RESUMO
The development and maturation of follicles are intricately linked to egg production and reproductive performance of chickens. Granulosa cells death directly affects the development and maturation of follicles, thereby impacting the reproductive performance of hens. Ferroptosis is a new type of cell death, it is unknown how it affects the growth and development of chicken follicles. In this study, RNA-seq analysis revealed significant differences in the expression of ferroptosis-related genes between normal follicles and atretic follicles, suggesting a potential role for ferroptosis in follicle growth and development. In addition, we found that ubiquitin-specific protease 13 (USP13) was significantly upregulated in atrophic follicles. Overexpression of USP13 results in depletion of glutathione (GSH), peroxidation of lipids, accumulation of iron, and activation of ferroptosis in chicken granulosa cells. In contrast, USP13 knockdown significantly inhibited ferroptosis events. Mechanistically, USP13 prevents the degradation of autophagy related 7 (ATG7) by deubiquitinating it, thereby enhancing the stability of ATG7 protein and ultimately promoting ferroptosis. In conclusion, this study elucidates the crucial role of the USP13-ATG7 axis in regulating ferroptosis in chicken follicle granulosa cells, thereby presenting a novel avenue for molecular breeding research in chickens.
Assuntos
Proteína 7 Relacionada à Autofagia , Proteínas Aviárias , Galinhas , Ferroptose , Células da Granulosa , Proteases Específicas de Ubiquitina , Animais , Feminino , Galinhas/genética , Células da Granulosa/fisiologia , Células da Granulosa/metabolismo , Ferroptose/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genética , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologia , UbiquitinaçãoRESUMO
The degenerative process of follicular atresia in hens naturally commences in granulosa cells, significantly impacting laying hens' reproductive performance. Past studies suggested that granulosa cell autophagy and apoptosis work together to cause follicular atresia. Recent research indicates that miRNA regulates granulosa autophagy and apoptosis, which contributes to the development of follicular atresia. However, the role of miR-302c-3p in follicular atresia and development remains unclear. In this study with the RNA-seq approach, we found that miR-302c-3p expression was significantly decreased in atrophic follicles, suggesting its involvement in the follicular atresia process. Following this, we performed in vitro studies to confirm that miR-302c-3p inhibits autophagy and apoptosis in chicken granulosa cells. Mechanistically, LATS2 is considered as the putative target gene of miR-302c-3p, and it has been demonstrated that LATS2 exerts a positive regulatory role in the modulation of autophagy and apoptosis in chicken granulosa cells. Furthermore, we verified the regulatory function of miR-302c-3p in chicken granulosa cells via the LATS2-YAP signaling pathway. Our results collectively demonstrates that miR-302c-3p targets LATS2 to modulate the YAP signaling pathway, impacting autophagy and apoptosis in granulosa cells leading to follicular atresia.
Assuntos
Apoptose , Autofagia , Galinhas , Células da Granulosa , MicroRNAs , Animais , Feminino , Células da Granulosa/fisiologia , Células da Granulosa/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Galinhas/genética , Transdução de Sinais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Regulação da Expressão Gênica , Proteínas de Sinalização YAP/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Atresia Folicular/genética , Atresia Folicular/fisiologiaRESUMO
The aim of the present study was to determine whether adipokines monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) can affect the functions of ovarian cells in cats. The addition of either MCP-1 or PAI-1 increased viability; promoted the accumulation of proliferation markers and progesterone and estradiol release; and decreased the accumulation of apoptosis markers in cultured feline granulosa cells. The present observations suggest that MCP-1 or PAI-1 can be physiological stimulators of ovarian granulosa cell functions.
