Effect of quercetin on granulosa cells development from hierarchical follicles in chicken.

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.

Pathway analysis of spermidine anti-oxidative stress and inducing autophagy in granulosa cells of Sichuan white geese.

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.

Three-dimensional architecture of granulosa cell derived from oocyte cumulus complex, revealed by FIB-SEM.

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.

Circadian clock gene BMAL1 regulates STAR expression in goose ovarian preovulatory granulosa cells.

The ovarian circadian clock plays a regulatory role in the avian ovulation-oviposition cycle. However, little is known regarding the ovarian circadian clock of geese. In this study, we investigated rhythmic changes in clock genes over a 48-h period and identified potential clock-controlled genes involved in progesterone synthesis in goose ovarian preovulatory granulosa cells. The results showed that BMAL1, CRY1, and CRY2, as well as 4 genes (LHR, STAR, CYP11A1, and HSD3B) involved in progesterone synthesis exhibited rhythmic expression patterns in goose ovarian preovulatory granulosa cells over a 48-h period. Knockdown of BMAL1 decreased the progesterone concentration and downregulated STAR mRNA and protein levels in goose ovarian preovulatory granulosa cells. Overexpression of BMAL1 increased the progesterone concentration and upregulated the STAR mRNA level in goose ovarian preovulatory granulosa cells. Moreover, we demonstrated that the BMAL1/CLOCK complex activated the transcription of goose STAR gene by binding to an E-box motif. These results suggest that the circadian clock is involved in the regulation of progesterone synthesis in goose ovarian preovulatory granulosa cells by orchestrating the transcription of steroidogenesis-related genes.

HD-sEVs in bovine follicular fluid regulate granulosa cell apoptosis and estradiol secretion through the autophagy pathway.

Follicular fluid (FF) is rich in extracellular vesicles (EVs), which have regulatory effects on follicular growth and oocyte development. EVs can be divided into two subtypes, i.e. HD-sEVs and LD-sEVs. In this study, HD-sEVs were successfully isolated from bovine follicular fluid (BFF) by density gradient ultracentrifugation. By western blot, quantitative polymerase chain reaction (qPCR), flow cytometry, transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA), this study found HD-sEVs promoted autophagy in bGCs by increasing the protein and mRNA expression of LC3II/LC3I ratio and Beclin1, and inhibiting the protein and mRNA expression of p62. HD-sEVs promoted mitophagy in bGCs by increasing the protein and mRNA expression of VDAC1, CTSD, and HSP60. Flow cytometry showed that HD-sEVs inhibited bGCs apoptosis rate. HD-sEVs promoted estradiol secretion by increasing steroidogenesis-associated proteins and mRNA, such as CYP19A, HSD3B in bGCs. HD-sEVs promoted autophagosome formation and mitochondrial structure swelling in bGCs, and decreased p-mTOR/mTOR ratio. The above phenomenon was reversed when wortmannin was added. Collectively, BFF HD-sEVs promote bGCs autophagy and mitophagy, inhibit bGCs apoptosis and promote estradiol secretion through the autophagy pathway-mTOR signaling pathway.

Mouse primary follicles experience slow growth rates after activation and progressive increases that influence the duration of the primary follicle phase†.

