Proliferation Of Ovarian Granulosa Cells Research Articles

P-591 Follicular fluid-derived exosomes rejuvenate ovarian aging through miR-320a-3p-mediated FOXQ1 inhibition

Abstract Study question Dose the follicular fluid-derived exosomes rejuvenate ovarian aging? Summary answer Follicular fluid-derived exosomes efficiently rescued ovarian function in aged mice, which was achieved via transfer of miR-320-3p to granulosa cells and subsequent Foxq1 inhibition. What is known already The ovary enters the malfunctional aging phase earlier and faster than other organs and systems. Ovarian aging is mainly characterized by a progressive decline in oocyte quantity and quality, which ultimately leads to female infertility. Various therapies have been established to cope with age-related ovarian dysfunction, among which exosome-based therapy has been considered a promising strategy that can benefit ovarian functions via multiple pathways. Study design, size, duration Ovarian granulosa cells of young C57BL/6J mice (6-8 w) and old C57BL/6J mice (40 w) were cultured in vitro. The follicular fluid exosomes of young mice and the same amount of PBS were respectively injected into the ovarian sac of old mice. Participants/materials, setting, methods Cell proliferation and apoptosis were detected by CCK8, flow cytometry and Western Blot. Mitochondrial function was assessed by measuring mitochondrial membrane potential, ATP levels, mtDNA copy numbers. The number of ovarian follicles were observed by HE staining. Senescence and telomere related gene expression levels were detected by RT-qPCR. 2-cell rates and blastocyst formation rates were observed after in vitro fertilization. The model mice were directly cooped up to observe the pregnat and litter sizes. Main results and the role of chance The follicular fluid exosomes from young mice can promote the proliferation of ovarian granulosa cells from old mice and improve mitochondrial function. When the follicular fluid exosomes from young mice were injected into the ovarian sac of old mice, the number of primordial and growing follicles in the ovaries of old mice were increased and the developmental ability of oocytes was improved. Limitations, reasons for caution Findings in mouse. Wider implications of the findings This study suggests that young follicular fluid exosomes may be involved in ovarian senescence by transferring miR-320a-3p targeting FOXQ1 into ovarian granulosa cells of aged mice. Trial registration number no

Treatment of polycystic ovary syndrome and its associated psychiatric symptoms with the Mongolian medicine Nuangong Qiwei Pill and macelignan

Ethnopharmacological relevanceThe Mongolian medicine Nuangong Qiwei Pill (NGQW) is a folk prescription with a long history of use by the Mongolian people. NGQW comprises seven Mongolian medicines, which have the effects of regulating and nourishing blood, warming the uterus, dispelling cold and relieving pain. For a long time, it has been used as a good remedy for gynecological diseases, with remarkable curative effects, favored by the majority of patients and recommended by doctors. Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disorder that can lead to menstrual disorders or infertility. In the gynecological classification of Mongolian medicine, polycystic ovary syndrome has not been distinguished in detail, and the mechanism of NGQW in the treatment of polycystic ovary syndrome has not been scientifically studied and standardized. Aim of the studyThe aim of this study was to clarify the mechanism of action of NGQW and macelignan in the treatment of PCOS and to provide a reference for the clinical application of these drugs. Materials and methodsThe effect of intragastric administration of NGQW and macelignan on PCOS model mice was observed. The mental status of mice was examined behaviorally, and serum hormone levels and oxidative stress parameters were measured by ELISA. Giemsa staining was used to detect the reproductive cycle, and HE staining was used to observe the ovarian status. Immunofluorescence staining was performed to observe the proliferation and apoptosis of ovarian granulosa cells. qRT‒PCR was conducted to measure the expression of IL-6, BAX, BCL-2, and estrogen synthesis-related genes in ovarian tissue and particle cells. ResultsIn the dehydroepiandrosterone (DHEA)-induced PCOS model mice, both NGQW and macelignan improved the estrous cycle; increased the estradiol (E2) content; lowered testosterone (T), progesterone (P) and luteinizing hormone (LH) levels; reduced the number of polycystic follicles; promoted granulosa cell proliferation; reduced granulosa cell apoptosis; and alleviated depression and anxiety. In addition, Nuangong Qiwei Pill and macelignan reduced the mRNA levels of the ovarian inflammatory factor IL-6; improved the disordered levels of the antioxidant indicators GSH, MDA, and SOD; and activated the TGF-β3 signaling pathway to increase the transcription of Cyp19a1, which increases estrogen secretion. ConclusionNGQW and macelignan can treat PCOS through the TGF-β3/Smad/Cyp19a1 signaling pathway to regulate the secretion ability of ovarian granulosa cells. Our research justifies the traditional use of NGQW to treat PCOS and enriches the scope of action of macelignan.

