Granulosa Cell Proliferation Research Articles

A Network Pharmacological Study on the Treatment of Diminished Ovarian Reserve with Nourishing Yin Formula

Objective To identify the principal constituents of Nourishing Yin Formula, analyze its active substances and effective targets for treating DOR, and explore its potential mechanism of action. Methods The primary active constituents of Nourishing Yin Formula and their putative targets for treating DOR were identified through a search of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (BATMAN-TCM), GeneCards, OMIM, and Uniprot databases. A protein-protein interaction network was constructed using the String database to identify shared targets between the drug and disease. These drug-disease shared targets were then subjected to GO analysis and KEGG pathway enrichment analysis using the DAVID database. Results After screening, the primary chemical constituents of Nourishing Yin Formula for treating DOR were identified, including progesterone, beta-sitosterol, 17-Beta-Estradiol, quercetin, kaempferol, etc Among the 52 critical targets for Nourishing Yin Formula in DOR treatment, the core targets were AKT1, INS, IL6, TNF, EGFR, ESR1, MYC, CTNNB1, NOS3, ERBB2, EDN1, and ESR2. Conclusions The potential mechanism of Nourishing Yin Formula in the treatment of DOR may involve the herbal compound's impact on AKT1, INS, IL6, TNF, and EGFR pathways. It regulates ovarian granulosa cell proliferation and apoptosis, and restores ovarian blood supply.

Testosterone-Induced H3K27 Deacetylation Participates in Granulosa Cell Proliferation Suppression and Pathogenesis of Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is the leading cause of infertility in reproductive-age women. Hyperandrogenism, polycystic ovaries and chronic anovulation are its typical clinical features. However, the correlation between hyperandrogenism and ovarian follicle growth aberrations remains undisclosed. To advance our understanding of the molecular alterations in ovarian granulosa cells (GCs) with excessive androgen, epigenetic changes and affected gene expression in human granulosa-lutein cells (hGCs) and immortalized human GCs (KGN) were evaluated. A PCOS mouse model induced by dihydrotestosterone (DHT) was also established. This study found excessive testosterone significantly decreased the acetylation of lysine 27 on Histone H3 (H3K27ac). H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) data showed down-regulated expression of cell cycle-related genes (CCND1/CCND3/PCNA), which was confirmed by real-time quantitative PCR and western blot. Testosterone application impeding cell proliferation was also proved by Ki-67 immunofluorescence and flow-cytometric analysis. Moreover, testosterone influenced CK2α nuclear translocation, which increased the phosphorylation level of histone deacetylase 2 (HDAC2). Inhibition of CK2α nuclear translocation or silenced HDAC2 expression efficiently retarded H3K27 acetylation. Meanwhile, PCOS mouse model experiments also demonstrated decreased H3K27ac and enhanced HDAC2 phosphorylation in GCs. Cell proliferation-related genes were also downregulated in PCOS mouse GCs. In conclusion, hyperandrogenism in human and mouse GCs caused H3K27Ac aberrations, which are associated with CK2α nuclear translocation and HDAC2 phosphorylation, participating in abnormal follicle development in PCOS patients.

Porcine granulosa cell transcriptomic analyses reveal the differential regulation of lncRNAs and mRNAs in response to all-trans retinoic acid in vitro.

The active metabolite of vitamin A, all-trans retinoic acid (ATRA), is involved in the proliferation and differentiation of granulosa cells, and promotes the follicular development, oocyte maturation, and ovulation in mammals. This study aims to investigate the ATRA induced potential long noncoding RNAs (lncRNAs) that regulate the expression of genes associated with granulosa cell proliferation and follicular development. The lncRNA and mRNA profiles of porcine granulosa cells from ATRA treatment and control group in vitro were constructed through RNA sequencing. Meanwhile, the sequencing data were verified using quantitative polymerase chain reaction (qPCR). A total of 86 differentially expressed lncRNAs and 128 differentially expressed genes (DEGs) were detected in granulosa cells after ATRA treatment. The qRT-PCR results were consistent with the RNA-seq data. Functional annotation analysis revealed that the DEGs were remarkably enriched in ovary function and reproduction which contained FoxO, Hippo, Oocyte meiosis, mTOR signaling pathway, as well as several pathways associated with hormone regulation like Oxytocin signaling pathway and Steroid hormone biosynthesis. Moreover, an interaction network of lncRNAs and their cis-target DEGs was constructed, and 7 differentially expressed lncRNAs and 6 cis-target DEGs were enriched in ovarian steroidogenesis and reproduction. These findings expand the lncRNA catalogue and provide a basis for further studies on the mechanism of ATRA-mediated lncRNA regulation of follicular development in pigs.