There are conflicting estimates of the duration of mouse primary follicle development. An accurate determination is needed for studies examining preantral follicle survival and mathematical modeling of folliculogenesis. Primary follicle granulosa cell proliferation rates are low and variable, which may explain the variation in duration estimates. In the present study, female C57Bl6/J mice were exposed to bromodeoxyuridine for 48 hours, to label the proliferating granulosa cells in a large proportion of primary follicles. The bromodeoxyuridine-containing water was then withdrawn and replaced with drug-free water and the mice were euthanized at 0, 1, 3, 6, 10, or 13 days post-bromodeoxyuridine withdrawal. Granulosa cells were bromodeoxyuridine labeled in 48% of primary follicles at day 0, but this decreased to 5% over the 13-day period, as the labeled primary follicles progressed to the secondary follicle stage. Curve-fitting estimated that the last of the bromodeoxyuridine-labeled primary follicles would progress to the secondary stage by 13.7 days. Mathematical models that assumed constant rates of primary follicle proliferation were fitted to the data, but the observed pattern of bromodeoxyuridine-labeled primary follicle disappearance could not be replicated. The level of immunoreactivity for bromodeoxyuridine and proliferating-cell nuclear antigen in primary follicles revealed follicles with no granulosa cell proliferation during the 48-h bromodeoxyuridine-exposure period had resumed proliferation 1 or 3 days later. Therefore, primary follicle granulosa cells proliferate after follicle activation, but proliferation rates gradually increase as the follicle develops. Prior estimates of primary follicle duration are inaccurate due to the assumption that follicles develop at a constant rate.

MicroRNA-129-1-3p attenuates autophagy-dependent cell death by targeting MCU in granulosa cells of laying hens under H2O2-induced oxidative stress.

The present study aimed to investigate the mechanism of microRNA-129-1-3p (miR-129-1-3p) in regulating hydrogen peroxide (H2O2)-induced autophagic death of chicken granulosa cell by targeting mitochondrial calcium uniporter (MCU). The results indicated that the exposure of hens' ovaries to H2O2 resulted in a significant elevation in reactive oxygen species (ROS) levels, as well as the apoptosis of granulosa cells and follicular atresia. This was accompanied by an upregulation of glucose-regulated protein 75 (GRP75), voltage-dependent anion-selective channel 1 (VDAC1), MCU, mitochondria fission factor (MFF), microtubule-associated protein 1 light chain 3 (LC3) I, and LC3II expression, and a downregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitofusin-2 (MFN2) expression. In hens' granulosa cells, a luciferase reporter assay confirmed that miR-129-1-3p directly regulates MCU. The induction of oxidative stress through H2O2 resulted in the activation of the permeability transition pore, an overload of calcium, depolarization of the mitochondrial membrane potential, dysfunction of mitochondria-associated endoplasmic reticulum membranes (MAMs), and ultimately, autophagic cell death. The overexpression of miR-129-1-3p effectively mitigated these H2O2-induced changes. Furthermore, miR-129-1-3p overexpression in granulosa cells prevented the alterations induced by H2O2 in the expression of key proteins that play crucial roles in maintaining the integrity of MAMs and regulating autophagy, such as GRP75, VDAC1, MFN2, PTEN-induced kinase 1 (Pink1), and parkin RBR E3 ubiquitin-protein ligase (Parkin). Together, these in vitro- and in vivo-based experiments suggest that miR-129-1-3p protects granulosa cells from oxidative stress-induced autophagic cell death by downregulating the MCU-mediated mitochondrial autophagy. miR-129-1-3p/MCU calcium signaling pathway may act as a new target to alleviate follicular atresia caused by oxidative stress in laying hens.

Hypoxia enhances steroidogenic competence of buffalo (Bubalus bubalis) granulosa cells.

Granulosa cells (GCs) synthesize estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo GCs remain unclear. In this study, the impacts of hypoxic conditions (5% oxygen) on estrogen synthesis in buffalo GCs were examined. The results showed that hypoxia improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3ß-HSD) and the secretion levels of estradiol in buffalo GCs. Hypoxic conditions promoted the sensitivity of buffalo GCs to FSH. Furthermore, inhibition of cAMP/PKA signaling pathway (H89, a cAMP/PKA signaling pathway inhibitor) reduced both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3ß-HSD) and the secretion levels of estradiol in hypoxia-cultured buffalo GCs. Besides, inhibition of cAMP/PKA signaling pathway lowered the responsiveness of buffalo GCs to FSH under hypoxic conditions. The present study indicated that hypoxia enhanced the steroidogenic competence of buffalo GCs principal by affecting cAMP/PKA signaling pathway and subsequent sensitivity of GCs to FSH.