Retinoic acid enhances ovarian steroidogenesis by regulating granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway

IntroductionOvarian steroidogenesis not only affects the embryonic development and pregnancy outcome, but also associates with many diseases in mammals and women. Exploring the nutrients and mechanisms influencing ovarian steroidogenesis is critical to maintaining the optimal reproductive performance, as well as guaranteeing body health. ObjectivesThis research aimed to explore the effect of retinol metabolism on ovarian steroidogenesis and the underlying mechanisms. MethodsComparative transcriptomic analysis of ovaries from normal and low reproductive performance sows were performed to identify the main causes leading to low fertility. The metabolites regulating steroid hormones synthesis were investigated in ovarian granulosa cells. Gene interference, overexpression, dual-luciferase reporter assays, chromatin immunoprecipitation and transcriptome analysis were further conducted to explore the underlying mechanisms of Aldh1a1 mediating ovarian steroidogenesis. ResultsTranscriptome analysis of ovaries from normal and low reproductive performance sows showed the significant differences in both retinol metabolism and steroid hormones synthesis, indicating retinol metabolism probably influenced steroid hormones synthesis. The related metabolite retinoic acid was furtherly proven a highly active and potent substance strengthening estrogen and progesterone synthesis in ovarian granulosa cells. For the first time, we revealed that retinoic acid synthesis in porcine and human ovarian granulosa cells was dominated by Aldh1a1, and required the assistance of Aldh1a2. Importantly, we demonstrated that Aldh1a1 enhanced the proliferation of ovarian granulosa cells by activating PI3K-Akt-hedgehog signaling pathways. In addition, Aldh1a1 regulated the expression of transcription factor MESP2, which targeted the transcription of Star and Cyp11a1 through binding to corresponding promoter regions. ConclusionOur data identified Aldh1a1 modulates ovarian steroidogenesis through enhancing granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway. These findings provide valuable clues for improving ovarian health in mammals.

LncRNA PWRN2 promotes polycystic ovary syndrome progression via epigenetically reducing ATRX by recruiting LSD1

Long non-coding RNA has been shown to mediate the progression of polycystic ovary syndrome (PCOS). However, the role and mechanism of Prader-Willi region nonprotein coding RNA 2 (PWRN2) in PCOS progression remain unclear. In our study, Sprague-Dawley rat was injected with dehydroepiandrosterone to mimic PCOS rat models. HE staining was used to assess the number of benign granular cells, and serum insulin and hormone levels were detected by ELISA kit. The expression of PWRN2 was examined by qRT-PCR. Ovarian granulosa cells (GCs) proliferation and apoptosis were examined by CCK-8 assay and flow cytometry. The protein levels of apoptosis markers and Alpha thalassemia retardation syndrome X-linked (ATRX) were determined by western blot. The interaction between lysine-specific demethylase 1 (LSD1) and PWRN2 or ATRX was confirmed by RIP and ChIP assay. Our data showed that PWRN2 was upregulated and ATRX was downregulated in the ovarium tissues and serum of PCOS rat. PWRN2 knockdown promoted GCs proliferation and inhibited apoptosis. In the mechanism, PWRN2 inhibited ATRX transcription by binding with LSD1. In addition, downregulation of ATRX also eliminated the effect of sh-PWRN2 on GCs growth. In conclusion, our data suggested that PWRN2 might restrain GCs growth to promote PCOS progression, which was achieved by binding with LSD1 to inhibit ATRX transcription.

CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9

BackgroundClock circadian regulator (CLOCK) is a core factor of the mammalian biological clock system in regulating female fertility and ovarian physiology. However, CLOCK's specific function and molecular mechanism in porcine granulosa cells (GCs) remain unclear. In this study, we focused on CLOCK’s effects on GC proliferation.ResultsCLOCK significantly inhibited cell proliferation in porcine GCs. CLOCK decreased the expression of cell cycle-related genes, including CCNB1, CCNE1, and CDK4 at the mRNA and protein levels. CDKN1A levels were upregulated by CLOCK. ASB9 is a newly-identified target of CLOCK that inhibits GC proliferation; CLOCK binds to the E-box element in the ASB9 promoter.ConclusionsThese findings suggest that CLOCK inhibits the proliferation of porcine ovarian GCs by increasing ASB9 level.

Effects of feeding exogenous vitamins and microelements on follicular development and molecular function of granulosa cells in donkeys

In mammalian ovarian physiology, vitamins and microelements are essential for follicular development, ovulation, and the synthesis and secretion of hormones and growth factors. However. the molecular mechanisms by which they influence maturation and growth of follicles in donkeys are still unknown. Here, we investigated how vitamins and microelements affected the development of follicles and the proliferation of ovarian granulosa cells (GCs). For this experiment, 40 jennies (average body weight: 292.946±33.543 kg, age: 3∼4 years) were randomly assigned into 4 groups (n=10) and fed 50% (A), 75% (B), 100% (C), and 150% (D) of a mixture of vitamins A, D and E and microelements once a day (dose 5 ml) for two consecutive months. After that, ultrasonography was applied to record the change in follicular dynamics in three estrous cycles. Using a transvaginal ultrasound-guided aspiration device to obtain follicular fluid (containing GCs) from the follicles (Ф=30mm-35mm), multi-omics analysis revealed its molecular mechanisms and physiological functions. The results showed that the time required for growing follicles to reach ovulation (Ф>40mm) was shortened in the vitamin D group compared to the three other groups(P<0.05). Of note, the follicles (Ф=30mm-35mm) in the vitamin C group had less discrete diameters and developed more evenly and had significantly more twin ovulations than the other three groups (P<0.05). The omics analysis showed that945 metabolites, including organic acids and derivatives (24.44%), lipids and lipid-like molecules (24.34%), organic heterocyclic compounds (13.44%), and benzenoids (12.06%) were identified in follicular fluid. The results also revealed 232, 207, 210 and 202 differential cumulative metabolites (DAMs) in group A vs. group B, group B vs. group C, group C vs. group D and group A vs. group C, respectively. The DAMs were most significantly enriched in protein digestion and absorption, ABC transporters, amino acid biosynthesis, mineral absorption, aspartate-arginine biosynthesis, and ovarian steroid synthesis process. Meanwhile, nine DEGs (INSR, CYP19A1, ALOX5, PLA2G4A, ADCY6, CYP1B1, PRKACB, CYP17A1, and PLA2G4F) associated with the secretory function of GCs were involved in the ovarian steroidogenesis pathway, and they were significantly correlated with estradiol (E2), cholesterol, progesterone (P4), and testosterone (T)(P<0.05). Furthermore, STEAP2, CYBRD1, ATP1B3, ATP1A1, SLC26A6, SLC5A1, SLC6A19, SLC40A1, and TRPV6 were closely associated with the mineral absorption of GCs, and they had a positive correlation with glycine and L-methionine (P<0.05). In conclusion, exogenous supplementation of jennies with vitamins A, D and E and micronutrients can contribute to follicular growth and ovulation and this was associated with the interaction and expression of key genes related to -metabolites in ovarian steroidogenesis and mineral absorption pathways.

Bisphenol A interferes with lncRNA Fhadlos2 and RUNX3 association in adolescent mouse ovary

Bisphenol A (BPA) has a number of adverse effects on the reproductive development of females. In particular, the mechanism of disruption of ovarian development in adolescent mice is still unclear. Based on transcriptome sequencing results, a differentially expressed lncRNA, Fhad1os2, was detected in the ovaries of BPA-exposed pubertal mice. In our study, the lncRNA Fhad1os2, localized in the ovarian granulosa cell cytoplasm, could regulate the proliferation of mouse ovarian granulosa cells. Mechanistically, the results of RNA pull-down experiments as well as mass spectrometry analysis showed that ERα, an interfering signaling molecule of BPA, could directly bind lncRNA Fhad1os2 and decrease the transcription of lncRNA Fhad1os2 in response to the estrogen-like effect of BPA. BPA exposure also caused abnormal lncRNA Fhad1os2 pulldown protein-related signaling pathways in the ovaries of adolescent mice. Furthermore, lncRNA Fhad1os2 interacted with RUNX3, a transcription factor related to follicle development and hormone synthesis. As a negative regulator, lncRNA Fhad1os2 transactivated the expression of Runx3, which in turn induced RUNX3 to positively regulate aromatase (Cyp19a1) expression in mouse ovarian granulosa cells and promote estrogen synthesis. In conclusion, our study indicates that BPA exposure interferes with ERα-regulated lncRNA Fhad1os2 interactions with RUNX3 in pubertal mice, affecting estrogen synthesis in mouse granulosa cells and contributing to premature ovarian maturation in pubertal mice.