Assuntos
Quimiocina CCL2 , Células da Granulosa , Inibidor 1 de Ativador de Plasminogênio , Animais , Gatos , Feminino , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Células da Granulosa/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Células Cultivadas , Proliferação de Células/fisiologia , Estradiol/metabolismo , Estradiol/farmacologia , Progesterona/metabolismo , Progesterona/farmacologia , Apoptose , Sobrevivência CelularRESUMO
Follicular fluid (FF) is rich in extracellular vesicles (EVs). EVs carries a variety of miRNA involved in regulating follicular development, the function of cells in follicles, primordial follicular formation, follicular recruitment and selection, follicular atresia, oocyte communication, granulosa cells (GCs) function and luteinization and other biological processes of follicular development. Previous studies in our laboratory have shown that bovine follicular fluid (bFF) high density-small extracellular vesicles (HD-sEVs)-miRNA was enriched in autophagy-related pathways. However, the mechanism of bFF EVs carrying miRNA regulating GCs autophagy is not clear. Thus, this study carried out a series of studies on the previous HD-sEVs sequencing data and miR-128-3p contained in bFF HD-sEVs. A total of 38 differentially expressed genes were detected by RNA-Seq after overexpression of miR-128-3p in bovine GCs (bGCs). Through cell transfection, Western blot (WB) and Immunofluorescence (IF), it was proved that overexpression of miR-128-3p could promote the expression of LC3 (microtubule-associated protein I light chain 3), inhibit p62, promote the number of autophagosome, promote the formation of autophagy lysosome and autophagy flow, and activate bGCs autophagy. MiR-128-3p inhibitor significantly inhibited the expression of LC3 and monodansylcadaverine (MDC) in bGCs, and promoted the expression of autophagy substrate p62, indicating that HD-sEVs-miR-128-3p could activate bGCs autophagy. In addition, through double luciferase assay, bioinformatics analysis, WB and RT-qPCR, it was concluded that bFF HD-sEVs-miR-128-3p could target TFEB (transcription factor EB) and FoxO4 (Forkhead box O4) and activate GCs autophagy.
Assuntos
Autofagia , Líquido Folicular , Células da Granulosa , MicroRNAs , Animais , Bovinos , MicroRNAs/genética , MicroRNAs/metabolismo , Células da Granulosa/fisiologia , Células da Granulosa/metabolismo , Feminino , Líquido Folicular/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Regulação da Expressão Gênica/fisiologiaRESUMO
In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.
Assuntos
Autofagia , Atresia Folicular , Células da Granulosa , Feminino , Animais , Atresia Folicular/fisiologia , Ovinos/fisiologia , Autofagia/fisiologia , Células da Granulosa/fisiologia , Ovário , Folículo Ovariano/fisiologia , ApoptoseRESUMO
The proliferation and death of granulosa cells (GCs) in poultry play a decisive role in follicular fate and egg production. The follicular fluid (FF) contains a variety of nutrients and genetic substances to ensure the communication between follicular cells. Exosomes, as a new intercellular communication, could carry and transport the proteins, RNA, and lipids to react on GCs, which had been found in FF of various domestic animals. Whether exosomes of FF in poultry play a similar role is unclear. In this study, geese, a poultry with low egg production, were chosen, and the effect of FF exosomes on the proliferation and death of GCs was investigated. Firstly, there were not only a large number of healthy small yellow follicles (HSYFs) but also some atresia small yellow follicles (ASYFs) in the egg-laying stage. Also, the GC layers of ASYFs became loose interconnections, inward detachment, and diminished survival rate than that of HSYFs. Besides, compared to HSYFs, the contents of E2, P4, and the mRNA expression levels of ferroptosis-related genes GPX4, FPN1, and FTH1 were significantly decreased, while COX2, NCOA4, VDAC3 mRNA were significantly increased, and the structure of mitochondrial cristae disappeared and the outer membrane broke in the GC layers of ASYFs. Moreover, the ROS, MDA, and oxidation levels in the GC layers of ASYFs were significantly higher than those of HSYFs. All these hinted that ferroptosis might result in a large number of GCs death and involvement in follicle atresia. Secondly, FF exosomes were isolated from HSYFs and ASYFs, respectively, and identified by TEM, NTA, and detection of exosome marker proteins. Also, we found the exosomes were phagocytic by GCs by tracking CM-Dil. Moreover, the addition of ASYF-FF exosomes significantly elevated the MDA content, Fe2+ levels, and the mitochondrial membrane potential (MMP) in GCs, thus significantly inhibiting the proliferation of GCs, which was restored by the ferroptosis inhibitor ferrostatin-1. Thirdly, the proteomic sequencing was performed between FF-derived exosomes of HSYFs and ASYFs. We obtained 1615 differentially expressed proteins, which were mainly enriched in the protein transport and ferroptosis pathways. Among them, HMOX1 was enriched in the ferroptosis pathway based on differential protein-protein interaction network analysis. Finally, the role of HMOX1 in regulating ferroptosis in GCs was further explored. The highly expressed HMOX1 was observed in the exosomes of ASYF-FF than that in HSYF-FF. Overexpression of HMOX1 increased ATG5, LC3II, and NCOA4 expression and reduced the expression of FTH1, GPX4, PCBP2, FPN1 in the ferroptosis pathway, also promoted intracellular Fe2+ accumulation and MDA surge, which drove ferroptosis in GCs. The effects of HMOX1 on ferroptosis could be blocked by its inhibitor Znpp. Taken together, the important protein HMOX1 was identified in FF, which could be delivered to GCs via exosomes, triggering ferroptosis and thus determining the fate of follicles.