Integrative analysis of RNA-seq and Ribo-seq reveals that lncRNA-GRN regulates chicken follicular atresia through miR-103-3p/FBXW7 axis and encoding peptide

Follicular atresia in chickens seriously reduced the egg production and economic benefits of chickens. LncRNA plays a key role in the process of follicular atresia. In this study, RNA-seq and Ribo-seq were performed on normal and atretic follicles of Dahen broilers to screen out lncRNAs that may regulate follicle atresia, and to study the molecular mechanisms of their regulation. GRN granulin precursor (lncGRN, ID: 101748909) was highly expressed in atretic follicles with translational ability. A molecular regulatory network of lncGRN/miR-103-3p/FBXW7 was constructed through bioinformatics analysis and dual luciferase reporting. LncGRN promoted the expression of FBXW7 by adsorption of miR-103-3p, thereby inhibiting the proliferation of chicken granulosa cells (GCs), promoting apoptosis of chicken GCs and inhibiting steroid hormone synthesis thus induced follicular atresia. Meanwhile, we also found a micropeptide named GRN-122aa derived by lncGRN which can promote follicular atresia. In conclusion, our study found that lncGRN promoted follicular atresia through the lncGRN/miR-103-3p/FBXW7 axis and the translation micropeptide GRN-122aa. This study provided new insight into the post-transcriptional regulation mechanism of lncGRN suggesting that lncGRN may act as a potential to regulate chicken follicle development, and provided a theoretical argument for further improving the egg production of chickens through molecular breeding.

TRF-Gly-GCC in Atretic Follicles Promotes Ferroptosis in Granulosa Cells by Down-Regulating MAPK1.

Follicle development refers to the process in which the follicles in the ovary gradually develop from the primary stage to a mature state, and most primary follicles fail to develop normally, without forming a dense granular cell layer and cell wall, which is identified as atretic follicles. Granulosa cells assist follicle development by producing hormones and providing support, and interference in the interaction between granulosa cells and oocytes may lead to the formation of atretic follicles. Ferroptosis, as a non-apoptotic form of death, is caused by cells accumulating lethal levels of iron-dependent phospholipid peroxides. Healthy follicles ranging from 4 to 5 mm were randomly divided into two groups: a control group (DMSO) and treatment group (10 uM of ferroptosis inducer erastin). Each group was sequenced after three repeated cultures for 24 h. We found that ferroptosis was associated with atretic follicles and that the in vitro treatment of healthy follicles with the ferroptosis inducer erastin produced a phenotype similar to that of atretic follicles. Overall, our study elucidates that tRF-1:30-Gly-GCC-2 is involved in the apoptosis and ferroptosis of GCs. Mechanistically, tRF-1:30-Gly-GCC-2 inhibits granulosa cell proliferation and promotes ferroptosis by inhibiting Mitogen-activated protein kinase 1 (MAPK1). tRF-1:30-Gly-GCC-2 may be a novel molecular target for improving the development of atretic follicles in ovarian dysfunction. In conclusion, our study provides a new perspective on the pathogenesis of granulosa cell dysfunction and follicular atresia.

Genome-wide variation study and inter-tissue communication analysis unveil regulatory mechanisms of egg-laying performance in chickens

Egg-laying performance is of great economic importance in poultry, but the underlying genetic mechanisms are still elusive. In this work, we conduct a multi-omics and multi-tissue integrative study in hens with distinct egg production, to detect the hub candidate genes and construct hub molecular networks contributing to egg-laying phenotypic differences. We identifiy three hub candidate genes as egg-laying facilitators: TFPI2, which promotes the GnRH secretion in hypothalamic neuron cells; CAMK2D, which promotes the FSHβ and LHβ secretion in pituitary cells; and OSTN, which promotes granulosa cell proliferation and the synthesis of sex steroid hormones. We reveal key endocrine factors involving egg production by inter-tissue crosstalk analysis, and demonstrate that both a hepatokine, APOA4, and an adipokine, ANGPTL2, could increase egg production by inter-tissue communication with hypothalamic-pituitary-ovarian axis. Together, These results reveal the molecular mechanisms of multi-tissue coordinative regulation of chicken egg-laying performance and provide key insights to avian reproductive regulation.