Role of Melatonin in Bovine Reproductive Biotechnology.

Melatonin has profound antioxidant activity and numerous functions in humans as well as in livestock and poultry. Additionally, melatonin plays an important role in regulating the biological rhythms of animals. Combining melatonin with scientific breeding management has considerable potential for optimizing animal physiological functions, but this idea still faces significant challenges. In this review, we summarized the beneficial effects of melatonin supplementation on physiology and reproductive processes in cattle, including granulosa cells, oocytes, circadian rhythm, stress, inflammation, testicular function, spermatogenesis, and semen cryopreservation. There is much emerging evidence that melatonin can profoundly affect cattle. In the future, we hope that melatonin can not only be applied to cattle, but can also be used to safely and effectively improve the efficiency of animal husbandry.

Can flaxseed, chia or puncture vine affect mare ovarian cell functions and prevent the toxic effect of the environmental contaminant toluene?

The aim of this in vitro study was to examine the potential effect of functional food plant extracts, namely, extracts of flaxseed (Linum usitatissimum L.), chia (Salvia hispanica) and puncture vine (Tribulus terrestris L.), on basic mare ovarian cell functions and their response to the environmental contaminant toluene. Mare granulosa cells were incubated with and without toluene (0, 0.02, 0.2 or 2.0 µg/mL) in the presence or absence of flaxseed, chia and puncture vine extracts (10 µg/mL). Markers of cell proliferation (accumulation of proliferating cell nuclear antigen, PCNA) and apoptosis (accumulation of bax), viability (Trypan blue extrusion) and the release of progesterone (P), oxytocin (OT) and prostaglandin F 2 alpha (PGF) were measured. Toluene reduced all other measured parameters except OT release. All the tested plants were able to reduce cell viability and the release of P and PGF, but they did not influence other indexes. Moreover, flaxseed mitigated toluene action on ovarian cell proliferation, apoptosis, OT and PGF, whilst puncture vine prevented and inverted toluene action on P and PGF ourput. Chia extract did not modify toluene action on any parameter. On the other hand, toluene was able to promote the inhibitory action of flaxseed on cell viability and P release and to prevent the inhibitory action of all the plant extracts on PGF release. The present study (1) is the first demonstration, that flaxseed, chia and puncture vine can directly suppress mare ovarian cell functions, (2) shows that toluene can suppress basic ovarian cell functions and modify the reproductive effect of food plants and (3) demonstrates the ability of flaxseed and puncture vine, but not of chia, to prevent some toxic effect of toluene on mare ovarian cell functions.

MicroRNA miR-152 can support ovarian granulosa cell functions and modify apigenin actions.

The study aimed to evaluate the involvement of apigenin, microRNA (miR)-152, and their interrelationships in the control of basic ovarian granulosa cell functions. The effects of apigenin (0, 10, and 100 µg/mL), miR-152 analogues or miR-152 inhibitor, and their combinations with apigenin on porcine granulosa cells were examined. Expression levels of miR-152, viability, proliferation, apoptosis, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analyzed. Apigenin increased the expression of miR-152, cell proliferation, and estradiol release and reduced apoptosis, progesterone, and IGF-I output. MicroRNA-152 analogues promoted cell viability and proliferation, as well as the release of progesterone, IGF-I, oxytocin, and prostaglandin E2; however, it inhibited apoptosis and estradiol output. miR-152 inhibitor had the opposite effect. Moreover, miR-152 analogues suppressed the effect of apigenin on cell apoptosis and estradiol release. These observations 1) confirm the involvement of apigenin in the control of basic ovarian cell functions; 2) are the first demonstration of importance of miR-152 in the control of these functions; 3) show the ability of apigenin to promote miR-152 expression and the ability of miR-152 to modify apigenin effects on ovarian cells.

Oocyte-cumulus cells crosstalk: New comparative insights.