Assuntos
Exossomos , Ferroptose , Atresia Folicular , Líquido Folicular , Gansos , Células da Granulosa , Heme Oxigenase-1 , Animais , Ferroptose/fisiologia , Feminino , Exossomos/metabolismo , Células da Granulosa/fisiologia , Células da Granulosa/metabolismo , Atresia Folicular/fisiologia , Líquido Folicular/metabolismo , Gansos/fisiologia , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genéticaRESUMO
Within the poultry industry, hens' reproductive performance is of great economic significance. The development and growth of follicles is a key aspect of hen egg production, and ovarian follicle growth and development are closely associated with granulosa cells (GCs) proliferation and the synthesis of steroid hormones. It has been confirmed by numerous studies that microRNAs (miRNAs) play important roles in the steroid hormone synthesis and proliferation of GCs. In this study, we examined the main miRNAs influencing hens' ability to reproduce, identified the miR-223 that is mainly expressed in atretic follicles based on sequencing, and investigated its role in GCs. Then, we used miR-223 mimic and inhibitor to knockdown or overexpress miR-223 expression. The result showed that miR-223 significantly inhibits both the steroid hormone synthesis and the proliferation of GCs. Subsequently, the results of the dual luciferase reporter experiment and bioinformatics prediction demonstrated that cysteine rich transmembrane BMP regulator 1 (CRIM1) was a downstream target gene of miR-223, and overexpression of miR-223 prevented CRIM1 expression. The function of CRIM1 was further investigated, and we observed a significant reduction in the synthesis of steroid hormones and the proliferation of GCs after transfection with CRIM1 siRNA. The opposite function of miR-223 was observed for CRIM1 in our study. Additionally, we demonstrated the involvement of the miR-223/CRIM1 axis in GCs through modulation of the AKT signaling pathway. Our data demonstrate the pivotal role of the miR-223 in the proliferation and steroid hormone synthesis of chicken GCs, which helps to explain how non-coding RNA (ncRNA) affects chicken reproductive function.
Assuntos
Proliferação de Células , Galinhas , Células da Granulosa , MicroRNAs , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Galinhas/genética , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/biossínteseRESUMO
Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFß) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual cells, we examined the effect of depleting the canonical TGFß mediator, SMAD4, in mouse granulosa cells. We observed a 20-50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with wild-type oocytes. Three-dimensional image analysis revealed that TZPs of SMAD4-depleted cells were longer than controls and more frequently oriented towards the oocyte. Strikingly, the transmembrane proteins, N-cadherin and Notch2, were reduced by 50% in SMAD4-depleted cells. SMAD4 may thus modulate a network of cell adhesion proteins that stabilize the attachment of TZPs to the oocyte, thereby amplifying signalling between the two cell types.