Preliminary construction of non-coding RNAs and ceRNA regulatory networks mediated by exosomes in porcine follicular fluid

BackgroundFollicles are fundamental units of the ovary, regulated intricately during development. Exosomes and ovarian granulosa cells (OGCs) play pivotal roles in follicular development, yet the regulatory mechanisms governing exosomes remain elusive. ResultsHigh-throughput sequencing was employed to evaluate the complete transcript expression profiles of six samples (three porcine ovarian granulosa cells-exosome co-culture samples (GCE) and three porcine ovarian granulosa cells (POGCs) samples). Differential expression analysis revealed 924 lncRNAs, 35 circRNAs, 49 miRNAs, and 9823 mRNAs in the GCE group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated enrichment of differentially expressed transcripts in pathways related to cell proliferation and apoptosis. Furthermore, a ceRNA regulatory network comprising 43 lncRNAs, 6 circRNAs, 11 miRNAs, and 126 mRNAs was constructed based on intergene co-expression correlations. Seven miRNAs associated with cell proliferation and apoptosis regulation were identified within this network, encompassing 92 subnet pairs as candidate genes for further exploration of exosome regulatory mechanisms. Additionally, preliminary verification at the cellular level demonstrated that exosomal miR-200b enhances the viability of POGCs. ConclusionsTranscriptome analysis unveiled a pivotal candidate ceRNA network potentially implicated in exosome-mediated regulation of granulosa cell proliferation and apoptosis, thereby influencing porcine follicular development. These findings offer insights into the molecular mechanisms of follicular fluid exosome regulation, encompassing both coding and non-coding RNA perspectives.

Influence of O-GlcNAcylation on KGN cell function

O-GlcNAcylation is a unique form of post-translational glycosylation that affects a variety of cytoplasmic and nuclear proteins of cells. Aberrant O-GlcNAcylation is characteristic of many cancers, and impacts cell proliferation, tumorigenicity and metabolism. O-GlcNAcylation occurs in granulosa cells of ovarian follicles, its expression differs between small (3-5 mm) and large (>8.5 mm) antral follicles, and its manipulation in vitro alters granulosa cell proliferation and metabolism. Here, the aim was to assess whether O-GlcNAcylation similarly occurs in cells from a type of granulosa cell tumor, specifically KGN cells, knowing these cells share functional features of granulosa cells of mature, preovulatory follicles (e.g., FSH-responsiveness and estradiol production). The immortal KGN cell line was utilized to conduct cell culture experiments for the detection and manipulation of O-GlcNAcylation. The cells were grown to confluency in serum containing medium and then sub-cultured in serum-free conditions for immunodetection of O-GlcNAcylation (n = 8 expts.), for cell proliferation (n = 3 expts) and for metabolism assays (n = 12 expts.). The KGN cells were also treated without or with small molecule inhibitors to directly enhance or impair O-GlcNAcylation. Immunoblotting confirmed O-GlcNAc expression in KGN cells, as well as the efficacy of Thiamet-G and OSMI-1 to augment (P < 0.05) and inhibit O-GlcNAcylation (P < 0.05), respectively. Only the inhibition of O-GlcNAcylation compromised KGN cell proliferation (P < 0.05), resulting in a 25 % reduction in proliferation compared to control conditions over a 72 h culture period. Seahorse XFe96 analysis measured effects of O-GlcNAcylation on cellular respiration in the KGN cells. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) provided indices of glycolysis and oxidative phosphorylation, respectively. During a glycolysis stress test, high glucose increased ECAR and decreased OCR (P < 0.05); oligomycin did not further affect ECAR (P > 0.05), but impaired OCR (P < 0.05); and 2-deoxy-d-glucose decreased ECAR (P < 0.05) without affecting OCR (P > 0.05). Comparatively, a mitochondrial stress test revealed oligomycin increased ECAR (P < 0.05) with a compensatory decrease in OCR (P < 0.05); FCCP increased both ECAR and OCR (P < 0.05); and rotenone + antimycin A decreased both ECAR and OCR (P < 0.05). Manipulation of O-GlcNAcylation in the KGN cells had no effect on ECAR (P > 0.05), but inhibited OCR (P < 0.05). Collectively, the results indicate O-GlcNAcylation occurs in KGN cells, its inhibition impairs cell proliferation, and while KGN cells rely upon both glycolysis and oxidative phosphorylation for cellular respiration, manipulation of O-GlcNAcylation acutely perturbs only oxidative phosphorylation, an effect observed previously with granulosa cells of large antral follicles.