Mammalian follicles are constituted of a complex structure composed of several layers of granulosa cells surrounding the oocyte and of theca cells that reside beneath its basement membrane. During folliculogenesis, granulosa cells separate into two anatomically and functionally distinct sub-types; the mural cells lining the follicle wall and the oocyte-surrounding cumulus cells, i.e. those in intimate metabolic contact with the oocyte. The cumulus cells connecting with the oocyte have trans-zonal cytoplasmic projections which, penetrating the zona pellucida, form the cumulus-oocyte complex. The connections through gap junctions allow the transfer of small molecules between oocyte and cumulus cells, such as ions, metabolites, and amino acids necessary for oocyte growth, as well as small regulatory molecules that control oocyte development. The bi-directional communication between the oocyte and cumulus cells is crucial for the development and functions of both cell types. Our current knowledge of the relationship between the oocyte and its surrounding cumulus cells continues to change as we gain a greater understanding of factors regulating oocyte development and folliculogenesis. This review will mainly focus on the reciprocal interaction between oocytes and cumulus cells during the latter stages of follicle development i.e. through antral development to periovulatory events including oocyte maturation, expansion, and degradation of the cumulus matrix.

Mitochondrial function and E2 synthesis are impaired following alteration of CLOCK gene expression in porcine ovarian granulosa cells.

Circadian locomotor output cycles kaput (CLOCK) is a critical component of the mammalian circadian clock system and regulates ovarian physiology. However, the functions and mechanisms of CLOCK in porcine granulosa cells (GCs) are poorly understood. The present study focused on CLOCK's effects on estradiol synthesis. Similarity analysis showed that CLOCK is highly conserved between pigs and other species. The phylogenetic tree analysis indicated that porcine CLOCK was most closely related to that in Arabian camels. CLOCK significantly reduced E2 synthesis in GCs. CLOCK reduced the expression of steroidogenesis-related genes at the mRNA and protein levels, including CYP19A1, CYP11A1, and StAR. CYP17A1 levels were significantly downregulated. We demonstrated that CLOCK dramatically decreased ATP content, mitochondrial copy number, and mitochondrial membrane potential (MMP) and increased reactive oxygen species levels in GCs. We observed that mitochondria were severely damaged with fuzzy and fractured cristae and swollen matrix. These findings suggest that mitochondrial function and E2 synthesis are impaired following the alteration of CLOCK gene expression in porcine ovarian GCs.

Beyond apoptosis: evidence of other regulated cell death pathways in the ovary throughout development and life.

BACKGROUND: Regulated cell death is a fundamental component of numerous physiological processes; spanning from organogenesis in utero, to normal cell turnover during adulthood, as well as the elimination of infected or damaged cells throughout life. Quality control through regulation of cell death pathways is particularly important in the germline, which is responsible for the generation of offspring. Women are born with their entire supply of germ cells, housed in functional units known as follicles. Follicles contain an oocyte, as well as specialized somatic granulosa cells essential for oocyte survival. Follicle loss-via regulated cell death-occurs throughout follicle development and life, and can be accelerated following exposure to various environmental and lifestyle factors. It is thought that the elimination of damaged follicles is necessary to ensure that only the best quality oocytes are available for reproduction. OBJECTIVE AND RATIONALE: Understanding the precise factors involved in triggering and executing follicle death is crucial to uncovering how follicle endowment is initially determined, as well as how follicle number is maintained throughout puberty, reproductive life, and ovarian ageing in women. Apoptosis is established as essential for ovarian homeostasis at all stages of development and life. However, involvement of other cell death pathways in the ovary is less established. This review aims to summarize the most recent literature on cell death regulators in the ovary, with a particular focus on non-apoptotic pathways and their functions throughout the discrete stages of ovarian development and reproductive life. SEARCH METHODS: Comprehensive literature searches were carried out using PubMed and Google Scholar for human, animal, and cellular studies published until August 2022 using the following search terms: oogenesis, follicle formation, follicle atresia, oocyte loss, oocyte apoptosis, regulated cell death in the ovary, non-apoptotic cell death in the ovary, premature ovarian insufficiency, primordial follicles, oocyte quality control, granulosa cell death, autophagy in the ovary, autophagy in oocytes, necroptosis in the ovary, necroptosis in oocytes, pyroptosis in the ovary, pyroptosis in oocytes, parthanatos in the ovary, and parthanatos in oocytes. OUTCOMES: Numerous regulated cell death pathways operate in mammalian cells, including apoptosis, autophagic cell death, necroptosis, and pyroptosis. However, our understanding of the distinct cell death mediators in each ovarian cell type and follicle class across the different stages of life remains the source of ongoing investigation. Here, we highlight recent evidence for the contribution of non-apoptotic pathways to ovarian development and function. In particular, we discuss the involvement of autophagy during follicle formation and the role of autophagic cell death, necroptosis, pyroptosis, and parthanatos during follicle atresia, particularly in response to physiological stressors (e.g. oxidative stress). WIDER IMPLICATIONS: Improved knowledge of the roles of each regulated cell death pathway in the ovary is vital for understanding ovarian development, as well as maintenance of ovarian function throughout the lifespan. This information is pertinent not only to our understanding of endocrine health, reproductive health, and fertility in women but also to enable identification of novel fertility preservation targets.