Assuntos
Células da Granulosa , Oócitos , Proteína Smad4 , Animais , Proteína Smad4/metabolismo , Proteína Smad4/genética , Oócitos/metabolismo , Oócitos/crescimento & desenvolvimento , Camundongos , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Receptor Notch2/metabolismo , Receptor Notch2/genética , Caderinas/metabolismo , Caderinas/genética , Pseudópodes/metabolismo , Pseudópodes/fisiologiaRESUMO
GATA4 plays a pivotal role in the reproductive processes of mammals. However, the research on GATA4 in goat ovary is limited. This study aimed to study the expression and function of GATA4 in goat ovary. Utilizing real-time PCR and western blot analysis, we studied the expression and regulatory mechanisms of GATA4 in goat ovary and granulosa cells (GCs). We found that GATA4 was expressed in all follicle types in the goat ovary, with significantly higher levels in GCs of larger follicles (>3 mm) compared to those in smaller follicles (<3 mm). Additionally, we demonstrated that human chorionic gonadotrophin (hCG) induced GATA4 mRNA expression via the activation of PKA, MEK, p38 MAPK, PKC, and PI3K pathways in vitro. Our study also showed that hCG suppressed the levels of miR-200b and miR-429, which in turn directly target GATA4, thereby modulating the basal and hCG-induced expression of GATA4. Functionally, we examined the effect of siRNA-mediated GATA4 knockdown on cell proliferation and hormone secretion in goat GCs. Our results revealed that knockdown of GATA4, miR-200b, and miR-429 suppressed cell proliferation. Moreover, knockdown of GATA4 decreased estradiol and progesterone production by inhibiting the promoter activities of CYP11A1, CYP19A1, HSD3B, and StAR. Collectively, our findings suggest a critical involvement of GATA4 in regulating goat GC survival and steroidogenesis.
Assuntos
Gonadotropina Coriônica , Fator de Transcrição GATA4 , Regulação da Expressão Gênica , Cabras , Células da Granulosa , Animais , Células da Granulosa/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Cabras/fisiologia , Feminino , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Gonadotropina Coriônica/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Progesterona/metabolismo , Células Cultivadas , Estradiol/farmacologia , Estradiol/metabolismo , Proliferação de CélulasRESUMO
The current understanding of the role of circular RNAs (circRNAs) in regulating ovarian functions is inadequate. To assess the impact of ciR-00596 and ciR-00646 on the regulation of basic porcine ovarian granulosa cell functions, we conducted upregulation (utilizing overexpressing vectors) and downregulation (utilizing shRNA vectors) of these circRNAs. The relative expression of both circRNAs, cell viability and proliferation (accumulation of PCNA, cyclin B1, and XTT-positive cells), cytoplasmic (accumulation of bax and caspase-3) and nuclear (DNA fragmentation) apoptosis, and the release of progesterone, testosterone, estradiol, IGF-I, and oxytocin were evaluated. Transfection of cells with the ciR-00596 overexpression vector resulted in increases in cell viability and proliferation and the release of progesterone and IGF-I, while it decreased the cytoplasmic and nuclear apoptosis, testosterone, estradiol, and oxytocin output. CiR-00596 inhibition had the opposite effects. The overexpression of ciR-00646 decreased cell viability and proliferation, and the release of progesterone, IGF-I, and oxytocin, while increasing cytoplasmic and nuclear apoptosis and the output of testosterone and estradiol. Our findings are the first to show the stimulatory action of ciR-00596 and the inhibitory effect of ciR-00646 on ovarian cell functions, including the cell cycle, apoptosis, and secretory activity.
Assuntos
Apoptose , Regulação para Baixo , Células da Granulosa , RNA Circular , Regulação para Cima , Animais , Feminino , RNA Circular/metabolismo , RNA Circular/genética , Suínos , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Ovário/metabolismo , Progesterona/metabolismo , Estradiol/metabolismo , Regulação da Expressão Gênica/fisiologiaRESUMO
The aim of the present study was to examine the influence of monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) on ovarian cell functions. Rabbit ovarian granulosa cells were cultured with or without MCP-1 or PAI-1 (at 0, 0.1, 1, or 10 ng/ml). Cell viability, proliferation, cytoplasmic apoptosis and release of progesterone and estradiol were measured by Cell Counting Kit-8 (CCK-8), BrdU incorporation, and cell death detection assays and ELISA. The addition of either MCP-1 or PAI-1 increased cell viability and proliferation and decreased apoptosis. MCP-1 promoted, while PAI-1 suppressed, progesterone release. Both MCP-1 and PAI-1 reduced estradiol output. The present results suggest that MCP-1 or PAI-1 can be physiological promoters of rabbit ovarian cell viability and proliferation, inhibitors of apoptosis and regulators of ovarian steroidogenesis.