Investigation of Fructus Ligustri Lucidi for Premature Ovarian Insufficiency based on a Network Pharmacology and Experiment Verifications.

Premature ovarian insufficiency [POI] is a disease characterized by a premature decline in ovarian function before the age of 40. In China, Ligustrum lucidum [FLL] has long been used to improve ovarian function and treat POI. This study aims to verify the effect of FLL on POI through network pharmacology, molecular docking, and in-vitro cell experiments. A total of 13 active substances were screened in FLL, including including quercetin, taxifolin, luteolin, kaempferol, and beta-sitosterol. Then, network analysis found that FLL may exert effects on POI through 10 targets, including AR, ESR1, ESR2, KDR, CYP19A1, CLPP, GC, MMP3, PPARG, and STS. According to GO and KEGG enrichment analysis, FLL is associated with mechanisms related to estrogen, including steroid hormone biosynthesis, ovarian steroidogenesis, and the estrogen signaling pathway. Molecular docking confirms the interaction between the active ingredients of FLL and CYP19A1, which encodes aromatase. CCK8 experiment confirmed that quercetin and taxifolin can enhance the proliferation of KGN granulosa cells, while quercetin, taxifolin, and kaempferol can inhibit the apoptosis of KGN granulosa cells. ELISA experiments have confirmed that quercetin, taxifolin, luteolin, and kaempferol can increase the synthesis of estradiol in KGN granulosa cells. WB confirms that quercetin can increase the expression level of CYP19A1 in KGN cells. FLL can improve the proliferation, apoptosis, and synthesis of estradiol in ovarian granulosa cells, and has the potential to treat POI.

Chronic stress causes ovarian fibrosis to impair female fertility in mice

ObjectiveChronic psychological stress is associated with impaired follicular development and ovarian dysfunction. Many aspects of this dysfunction and the underlying mechanisms remain unclear. Using a chronic unpredictable mild stress (CUMS) mouse model, we investigate the influence of chronic stress on ovarian function and explore potential mechanisms. MethodsA CUMS mouse model was constructed over eight months, covering the period from sexual maturity to the onset of declining fertility in mice. At the end of the 2nd, 4th, 6th, and 8th months of exposure to CUMS, behavioral and physiological assays, including the sucrose preference test, tail suspension test, and serum corticosterone levels, were conducted to validate the effectiveness of the stress model. Fertility and ovarian function were assessed by analyzing the estrous cycle, number of offspring, sex hormone levels, follicle counts, granulosa cell proliferation and apoptosis, and the expression levels of fibrosis markers. Furthermore, proteomic analyses were performed on the ovaries to investigate the molecular mechanisms of ovarian fibrosis induced by CUMS. ResultsWith continued CUMS exposure, there was a gradual decline in both the ovary-to-body weight ratio and the number of offspring. Moreover, the percentage of atretic follicles was notably higher in the CUMS-exposed groups compared to the control groups. It is noticeable that CUMS triggered granulosa cell apoptosis and halted proliferation. Additionally, increased expression of α-SMA and Collagen I in the ovaries of CUMS-exposed mice indicated that CUMS could induce ovarian fibrosis. Proteomic analysis provided insights into the activation of specific biological processes and molecules associated with fibrosis induced by chronic stress. ConclusionsOur results strongly suggest that exposure to CUMS induces ovarian fibrosis, which influences follicular development and ultimately contributes to fertility decline. These findings offer novel perspectives on the impact of chronic stress on ovarian dysfunction.

Paeoniflorin Promotes Ovarian Development in Mice by Activating Mitophagy and Preventing Oxidative Stress.