miR-199-3p suppresses cellular migration and viability and promotes progesterone production in goose ovarian follicles before selection through regulating ITGB8 and other ECM-related genes.

1. The extracellular matrix (ECM) constitutes the basal lamina and the area between follicular cells. Remodelling the ECM is believed to be a key event in follicular development, especially during selection, and plays an important role in cell migration, survival, and steroidogenesis. miR-199-3p is differentially expressed in the goose granulosa layer during follicular selection and is reported to play a primary role in inhibiting cell migration and invasion. Nevertheless, the effect of miR-199-3p on ovarian follicles and its role in follicular cellular migration is not understood.2. In this study, qRT-PCR assays revealed that miR-199-3p was differentially expressed in the granulosa layer from goose ovarian follicles before and after follicular selection. Additionally, miR-199-3p overexpression in cultured granulosa cells (GCs) from goose pre-hierarchical follicles significantly suppressed cell viability and migration. It elevated the concentration of progesterone and the expression of key progesterone production genes. Furthermore, miR-199-3p overexpression in the GCs of goose pre-hierarchical follicles inhibited the expression of ECM-related genes (ITGB8, MMP9 and MMP15) yet promoted the expression of another two ECM-related genes (COL4A1 and LAMA1). Finally, dual-fluorescence reporter experiments on 293T cells established the direct targeting of ECM gene ITGB8 by miR-199-3p.3. In conclusion, miR-199-3p may participate in granulosa cell migration, viability, and steroidogenesis in goose ovarian follicles before selection by modulating ITGB8 and other ECM-related genes.

Molecular characterisation and expression profile of the PRLR gene during goose ovarian follicle development.

1. Although PRL-PRLR signalling plays important roles in regulating avian reproduction, there is a paucity of information regarding the functional significance of PRLR in goose ovarian follicle development.2. The full-length 2,496 bp coding sequence of PRLR was obtained from Sichuan White goose (Anser cygnoides) for the first time and was seen to encode a polypeptide containing 831 amino acids. Goose PRLR shares similar sequence characteristics and conserved functional domains to other avian species and was phylogenetically clustered into the avian clade.3. The qPCR results suggested that the mRNA levels of PRLR significantly increased in primary follicles during weeks 3 to 4 of age and were higher in secondary- than in primordial follicles at week 5 post-hatching, which suggested that the PRLR-mediated signalling could be involved in regulation of early folliculogenesis.4. The PRLR mRNA was expressed at the highest levels in the prehierarchical 8-10 mm granulosa layers throughout goose ovarian follicle development, indicating a role for PRLR in the process of follicle selection.5. PRLR mRNA was differentially expressed in the three cohorts of in vitro cultured granulosa cells harvested from different sized goose ovarian follicles, which suggested that PRLR was involved in regulating granulosa cell functions depending on the stage of follicle development. These data provide novel insights into the role of PRLR during goose ovarian follicle development, although the underlying mechanisms await further investigations.