Assuntos
Apoptose , Quimiocina CCL2 , Células da Granulosa , Inibidor 1 de Ativador de Plasminogênio , Progesterona , Animais , Feminino , Coelhos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Apoptose/efeitos dos fármacos , Progesterona/farmacologia , Estradiol/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células CultivadasRESUMO
Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that have been implicated in mediating granulosa cell (GC) proliferation and apoptosis. CircRAB11A was found to have a significantly higher expression in normal follicles compared to atrophic follicles. In this study, we determined that the knockdown of circRAB11A resulted in the inhibition of proliferation and promotion of apoptosis in GCs of chicken. Moreover, circRAB11A was found to act as a sponge for miR-24-5p, both member RAS oncogene family (RAB11A) and epidermal growth factor receptor (EGFR) were revealed to be targets of miR-24-5p through a dual-luciferase reporter assay. RAB11A or EGFR promoted proliferation and suppressed apoptosis in GCs through the phosphatidylinositol-kinase (PI3K)/AKT or extracellular signal-regulated kinase (ERK)1/2 pathway. These findings suggest that circRAB11A may function as a competing endogenous RNA (ceRNA) by targeting the miR-24-5p/RAB11A and miR-24-5p/EGFR axes and activating the ERK1/2 and PI3K/AKT pathways, offering a potential avenue for exploring the mechanism of follicle development.
Assuntos
Apoptose , Proliferação de Células , Galinhas , Receptores ErbB , Células da Granulosa , MicroRNAs , RNA Circular , Proteínas rab de Ligação ao GTP , Animais , Células da Granulosa/fisiologia , Células da Granulosa/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Galinhas/genética , Feminino , RNA Circular/genética , RNA Circular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genéticaRESUMO
Granular cell apoptosis is a key factor leading to follicular atresia and decreased laying rate in aged laying hens. Endoplasmic reticulum stress (ERS) induced cell apoptosis is a new type of apoptosis pathway. Previous studies have shown that the ERS pathway is involved in the regulation of follicular development and atresia, and can be regulated by mTOR. Melatonin (MEL) can protect the normal development of follicles, but the precise mechanism by which MEL regulates follicular development is not yet clear. So, we investigated the potential relationship between MEL and ERS and mTOR signaling pathway in vivo through intraperitoneal injection of MEL in aged laying hens. The results show that the laying rate, ovarian follicle number, plasma MEL, E2, LH, FSH concentrations, as well as the mRNA expression of mTOR signaling-associated genes TSC1, TSC2, mTOR, 4E-BP1, and S6K in old later-period chicken control (Old-CN) group was significantly decreased (P < 0.01). In contrast, the ERS-related of plasma and granular cell layer mRNA expression of Grp78, CHOP, and Caspase-3 was significantly increased (P < 0.01). While both of the effects were reversed by MEL. Then, aging granulosa cells were treated with MEL in vitro, followed by RNA seq analysis, and it was found that 259 and 322 genes were upregulated and downregulated. After performing GO enrichment analysis, it was found that DEGs significantly contribute to the biological processes including cell growth and apoptosis. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to mTOR signaling and endoplasmic reticulum (ER) stress, involving genes such as GRB10, SGK1, PRKCA, RPS6KA2, RAF1, PIK3R3, FOXO1, DERL3, HMOX1, TLR7, VAMP7 and INSIG2. The obtained results of RT-PCR showed consistency with the RNA-Seq data. In summary, the underlined results revealed that MEL has significantly contributed to follicular development via activating the mTOR signaling pathway-related genes and alleviating ERS-related genes in laying hens. The current study provides a theoretical background for enhancing the egg-laying capability of hens and also providing a basis for elucidating the molecular mechanism of follicular selection.