During the development of animal organs, various adverse stimuli or toxic environments can induce oxidative stress and delay ovarian development. Paeoniflorin (PF), the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., has protective effects on various diseases by preventing oxidative stress. However, the mechanism by which PF attenuates oxidative damage in mouse ovaries remains unclear. We evaluated the protective effects of PF on ovaries in an H2O2-induced mouse oxidative stress model. The H2O2-induced mouse ovarian oxidative stress model was used to explore the protective effect of PF on ovarian development. Histology and follicular development were observed. We then detected related indicators of cell apoptosis, oxidative stress, and autophagy in mouse ovaries. We found that PF inhibited H2O2-induced ovarian cell apoptosis and ferroptosis and promoted granulosa cell proliferation. PF prevented oxidative stress by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels. In addition, the autophagic flux of ovarian cells was activated and was accompanied by increased lysosomal biogenesis. Moreover, PF-mediated autophagy was involved in clearing mitochondria damaged by H2O2. Importantly, PF administration significantly increased the number of primordial follicles, primary follicles, secondary follicles, and antral follicles. PF administration improved ovarian sizes compared with the H2O2 group. The present study suggested that PF administration reversed H2O2-induced ovarian developmental delay and promoted follicle development. PF-activated mitophagy is crucial for preventing oxidative stress and improving mitochondrial quality.

Plasma-derived exosome miR-10a-5p promotes premature ovarian failure by target BDNF via the TrkB/Akt/mTOR signaling pathway

Premature ovarian failure (POF) is characterized by a significant decline in the ovarian follicle pool and oocyte reserve, alongside an increase in the number of low-quality oocytes and apoptosis of granulosa cells (GCs). Exosome-derived miRNA plays a regulatory role in crucial cellular activities and contributes to the onset and progression of POF. In this study, we successfully established a rabbit model of POF and conducted in vitro and in vivo experiments that confirmed DiI-labeled Pla-Exos (exosomes derived from plasma) could enter the follicle through blood circulation, with GCs capable of uptaking these exosomes. Our RNA-seq analysis revealed elevated expression of miR-10a-5p in Pla-Exos from POF rabbits. Moreover, our findings demonstrate that exosomal miR-10a-5p suppresses GCs proliferation and induces apoptosis via the mitochondrial pathway. Additionally, exosomal miR-10a-5p inhibits the TrkB/Akt/mTOR signaling pathway by downregulating BDNF expression, thereby modulating the expression levels of proteins and genes associated with the cell cycle, follicle development, and GCs senescence. In conclusion, our study highlights the role of Pla-Exos miR-10a-5p in promoting rabbit POF through the TrkB/Akt/mTOR signaling pathway by targeting BDNF. These findings provide new insights into potential therapeutic targets for POF, offering valuable references for addressing concerns related to female reproductive function.

Effects and action mechanism of gonadotropins on ovarian follicular cells: A novel role of Sphingosine-1-Phosphate (S1P). A review

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control antral follicular growth by regulating several processes, such as the synthesis of hormones and signaling molecules, proliferation, survival, apoptosis, luteinization, and ovulation. To exert these effects, gonadotropins bind to their respective Gs protein-coupled receptors, activating the protein kinase A (PKA) pathway or recruiting Gq proteins to activate protein kinase C (PKC) signaling. Although the action mechanism of FSH and LH is clear, recently, it has been shown that both gonadotropins promote the synthesis of sphingosine-1-phosphate (S1P) in granulosa and theca cells through the activation of sphingosine kinase 1. Moreover, the inhibition of SPHKs reduces S1P synthesis, cell viability, and the proliferation of follicular cells in response to gonadotropins, and the addition of S1P to the culture medium increases the proliferation of granulosa and theca cells without apparent effects on sexual steroid synthesis. Therefore, we consider that S1P is a crucial signaling molecule that complements the canonical gonadotropin pathway to promote the proliferation and viability of granulosa and theca cells.