MicroRNA-646 inhibits the proliferation of ovarian granulosa cells via insulin-like growth factor 1 (IGF-1) in polycystic ovarian syndrome (PCOS).

Polycystic ovarian syndrome (PCOS) is a common endocrinopathy in women. MicroRNAs (miRNAs) have been proven to play a crucial role in balancing the proliferation and apoptosis of granulosa cells (GCs) in PCOS. The miRNA of PCOS was screened by bioinformatics analysis, and microRNA 646 (miR-646) was found to be involved in insulin-related pathways by enrichment analysis. The cell counting kit-8 (CCK-8), cell colony formation, and the 5-ethynyl-2'-deoxyuridine (EdU) assays were used to explore the effect of miR-646 on proliferation of GCs, flow cytometry was used to measure the cell cycle and apoptosis, and Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to explore the biological mechanism of miR-646. The human ovarian granulosa cells KGN were selected by measuring the miR-646 and via insulin-like growth factor 1 (IGF-1) levels and used for cell transfection. Overexpressed miR-646 inhibited KGN cell proliferation, and silenced miR-646 advanced it. Most cells were arrested in the S phase of cell cycle with overexpressed-miR-646, while after silencing miR-646, cells were arrested in the G2/M phase. And the miR-646 mimic raised apoptosis in KGN cells. Also, a dual-luciferase reporter proved the regulation effect of miR-646 on IGF-1, miR-646 mimic inhibited IGF-1, and miR-646 inhibitor advanced IGF-1. The cyclin D1, cyclin-dependent kinase 2 (CDK2), and B-cell CLL/lymphoma 2 (Bcl-2) levels were inhibited with overexpressed-miR-646, while silenced-miR-646 promoted their expression, and the bcl-2-like protein 4 (Bax) level was the opposite. This study found that silenced-IGF1 antagonized the promotive effect of the miR-646 inhibitor on cell proliferation. MiR-646 inhibitor treatment can promote the proliferation of GCs by regulating the cell cycle and inhibiting apoptosis, while silenced-IGF-1 antagonizes it.

N-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n-3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n-3 PUFA addition and showed that n-3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n-3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n-3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n-3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n-3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.

Human Pluripotent Stem Cell-Mesenchymal Stem Cell-Derived Exosomes Promote Ovarian Granulosa Cell Proliferation and Attenuate Cell Apoptosis Induced by Cyclophosphamide in a POI-like Mouse Model.

Premature ovarian insufficiency (POI) is a complex disease which causes amenorrhea, hypergonadotropism and infertility in patients no more than 40 years old. Recently, several studies have reported that exosomes have the potential to protect ovarian function using a POI-like mouse model induced by chemotherapy drugs. In this study, the therapeutic potential of exosomes derived from human pluripotent stem cell-mesenchymal stem cells (hiMSC exosomes) was evaluated through a cyclophosphamide (CTX)-induced POI-like mouse model. POI-like pathological changes in mice were determined by serum sex-hormones levels and the available number of ovarian follicles. The expression levels of cellular proliferation proteins and apoptosis-related proteins in mouse ovarian granulosa cells were measured using immunofluorescence, immunohistochemistry and Western blotting. Notably, a positive effect on the preservation of ovarian function was evidenced, since the loss of follicles in the POI-like mouse ovaries was slowed. Additionally, hiMSC exosomes not only restored the levels of serum sex hormones, but also significantly promoted the proliferation of granulosa cells and inhibited cell apoptosis. The current study suggests that the administration of hiMSC exosomes in the ovaries can preserve female-mouse fertility.

