCR, and the P4 levels in the cell supernatants were measured by radioimmunoassay (RIA). Both WT1(+KTS) and WT1(-KTS) significantly decreased P4 secretion due to a reduction in FSHR, STAR and CYP11A1 mRNA levels. Western blotting revealed that ERK1/2 and BRAF phosphorylation levels were suppressed after overexpression of WT1(+KTS) or WT1(-KTS), whereas total protein and mRNA levels were not significantly changed. In addition, CREB protein and phosphorylation levels were inhibited after overexpression of WT1(+KTS) or WT1(-KTS). In conclusion, WT1(+KTS) and WT1(-KTS) inhibited CREB protein activity and significantly reduced FSHR, STAR and CYP11A1 mRNA levels, which subsequently suppressed FSH-induced P4 secretion in preovulatory GCs by modulating ERK1/2 signaling.

The opposite effects of VGLL1 and VGLL4 genes on granulosa cell proliferation and apoptosis of hen ovarian prehierarchical follicles

Transcription cofactors Vestigial like family (VGLL) members consisting of four homologs (VGLL1-4) are associated with cell growth and metastasis in mammals, among which VGLL1 gene has been documented to possess tumorigenic functions in various types of tumor, and VGLL4 acts as a new tumor suppressor; likewise several studies indicated that they potentially play a role in the regulation of ovary growth and function. However, the biological effects of chicken VGLL1 and VGLL4 on the proliferation, apoptosis, and steroidogenesis of the granulosa cells (GCs) during ovarian follicle development remain unknown now. This study found that VGLL1 and VGLL4 genes present divergent expression patterns of the transcripts in the GCs of various sized prehierarchical follicles (PFs) before follicle selection. Specific small interfering RNA (siRNA) was employed to elucidate the exact roles of VGLL1 and VGLL4 in regulating the PF development of the hen ovary. The results demonstrated that the mRNA expression levels of the steroidogenic-related enzyme steroidogenic acute regulatory protein (STAR) gene and the cell proliferation-related factors B-cell lymphoma-2 (BCL2), and cyclin D1 (CCND1) genes were significantly down-regulated in the cells with VGLL1 silence but remarkably up-regulated in the cells lacking VGLL4. Whereas the expression level of the cell apoptosis biomarker caspase-3 (CASP3) transcript was noticeably enhanced in the GCs without VGLL1 but significantly decreased in the GCs deprived of VGLL4. Further results showed that the siRNA-mediated silence of VGLL1 caused a significant increase in apoptosis with a reduction in the proliferation of GCs. Nevertheless, knockdown of VGLL4 resulted in a remarkable decrement in apoptosis but a memorable augment in proliferation of the GCs. Taken together, this study proved that VGLL1 promotes cell proliferation and steroidogenesis but inhibits apoptosis. In contrast, VGLL4 stimulates GC apoptosis while suppressing the GC proliferation and steroidogenesis in the hen ovarian follicles. We conluded that VGLL1 and VGLL4 affect oppositely the ovarian prehierarchical follicle development by the different regulatory manner in the GC proliferation and apoptosis of chicken ovary.

Transcriptome comparative analysis of ovarian follicles reveals the key genes and signaling pathways implicated in hen egg production