The inhibition roles of RAB23 gene in granulosa cell proliferation and progesterone synthesis of hen ovarian prehierarchical follicles

ABSTRACT 1. The proliferation of granulosa cells is vital for the development and recruitment of hen ovarian prehierarchical follicles (PF). The RAB23 protein is a member of the Rab family, belonging to the GTPase family. This study studied the regulatory roles of the RAB23 gene in PF. 2. The expression of RAB23 was significantly increased in granulosa cells (GC) during PF growth and was highest in GC at 6–8 mm diameter (p < 0.05). The RAB23 protein was mainly expressed in the GC, oocytes (OC) as well as somatic cells (SC) of the PF. 3. The mRNA expression of FSHR, CCND1,CYP11A1, StAR and HSD3B1 was significantly increased in the siRNA RAB23 group (p < 0.05). Additionally, protein expression of FSHR, CCND1, CYP11A1, HSD3B1 was significantly increased (p < 0.05) after GC were transfected with RAB23-specific siRNA. Protein expression of StAR in the siRNA RAB23 group was numerically higher than that in the positive control (PC) and negative control (NC) groups. The GC proliferation rate and progesterone synthesis of the prehierarchical follicles in hen ovaries were markedly increased in vitro (p < 0.05). 4.This study revealed that RAB23 might play an inhibitory role in GC proliferation and progesterone synthesis during the prehierarchical follicles development in vitro.

Flavonoid Supplementation Is Beneficial for Polycystic Ovary Syndrome: A Systematic Review and Meta-analysis.

Polycystic ovary syndrome (PCOS) is a prevalent hormonal imbalance that predominantly affects women in their reproductive years. Previous studies have yielded conflicting conclusions. This is an updated meta-analysis aiming to explore the connection between flavonoid supplementation and PCOS. Seven databases were searched: Cochrane Library, PubMed, Web of Science, Embase, Wanfang, China Science and Technology Journal, and China National Knowledge Infrastructure, spanning from their inception to April 15, 2024. Two authors independently searched the databases using the search terms. Following strict inclusion criteria, 8 papers were ultimately included. This updated meta-analysis suggests that flavonoid supplementation could enhance follicular development, promote the proliferation and differentiation of follicular granulosa cells, elevate estradiol levels, and mitigate testosterone, C-reactive protein, and ovarian index levels. This analysis suggests that dietary flavonoids could potentially alleviate symptoms associated with PCOS. PROSPERO registration no. CRD42022382912.

Circ_0043314 Modulates Proliferation and Apoptosis of Ovarian Granulosa Cells in Polycystic Ovarian Syndrome via the MicroRNA-146b-3p/Apelin 13 Axis.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. At present, the pathogenesis has not been clarified, and the clinical application of drugs and lifestyle intervention may not prevent disease progression. This study aimed to investigate how circ_0043314 regulates ovarian granulosa cell biological functions to provide a theoretical basis for the treatment of patients with PCOS. MicroRNA (miR)-146b-3p/Apelin 13 axis was used to investigate the mechanism by which circ_0043314 regulated ovarian granulosa cell proliferation and apoptosis in PCOS via miR-146b-3p/Apelin 13 axis. Participants/Materials, Methods: Ovarian tissues (cortical tissues) from 35 PCOS patients and 35 normal controls, as well as HEK293T and human ovarian granulosa cell line (KGN, COV434), were included in this study. We examined the expression levels of circ_0043314, miR-146b-3p, and Apelin 13 in PCOS tissues. Ovarian granulosa cells were transfected with corresponding plasmids to clarify the influence of circ_0043314, miR-146b-3p, or Apelin 13 on proliferation and apoptosis of ovarian granulosa cells through MTT and flow cytometry assays. Moreover, the relationships among circ_0043314, miR-146b-3p, and Apelin 13 were analyzed through dual-luciferase and RNA immunoprecipitation assays. Circ_0043314 and Apelin 13 were highly expressed and miR-146b-3p was lowly expressed in ovarian tissues of PCOS compared with non-PCOS controls. Downregulation of circ_0043314 or upregulation of miR-146b-3p hindered ovarian granulosa cell proliferation and advanced its apoptosis. Downregulation of miR-146b-3p reversed the impacts of downregulation of circ_0043314, and overexpression of Apelin 13 counteracted the influences of upregulation of miR-146b-3p in ovarian granulosa cells. Mechanically, circ_0043314 could bind to miR-146b-3p, and miR-146b-3p directly targeted and modulated Apelin 13 expression. This study was limited by the lack of animal experiments. Our data demonstrated that circ_0043314 enhances ovarian granulosa cell proliferation and suppresses its apoptosis via miR-146b-3p/Apelin 13 axis.

Neuromedin S regulates goat ovarian granulosa cell proliferation and steroidogenesis via endoplasmic reticulum Ca2+-YAP1-ATF4-c-Jun pathway.