Retraction Note: MiR-140 targets RAP2A to enable the proliferation of insulin-treated ovarian granulosa cells

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s13048-020-0611-4.

Effects of N-Acetylcysteine on the Proliferation, Hormone Secretion Level, and Gene Expression Profiles of Goat Ovarian Granulosa Cells.

The purpose of this paper was to investigate the effects of N-acetylcysteine (NAC) on the proliferation, hormone secretion, and mRNA expression profiles of ovarian granulosa cells (GCs) in vitro. A total of 12 ovaries from 6 follicular-stage goats were collected for granulosa cell extraction. The optimum concentration of NAC addition was determined to be 200 μM via the Cell Counting Kit 8 (CCK-8) method. Next, GCs were cultured in a medium supplemented with 200 μM NAC (200 μM NAC group) and 0 μ M NAC (control group) for 48 h. The effects of 200 μM NAC on the proliferation of granulosa cells and hormones were studied by 5-ethynyl-2'-deoxyuridine (EdU) assay and enzyme-linked immunosorbent assay (ELISA). mRNA expression was analyzed by transcriptome sequencing. The results indicate that 200 μM NAC significantly increased cell viability and the proportion of cells in the S phase but promoted hormone secretion to a lesser degree. Overall, 122 differentially expressed genes (DEGs) were identified. A total of 51 upregulated and 71 downregulated genes were included. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the most DEGs were enriched in terms of cell growth regulation, cell growth, neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, the cAMP-signaling pathway, and the Wnt-signaling pathway. Seven genes related to granulosa cell proliferation were screened, IGFBP4, HTRA4, SST, SSTR1, WISP1, DAAM2, and RSPO2. The above results provide molecular theoretical support for NAC as a feed additive to improve follicle development and improve reproductive performance in ewes.

Isorhamnetin protects zearalenone-induced damage via the PI3K/Akt signaling pathway in porcine ovarian granulosa cells.

Zearalenone (ZEA) is widely derived from moldy cereal grain, which has adverse effects on animal reproduction. In particular, pigs are more sensitive to ZEA-induced toxicity than other animals. Isorhamnetin has extensive pharmacological activity. However, the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear. This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism. Two experiments were conducted, where a minimum of 3 biological replicates were used for each treatment. In Exp. 1, ovarian granulosa cells were treated with different concentrations of isorhamnetin (1, 5, 10, 20 and 30μmol/L) and ZEA (0, 10, 30, 60, 90 and 120μmol/L) for 24h. Our results indicated that 60μmol/L ZEA (half-maximal inhibitory concentration value) and 20μmol/L isorhamnetin (the most effective concentration against ZEA-induced cytotoxicity) were optimum concentrations. In Exp. 2, ovarian granulosa cells were treated with isorhamnetin (20μmol/L) for 2h, before treatment with ZEA (60μmol/L) for 24h. Apoptosis, endoplasmic reticulum stress, oxidative stress, proliferation and hormone secretion of ovarian granulosa cells were detected. Our findings showed that isorhamnetin suppressed (P<0.05) ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins (B-cell lymphoma-2 [Bcl-2], Bcl2-associated x [Bax] and cleaved caspase-3 [C-Casp3]). Changes in intracellular Ca2+ levels and C/EBP homologous protein (CHOP), recombinant activating transcription factor 6 (ATF6), glucose regulated protein78 kD (GRP78) indicated that isorhamnetin rescued (P<0.05) ZEA-induced endoplasmic reticulum stress. Furthermore, isorhamnetin prevented (P<0.05) ZEA-induced oxidative stress via the mitogen-activated protein kinase (P38) signaling pathway. Mechanistically, isorhamnetin stimulated (P<0.05) the expression of proliferating cell nuclear antigen (PCNA) and cyclin D, thereby increasing the ratio of S phase cells in response to ZEA-induced apoptosis via phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway. Isorhamnetin also recovered (P<0.05) ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins (follicle-stimulating hormone receptor [FSHR] and cytochrome P450 family 19 subfamily a member 1 [CYP19A1]). Collectively, these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage, which promotes proliferation, alleviates apoptosis, endoplasmic reticulum stress, oxidative stress, and steroidogenesis disorder.