Exploration the role of INHBA in Hu sheep granulosa cells using RNA-Seq.

Activin/inhibin is an important factor for the fecundity of Hu sheep, and it is involved in follicular development in ovaries. Inhibin subunit beta A (INHBA) participates in the synthesis of activin A and inhibin A. In this study, we also noted a positive correlation between INHBA level and the secretion of both activin A and inhibin A in culture medium. Nevertheless, both knockdown and overexpression of INHBA downregulated the expression of Inhibin Subunit Alpha (INHA). Based on RNA-Sequencing, we further examined the effect and molecular mechanism of INHBA knockdown in GCs on mRNA expression. A total of 1,687 differentially expressed genes (DEGs) were identified (Fold change ≥ 2; False-discovory-rates (FDR) ≤ 0.01), of which 602 genes were upregulated and 1,087 genes were downregulated in the INHBA interference group compared with the control groups. Gene Ontology (GO) enrichment indicated that these DEGs were mainly involved in the regulation of cell cycle, protein serine/threonine kinase activity, and actin cytoskeleton reorganization. Moreover, DEGs were significantly enriched in 40 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including P53, progesterone-mediated oocyte maturation, and PI3K-AKT signaling pathways. We also noted a positive correlation between INHBA level and many PI3K/Akt/mTOR pathway-related genes at the gene or/and protein expression. Overall, this study may contribute to a better understanding of the roles of INHBA on GCs of prolific sheep, as well as the molecular effect of low INHBA expression on GCs, clarifying some reproductive failures.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells.

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4-6 mm; <0.5 ng/mL) (ii) F2 (7-9 mm; 0.5-5 ng/mL) (iii) F3 (10-13 mm; 5-40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

PHB2 binds to ERß to induce the autophagy of porcine ovarian granulosa cells through mTOR phosphorylation.

Autophagy of ovarian granulosa cells is one of the reasons which results in follicular atresia. PHB2 regulates many fundamental biological processes and is pivotal in the mitophagy of cells; nevertheless, the autophagy in the porcine ovary and how PHB2 regulates the follicular cells are unknown. Here we report a protein complex that induces autophagy in porcine granulosa cells (PGCs) through the direct interaction of ERß and PHB2. In this study, we aimed to elucidate the autophagy and the role of PHB2 in porcine ovaries using porcine primary ovarian granulosa cells (PGCs). The results showed that PHB2 induces PGCs autophagy because of the change in related genes and protein expression levels. In addition, the results of Co-IP and the distribution of the combination of PHB2 and ERß showed that this complex is also indicated as an essential role of PHB2 in PGCs autophagy. Based on our results, it can be concluded that PHB2 combined with ERß induces PGCs autophagy by targeting the mTOR pathway. This study pinpoints a novel regulatory mechanism of autophagy and demonstrates the existence of a protein complex that may underlie its roles in autophagy in PGCs.

[Research Progress in the Regulation of Follicle Development by Melatonin].

Melatonin,an endocrine hormone synthesized by the pineal gland,plays an important role in the reproduction.The growth and development of follicles is the basis of female mammalian fertility.Follicles have a high concentration of melatonin.Melatonin receptors exist on ovarian granulosa cells,follicle cells,and oocytes.It regulates the growth and development of these cells and the maturation and atresia of follicles,affecting female fertility.This paper reviews the protective effects and regulatory mechanisms of melatonin on the development of ovarian follicles,granulosa cells,and oocytes and makes an outlook on the therapeutic potential of melatonin for ovarian injury,underpinning the clinical application of melatonin in the future.