Assuntos
Galinhas , Estresse do Retículo Endoplasmático , Melatonina , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Feminino , Melatonina/farmacologia , Melatonina/administração & dosagem , Galinhas/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genética , Ovário/efeitos dos fármacos , Ovário/fisiologia , Envelhecimento , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologiaRESUMO
Follicular atresia in chickens reduces the number of follicles that can further develop, leading to decrease egg laying. Endoplasmic reticulum stress (ERS) can initiate a unique pathway inducing the apoptosis of follicular granulosa cells, thus reducing egg laying. Melatonin (MEL) is involved in the regulation of follicle development, ovulation, and oocyte maturation, and is closely related to follicle fate. Mammalian target of Rapamycin (mTOR) signaling pathway plays an important role in cell growth regulation, and that there is a possible crosstalk between melatonin and mTOR activity in granular cells maturation and ovulation. This study aimed to investigate whether MEL inhibits ERS and follicular granulosa cell apoptosis by regulating ATF4 to activate mTOR signaling pathway in chickens. Frist, we established an in vitro ERS cell model using tunicamycin (TM). The results showed that different concentrations of TM exhibited dose-dependent inhibition of cell activity and induction of granulosa cells (P<0.01). Therefore, we chose 5 µg/mL of TM and a treatment time for 6 h as the optimal concentration for the following experiments. Then we investigate whether melatonin can inhibit ERS. TM treatment decreased the cell viability and Bcl-2 expression, increasing ROS levels and the mRNA expression of Grp78, ATF4, CHOP, PERK, eIF-2α, and BAX (P<0.01), whereas TM+MEL treatment significantly inhibited these changes (P<0.01). Then we explored whether melatonin protects follicular granulosa cells from ERS-induced apoptosis through the mammalian target of rapamycin (mTOR) signaling pathway by regulating ATF4, we found that ATF4 knockdown inhibited ERS by decreasing the expression of ERS-related genes and proteins and activating mTOR signaling pathway by increasing the protein expression of p4E-BP1 and pT389-S6K (P<0.001), while these changes were promoted by TM+si-ATF4+MEL treatment (P<0.01). These results indicate that MEL could alleviate TM-induced ERS by regulating ATF4 to activate mTOR signaling pathway in follicular granulosa cells, thus providing a new perspective for prolonging the laying cycle in chickens.
Assuntos
Fator 4 Ativador da Transcrição , Apoptose , Proteínas Aviárias , Galinhas , Estresse do Retículo Endoplasmático , Células da Granulosa , Melatonina , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Melatonina/farmacologia , Feminino , Galinhas/fisiologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genética , Tunicamicina/farmacologiaRESUMO
Egg production is an economically important trait in poultry breeding and production. Follicular development was regulated by several hormones released and genes expressed in the granulosa cells, impacting the egg production and fecundity of hens. However, the molecular functions of these candidate genes that modulate these processes remain largely unknown. In the present study, bioinformatics analyses were performed to identify the candidate genes related to egg production in the ovarian tissue of White Leghorns with high egg production and Beijing You chicken with low egg production during sexual maturity and peak laying periods. The ovarian granulosa cells were used to assess the function of CYP21A1 by transfecting with CYP21A1-specific small interfering RNAs (siRNAs) and overexpression plasmids. We identified 514 differentially expressed genes (|Log2(fold change) | >1, P <0.05) between the 2 chicken breeds in both laying periods. Among these genes, CYP21A1, which is involved in the steroid hormone biosynthesis pathway was consistently upregulated in White Leghorns. Weighted gene co-expression network analysis (WGCNA) further suggested that CYP21A1 was a hub gene, which could positively respond to treatment with follicle stimulation hormone (FSH), affecting egg production. The interference of CYP21A1 significantly inhibited cell proliferation and promoted cell apoptosis. Overexpression of CYP21A1 promotes cell proliferation and inhibits cell apoptosis. Furthermore, the interference with CYP21A1 significantly downregulated the expression of STAR, CYP11A1, HSD3B1, and FSHR and also decreased the synthesis of progesterone (P4) and estradiol (E2) in granulosa cells. Overexpression of CYP21A1 increased the synthesis of P4 and estradiol E2 and the expression of steroid hormone synthesis-related genes in granulosa cells. Our findings provide new evidence for the biological role of CYP21A1 on granulosa cell proliferation, apoptosis, and steroid hormone synthesis, which lays the theoretical basis for improving egg production.