Polymorphism within the GATA binding protein 4 gene is significantly associated with goat litter size

GATA binding protein 4 (GATA4) is a typical transcription binding factor, and its main functions include regulating the proliferation, differentiation and apoptosis of ovarian granulosa cells, promoting spermatogenesis and sex differentiation, implying that this gene have possibly roles in animal reproduction. This study aims to detect five potential insertion/deletions (indels) of the GATA4 gene in 606 healthy unrelated Shaanbei white cashmere (SBWC) goats and analyze its association with the litter size. The electrophoresis and DNA sequencing identified two polymorphic indels (e.g., P4-Del-8bp and P5-Ins-9bp indel). Then T-test analysis showed that P4-Del-8bp was significantly correlated with litter size (p = 0.022) because of two different genotypes detected, e.g., insertion-deletion (ID) and deletion-deletion (DD), and the average litter size of individuals with DD genotype goats was higher than that of others. However, there was no correlation between P5-Ins-9bp and lambing of goats. Chi-square (X 2) test found that the distribution of and P4-Del-8bp genotypes (X 2 = 6.475, p = 0.011) was significantly different between single and multiple-lamb groups, while P5-Ins-9bp (X 2 = 0.030, p = 0.862) was not. Therefore, these findings revealed that P4-Del-8bp polymorphism of goat GATA4 gene was a potential molecular marker significantly associated with litter size, which can be used for the marker-assisted selection (MAS) breeding to improve goat industry.

Fibroblast growth factor 21 (FGF21) promotes porcine granulosa cell estradiol production and proliferation via PI3K/AKT/mTOR signaling

The proliferation and steroidogenesis of mammalian ovarian granulosa cells (GCs) are related to follicular development. Previous studies found that fibroblast growth factor 21 (FGF21) regulated female fertility through the hypothalamic–pituitary–gonad axis. However, FGF21 receptors are expressed on GCs, so we speculate that it might affect female reproduction by regulating their physiological activities. Here, we showed that FGF21, fibroblast growth factor receptor-1(FGFR1), and beta-klotho (KLB) were expressed in porcine GCs. ELISA assays showed that estradiol (E2) production was increased significantly when treating GCs with recombinant FGF21 (rFGF21). In addition, rFGF21 upregulated the mRNA and protein levels of E2 synthesis-related genes including StAR, CYP11A1, and CYP19A1 in porcine GCs. Correspondingly, FGF21 siRNA inhibited E2 levels and its synthesis-related gene expression. After rFGF21 treatment, CCK8 showed increased cell viability, and flow cytometry showed that the number of S phase increased, and cycle-related genes also increased. However, treatment with FGF21 siRNA to porcine GCs suppressed the cell cycle, viability, and EdU positive cell number. Consequently, FGF21/FGFR1/KLB forms a complex to activate the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway and further promote the proliferation and E2 synthesis in porcine GCs. Collectively, these findings suggests that FGF21 regulates porcine ovarian folliculogenesis.

Whole-genome sequence association study identifies cyclin dependent kinase 8 as a key gene for the number of mummified piglets.

Pigs, an ideal biomedical model for human diseases, suffer from about 50% early embryonic and fetal death, a major cause of fertility loss worldwide. However, identifying the causal variant remains a huge challenge. This study aimed to detect single nucleotide polymorphisms (SNPs) and candidate genes for the number of mummified (NM) piglets using the imputed whole-genome sequence (WGS) and validate the potential candidate genes. The imputed WGS was introduced from genotyping-by-sequencing (GBS) using a multi-breed reference population. We performed genome-wide association studies (GWAS) for NM piglets at birth from a Landrace pig populatiGWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase on. A total of 300 Landrace pigs were genotyped by GBS. The whole-genome variants were imputed, and 4,252,858 SNPs were obtained. Various molecular experiments were conducted to determine how the genes affected NM in pigs. A strong GWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase 8 (CDK8) gene, which plays a crucial role in embryonic retardation and lethality. Based on the molecular experiments, we found that Y-box binding protein 1 (YBX1) was a crucial transcription factor for CDK8, which mediated the effect of CDK8 in the proliferation of porcine ovarian granulosa cells via transforming growth factor beta/small mother against decapentaplegic signaling pathway, and, as a consequence, affected embryo quality, indicating that this pathway may be contributing to mummified fetal in pigs. A powerful imputation-based association study was performed to identify genes associated with NM in pigs. CDK8 was suggested as a functional gene for the proliferation of porcine ovarian granulosa cells, but further studies are required to determine causative mutations and the effect of loci on NM in pigs.