BackgroundOvarian follicle development plays an important role in determination of poultry egg production. The follicles at the various developmental stages possess their own distinct molecular genetic characteristics and have different biological roles in chicken ovary development and function. In the each stage, several genes of follicle-specific expression and biological pathways are involved in the vary-sized follicular development and physiological events. Identification of the pivotal genes and signaling pathways that control the follicular development is helpful for understanding their exact regulatory functions and molecular mechanisms underlying egg-laying traits of laying hens.ResultsThe comparative mRNA transcriptomic analysis of ovarian follicles at three key developmental stages including slow growing white follicles (GWF), small yellow follicles (SYF) of recruitment into the hierarchy, and differentiated large yellow follicles (LYF), was accomplished in the layers with lower and higher egg production. Totally, 137, 447, and 229 of up-regulated differentially expressed genes (DEGs), and 99, 97, and 157 of down-regulated DEGs in the GWF, SYF and LYF follicles, including VIPR1, VIPR2, ADRB2, and HSD17B1 were identified, respectively. Moreover, NDUFAB1 and GABRA1 genes, two most promising candidates potentially associated with egg-laying performance were screened out from the 13 co-expressed DEGs in the GWF, SYF and LYF samples. We further investigated the biological effects of NDUFAB1 and GABRA1 on ovarian follicular development and found that NDUFAB1 promotes follicle development by stimulating granulosa cell (GC) proliferation and decreasing cell apoptosis, increases the expression of CCND1 and BCL-2 but attenuates the expression of caspase-3, and facilitates steroidogenesis by enhancing the expression of STAR and CYP11A1. In contrast, GABRA1 inhibits GC proliferation and stimulates cell apoptosis, decreases the expression of CCND1, BCL-2, STAR, and CYP11A1 but elevates the expression of caspase-3. Furthermore, the three crucial signaling pathways such as PPAR signaling pathway, cAMP signaling pathway and neuroactive ligand-receptor interaction were significantly enriched, which may play essential roles in ovarian follicle growth, differentiation, follicle selection, and maturation.ConclusionsThe current study provided new molecular data for insight into the regulatory mechanism underlying ovarian follicle development associated with egg production in chicken.

Fibromodulin is involved in autophagy and apoptosis of granulosa cells affecting the follicular atresia in chicken

Follicular atresia is an important cause of reproductive decline in egg-laying hens. Therefore, a better understanding of the regulation mechanism of follicle atresia in poultry is an important measure to maintain persistent high egg performance. However, how the role of the regulatory relationship between autophagy and apoptosis in the intrafollicular environment affects the follicular atresia of chickens is remain unclear. The objective of this study was to explore the regulatory molecular mechanisms in regard to follicular atresia. 20 white leghorn layers (32-wk-old) were equally divided into 2 groups. The control group was fed freely, and the experimental group induced follicular atretic by fasting for 5 d. The results showed that the expression of prolactin (PRL) levels was significantly higher in the fasted hens, while the levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were lower. Most importantly, RNA sequencing, qPCR, and Western blotting detected significantly elevated levels of autophagy and apoptosis markers in atresia follicles. Interestingly, we found that fibromodulin (FMOD) levels was significantly lower in follicles from fasted hens and that this molecule had an important regulatory role in autophagy. FMOD silencing significantly promoted autophagy and apoptosis in granulosa cells, resulting in hormonal imbalance. FMOD was found to regulate autophagy via the transforming growth factor beta (TGF-β) signaling pathway. Our results suggest that the increase in autophagy and the imbalance in internal homeostasis cause granulosa cell apoptosis, leading to follicular atresia in the chicken ovary. This finding could provide further insight into broodiness in chicken and provide avenues for further improvements in poultry production.

UBE2I stimulates female gonadal differentiation in chicken (Gallus gallus) embryos

Without known analogous sex-determining factors like SRY (sex determining region Y) in mammals, the chicken (Gallus gallus) sex determination mechanism still remains unclear, which highly restricts the biological research on chicken development and poultry single-sex reproduction. Here we not only characterized a new female-biased gene UBE2I and identified the expression pattern by qRT-PCR, but also described the functional role of UBE2I in the gonadal development of chicken embryos. Results showed that UBE2I exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads and robust expression in ovaries of newborn chickens. Most importantly, we successfully developed an effective method to interfere or overexpress UBE2I in chicken embryos through the intravascular injection. The qRT-PCR analysis showed that the sex-related genes (FOXL2, CYP19A1 and HINTW) in females were upregulated (P<0.05) under the overexpression of UBE2I and the sex-related genes (SOX9, DMRT1 and WT1) in females were downregulated (P<0.05) after interfering UBE2I. Furthermore, the change of UBE2I expression was associated with the level of estradiol and its receptors (AR and ESR), which suggests that UBE2I is necessary to initiate the female-specific development in chickens. In conclusion, this work demonstrates that UBE2I is a crucial sex differentiation-related gene in the embryonic development of chickens, which provides insights for further understanding the mechanism of sex determination in chickens.