Assuntos
Galinhas , Perfilação da Expressão Gênica , Células da Granulosa , Animais , Feminino , Galinhas/genética , Galinhas/fisiologia , Células da Granulosa/metabolismo , Células da Granulosa/fisiologia , Perfilação da Expressão Gênica/veterinária , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Ovário/metabolismo , Hormônios Esteroides Gonadais/biossíntese , Hormônios Esteroides Gonadais/metabolismo , Transcriptoma , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologiaRESUMO
Chicken ovarian follicle development is regulated by complex and dynamic gene expression. Nuclear receptor 5A1 and 5A2 (NR5A1 and NR5A2, respectively) are key genes that regulate steroid hormone production and gonadal development in mammals; however, studies on follicular development in the chicken ovary are scarce. In this study, we investigated the functions of NR5A1 and NR5A2 on follicle development in chickens. The results showed that the expression of NR5A1 and NR5A2 was significantly higher in small yellow follicles and F5. Furthermore, the expression of NR5A1 and NR5A2 was significantly higher in follicular tissues of peak-laying hens (30 wk) than in follicular tissues of late-laying hens (60 wk), with high expression abundance in granulosa cells (GC). The overexpression of NR5A1 and NR5A2 significantly promoted proliferation and inhibited apoptosis of cultured GC; upregulated STAR, CYP11A1, and CYP19A1 expression and estradiol (E2) and progesterone (P4) synthesis in GC from preovulatory follicles (po-GC); and increased STAR, CYP11A1, and CYP19A1 promoter activities. In addition, follicle-stimulating hormone treatment significantly upregulated NR5A1 and NR5A2 expression in po-GC and significantly promoted FSHR, CYP11A1, and HSD3B1 expression in GC from pre-hierarchical follicles and po-GC. The core promoter region of NR5A1 was identified at the -1,095- to -483-bp and -2,054- to -1,536-bp regions from the translation start site (+1), and the core promoter region of NR5A2 was at -998 to -489 bp. Two single nucleotide polymorphisms (SNP) were identified in the core promoter region of the NR5A1 gene, which differed between high- and low-yielding chicken groups. Our study suggested that NR5A1 and NR5A2 promoted chicken follicle development by promoting GC proliferation and E2 and P4 hormone synthesis and inhibiting apoptosis. Moreover, we identified the promoter core region or functional site that regulates NR5A1 and NR5A2 expression.
Assuntos
Apoptose , Proteínas Aviárias , Proliferação de Células , Galinhas , Células da Granulosa , Folículo Ovariano , Animais , Feminino , Galinhas/genética , Células da Granulosa/fisiologia , Células da Granulosa/metabolismo , Folículo Ovariano/fisiologia , Folículo Ovariano/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Fator Esteroidogênico 1/genética , Fator Esteroidogênico 1/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/biossínteseRESUMO
1. The ferritin heavy chain (FHC) has a vital impact on follicular development in geese, due to its ability to regulate apoptosis of granulosa cells (GCs) and follicular atresia. However, its specific regulatory mechanisms remain unclear. The present study characterised how FHC regulates oxidative stress, cell proliferation and apoptosis in goose GCs by interfering with and overexpressing the FHC gene.2. After 72 h of interference with FHC expression, the activity of GCs decreased remarkably (p < 0.05), reactive oxygen species (ROS) levels and the expression levels of antioxidant enzyme genes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) increased significantly (p < 0.05). The overexpression of FHC for 72 h was found to significantly reduce the expression of CAT and SOD genes (p < 0.05).3. Interfering with FHC expression revealed that the expression levels of the cell proliferation gene Aurora kinase A (AURORA-A) were significantly decreased (p < 0.05), while the expression levels of the apoptosis genes B-cell lymphoma-2 (BCL-2) and cysteine aspartate-specific protease 8 (CASPASE 8) increased (p < 0.05). Further research has shown that, when interfering with FHC expression for 72 h, apoptosis rate increased by 1.19-fold (p < 0.05), but the current data showed a lower apoptosis rate after FHC overexpression by 59.41%, 63.39%, and 52.31% at three different treatment times (p < 0.05).4. In conclusion, FHC improved the antioxidant capacity of GCs, promotes GCs proliferation, and inhibits GCs apoptosis of ovarian follicles in Sichuan white geese.