Neuromedin S (NMS) plays key roles in reproductive regulation, while its function and mechanism in follicular development remain unclear. The current study aims to investigate the specific role and mechanisms of NMS and its receptors in regulating the proliferation and steroidogenesis of ovarian granulosa cells (GCs). Phenotypically, a certain concentration of NMS addition promoted the proliferation and estrogen production of goat GCs, accompanied by an increase in the G1/S cell population and upregulation of the expression levels of cyclin D1, cyclin dependent kinase 6, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450, family 11, subfamily A, polypeptide 1, while the effects of NMS treatment were effectively hindered by knockdown of neuromedin U receptor type 2 (NMUR2). Mechanistically, activation of NMUR2 with NMS maintained endoplasmic reticulum (ER) calcium (Ca2+) homeostasis by triggering the PLCG1-IP3R pathway, which helped preserve ER morphology, sustained an appropriate level of endoplasmic reticulum unfoldedprotein response (UPRer), and suppressed the nuclear translocation of activating transcription factor 4. Moreover, NMS maintained intracellular Ca2+ homeostasis to activate the calmodulin 1-large tumor suppressor kinase 1pathway, ultimately orchestrating the regulation of goat GC proliferation and estrogen production through the Yes1 associated transcriptional regulator-ATF4-c-Jun pathway. Crucially, the effects of NMS were mitigated by concurrent knockdown of the NMUR2 gene. Collectively, these data suggest that activation of NMUR2 by NMS enhances cell proliferation and estrogen production in goat GCs through modulating the ER and intracellular Ca2+ homeostasis, leading to activation of the YAP1-ATF4-c-Jun pathway. These findings offer valuable insights into the regulatory mechanisms involved in follicular growth and development, providing a novel perspective for future research.

Interaction of the transforming growth factor-β and Notch signaling pathways in the regulation of granulosa cell proliferation.

The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

P-640 Dynamics of IGF signalling during the ovulatory peak in women undergoing ovarian stimulation

Abstract Study question How is the IGF system expressed and regulated during the midcycle surge in women? Summary answer The IGF system becomes downregulated after ovulation trigger (OT) highlighted by a significant upregulation of the inhibitor, stanniocalcin-1, and a downregulation of protease-induced IGF release. What is known already IGF signalling is known to affect human ovarian follicular function during growth and development. Particularly during the preovulatory stage, IGF activity appears to be highly significant and has been proposed as one of the primary factors contributing to the remarkable mitotic activity observed in the granulosa cells (GCs). However, the role and regulation of the IGF system is unknown during the ovulatory peak, which is characterized by profound changes in GC function. Study design, size, duration Follicular fluid (FF) and GCs were collected from fifty women during the ovulatory peak from two specific time-points. One sample was obtained before oocyte pick up (OPU): before OT (T = 0 h (n = 23)), at 12 h (n = 10), 17 h (n = 6), or 32 h (n = 11) after OT, and one sample was obtained at OPU 36 h after OT (n = 50). Participants/materials, setting, methods Fifty women receiving fertility treatment at the Fertility Clinic, Department of Gynaecology and Obstetrics, Holbæk Hospital, Denmark, were included. For ethical reasons, only women who had developed more than eight mature follicles at their final control visit before OT were approached and asked for possible participation. Gene expression profiles were assessed by microarray analysis of GCs. Intrafollicular levels of IGF-related proteins were assessed with immunoassays and a proteinase assay was used to quantify activity. Main results and the role of chance This study presents a novel insight into the dynamics of IGF bioavailability within human ovarian follicles during the midcycle surge of gonadotropins. Notably, this investigation reports a significant downregulation of genes promoting IGF signalling, i.e., IGF-2, PAPPA, and IRS1, along with a concurrent upregulation of genes associated with IGF binding and inactivation, including IGFBPs, IGF2R, and STC1. Particularly, the substantial increase in the inhibitor, stanniocalcin-1 (STC1), at both gene and protein levels likely plays a key role in the downregulation of IGF signalling. Additionally, this study reveals a marked reduction in intrafollicular PAPP-A proteolytic activity after OT, further emphasizing the importance of PAPP-A within human ovaries. In addition, this study indicates a pronounced decline in GC mitotic activity during the midcycle surge, as evidenced by the downregulation of proliferation markers, including MKI67, E2F1, E2F8, and FOXM1, highlighting a substantial decrease in GC proliferation rate during this critical phase. Previous research has demonstrated that IGF signalling stimulates the proliferation and differentiation of cultured human GCs. Therefore, the significant changes in IGF bioavailability observed in human GCs during the ovulatory surge suggest an essential role of IGF signalling in controlling the proliferation and differentiation of GCs within the preovulatory follicle. Limitations, reasons for caution This study highlights the need for future functional studies to confirm a direct mechanism to account for the abrupt cessation of GC proliferation. Differences between using hCG or GnRH agonist for final maturation of follicles were not shown, thus future studies should be powered to reveal effects of the protocols. Wider implications of the findings These data suggest that downregulation of IGF signalling mediated by increased STC1 expression is instrumental for the sudden cessation in GC proliferation and onset of differentiation during the ovulatory peak. Furthermore, this study implies importance of IGF-independent functions of STC1, especially during the ovulatory peak, where elevated levels are observed. Trial registration number Not applicable