Assuntos
Apoferritinas , Apoptose , Proliferação de Células , Gansos , Células da Granulosa , Estresse Oxidativo , Animais , Feminino , Gansos/fisiologia , Células da Granulosa/fisiologia , Apoferritinas/genética , Apoferritinas/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
1. The bioflavonoid quercetin is a biologically active component, but its functional regulation of granulosa cells (GCs) during chicken follicular development is little studied. To investigate the effect of quercetin on follicular development in laying hens, an in vitro study was conducted on granulosa cells from hierarchical follicles treated with quercetin.2. The effect of quercetin on cell activity, proliferation and apoptosis of granulosa cells was detected by CCK-8, EdU and apoptosis assays. The effect on progesterone secretion from granulosa cells was investigated by enzyme-linked immunosorbent assay (ELISA). Expression of proliferating cell nuclear antigen (PCNA) mRNA and oestrogen receptors (ERs), as well as the expression of steroid acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA during progesterone synthesis, were measured by real-time quantitative polymerase chain reaction (RT-qPCR). PCNA, StAR and CYP11A1 protein expression levels were detected using Western blotting (WB).3. The results showed that treatment with quercetin in granulosa cells significantly enhanced cell vitality and proliferation, reduced apoptosis and promoted the expression of gene and protein levels of PCNA. The levels of progesterone secretion increased significantly following quercetin treatment, as did the expression levels of StAR and CYP11A1 using the Western Blot (WB) method.4. The mRNA expression levels of ERα were significantly upregulated in the 100 ng/ml and 1000 ng/ml quercetin-treated groups, while there was no significant difference in expression levels of ERß mRNA.
Assuntos
Galinhas , Progesterona , Feminino , Animais , Progesterona/metabolismo , Progesterona/farmacologia , Galinhas/genética , Quercetina/farmacologia , Quercetina/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/farmacologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Células da Granulosa/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Spermidine, a natural polyamine, has been proven antioxidant function, but its pathway and mechanism of action remain unclear. Based on the oxidative stress model by 3-nitropropionic acid (3-NPA), the study explored the pathways by spermidine to rescue oxidative stress via autophagic process in goose granulosa cells by RNA-seq and RNA interference. In transcriptional regulation, in addition to KEGG pathways related to cell proliferation and differentiation, lots of KEGG pathways associated with inflammation, metabolism, and signaling were also significantly enriched in 3-NPA vs. 3-NPA + spermidine treatments. Six key genes (JUN, CD44, KITLG, RND2, BMP4 and KALRN) involved in spermidine-mediated anti-oxidative stress were screened. Furthermore, the experimental results showed that spermidine (80 µmol/L) significantly increased autophagic gene expression in goose granulosa cells, while EP300-siRNA or MAP1S-siRNA also significantly increased autophagic process. The autophagic gene expressions were no difference between EP300-siRNA and EP300-siRNA + spermidine treatments, although spermidine significantly increased autophagic process of granulosa cells compared to MAP1S-siRNA alone. In addition, inhibition of mTOR pathway significantly increased autophagic gene expression, which was further enhanced by spermidine in combined with mTOR inhibitor. These results suggest that spermidine can alleviate oxidative stress by inducing autophagy regulated by EP300, MAP1S and mTOR as well as regulating other independent gene expressions in goose granulosa cells.
Assuntos
Gansos , Espermidina , Feminino , Animais , Gansos/metabolismo , Espermidina/farmacologia , Espermidina/metabolismo , Células da Granulosa/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Estresse Oxidativo , Autofagia , RNA Interferente PequenoRESUMO
The oocyte cumulus complex is mainly composed of an oocyte, the perivitelline space, zona pellucida and numerous granulosa cells. The cumulus granulosa cells (cGCs) provide a particularly important microenvironment for oocyte development, regulating its growth, maturation and meiosis. In this study, we studied the internal structures and cell-to-cell connections of mouse cGCs using focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed three-dimensional models to display characteristic connections between the oocyte and cGCs, and to illustrate various main organelles in cGCs together with their interaction relationship. A special form of cilium identified in granulosa cell was never reported in previous literature.