P-649 Unconventional action of Luteinizing Hormone

Abstract Study question Has Luteinizing Hormone (LH) a role in the activation/survival of follicles of the primary ovarian reserve? Summary answer LH promotes survival and perhaps activation/growth of primordial and primary follicles, proliferation of granulosa cells of secondary follicles, reduces follicle atresia at later growing stages. What is known already In the mammalian ovary, Primordial Follicles (PMFs) constitutes the primary ovarian reserve, which declines over time due to physiological events such as atresia, recruitment, and ovulation. While in the adult, the role of LH in steroidogenesis and ovulation is well established, recent work from our laboratory has shown that under chemotherapeutic treatments, LH supports PMFs survival in prepuberal mouse ovary. Study design, size, duration LH effects on follicle dynamics and some activities of pre-granulosa cells were investigated in vitro and in vivo. In vitroexperiments were performed on PD4 GFP/Kit mice ovarian fragments and on PD8 CD1 mice ovarian somatic cells cultured up to six days in continuous presence of 0.2, 0.5, 1IU LH. In vivo experiments were performed on 9 PD4 and 8 PD28 CD1 mice receiving an intra-peritoneal injection for eight consecutive days of 1-5IU LH. Participants/materials, setting, methods Number and diameters of the GFP stained oocyte were counted in the ovarian fragments. Kit-Ligand and LH-R mRNA expression levels were analyzed by qPCR in pre-granulosa cells. For in vivo experiments, ovarian volume was measured by Micro-Ct and follicle counts were performed on histological sections. Estrous cycle phases were monitored by vaginal smear during the 8 days of treatment. Statistics was performed using Student’s t-test. Main results and the role of chance In fragments of PD4 mouse ovaries cultured in vitro for six days, 1IU LH significantly increased the number of oocytes with a diameter &amp;gt;40 mm (CTRL 17.7% vs LH 36.07%, p &amp;lt; 0,05), suggesting activation of PMFs. Moreover, the hormone increased the expression of mRNA for LHR and KL in the somatic ovarian cells and promoted their proliferation in vitro. The effects of 1IU LH on PD4 females can be summarized as follows: 1. Ovary volume increase (CTRL 0.3 mm3 vs LH 0.36 mm3, p = 0.07), 2. higher percent of PMFs (CTRL 68.227% vs LH 78.29%, p &amp;lt; 0,05) associated with lower percent of primary (CTRL 7.2% vs LH 4.05%, p &amp;lt; 0.04) and growing follicles (CTRL 14.18% vs LH 8.56%, p = 0.1), 3. higher number of granulosa cell layers (&amp;gt; three layers) in secondary follicles (CTRL 42.8% vs LH 54.6%), and 4. lower percent of atretic follicles (CTRL 2.64% vs LH 1.72%, p = 0.1). Similar results were obtained on 28 dpp mice. Morever, we observed that corpora lutea were present only in ovaries of LH-treated females and that all such females were in estrus. Limitations, reasons for caution The study was conducted only in mice. Wider implications of the findings These studies have highlighted new possible roles of LH on follicle classes (primordial/primary) whose development is considered gonadotropin independent. The results, if confirmed in humans, imply that LH might be used as a treatment for patients with ovarian disorders involved the survival/dynamics of the ovarian reserve. Trial registration number NA