The expression of microRNA-197-3p regulates the proliferation of ovarian granulosa cells through CUL3 in polycystic ovarian syndrome.

MicroRNAs (miR) have been shown to exhibit marked dysregulation in polycystic ovarian syndrome (PCOS). Additionally, granulosa cell proliferation and apoptosis are important in PCOS pathogenesis. Considering this, the current study aimed at characterization of the regulatory function of miR-197-3p on the growth and proliferation of ovarian granulosa cells at the molecular level. The results showed that miR-197-3p is significantly repressed (P<0.05) in PCOS tissues and granulosa cells. Granulosa cell proliferation was significantly (P<0.05) inhibited by miR-197-3p overexpression via induction of apoptosis. In silico analysis predicted cullin 3 (CUL3) as the regulatory target of miR-197-3p, which was also confirmed by the dual luciferase reporter assay. CUL3 was overexpressed in KGN ovarian granulosa cells, and its silencing mimicked the tumor suppressive effects of miR-197-3p. Taken together, miR-197-3p exhibits a negative regulatory role in regulating granulosa ovarian cell proliferation via posttranscriptional repression of CUL3 in PCOS.

Chemotherapy-Related Structural Changes in Cancer: Effect of GnRH Antagonist in the Ovarian Follicles

Objectives: The adverse effect of chemotherapy on the proliferation of granulosa cells has been indicated in recent studies. Gonadotropin-releasing hormone (GnRH) antagonists exert protective effects on granulosa cells against the side effects of chemotherapy. In the present study, we aimed to evaluate the impact of cetrorelix on the proliferation of ovarian granulosa cells following administration of thiotepa in the ovaries of female mice. Materials and Methods: In this experimental study, 30 adult Balb/c female mice (5-8 weeks old, weighing 24-28 g) were divided into three groups (n=10/ each group) (Control group, T. group, and C. group). T. group received 2.5 mg/kg of thiotepa for four consecutive days. The C. group received cetrorelix (0.25 mg/kg) before and at the same time as thiotepa administration and a week after the end of thiotepa administration. Ovaries were used for quantitative and immunohistochemical studies at the end of the investigation. Results: The mean numbers of follicles such as primordial, primary, secondary, and tertiary significantly decreased in the T. group than control group (P=0.02). Cetrorelix treatment exerted a protective effect against thiotepa-induced damage by increasing the mean numbers of follicles in the ovarian cortex (P=0.04). Conclusions: As a GnRH antagonist, cetrorelix can be considered as one of the effective drugs to protect the granulosa cells against chemotherapy-induced damages in cancer disease.

Effect of Upregulation of Transcription Factor TFDP1 Binding Promoter Activity Due to RBP4 g.36491960G>C Mutation on the Proliferation of Goat Granulosa Cells

Retinol-binding protein 4 (RBP4), a member of the lipocalin family, is a specific carrier of retinol (vitamin A) in the blood. Numerous studies have shown that RBP4 plays an important role in mammalian embryonic development and that mutations in RBP4 can be used for the marker-assisted selection of animal reproductive traits. However, there are few studies on the regulation of reproduction and high-prolificacy traits by RBP4 in goats. In this study, the 5′ flanking sequence of RBP4 was amplified, and a G>C polymorphism in the promoter region -211 bp (g.36491960) was detected. An association analysis revealed that the respective first, second and third kidding number and mean kidding number of nanny goats with CC and GC genotypes (2.167 ± 0.085, 2.341 ± 0.104, 2.529 ± 0.107 and 2.189 ± 0.070 for CC and 2.052 ± 0.047, 2.206 ± 0.057, 2.341 ± 0.056 and 2.160 ± 0.039 for GC) were significantly higher (p < 0.05) than those with the GG genotype (1.893 ± 0.051, 2.027 ± 0.064, 2.107 ± 0.061 and 1.74 ± 0.05). The luciferase assay showed that luciferase activity was increased in C allele individuals compared with that in G allele individuals. A competitive electrophoretic mobility shift assay (EMSA) showed that individuals with the CC genotype had a stronger promoter region binding capacity than those with the GG genotype. In addition, transcription factor prediction software showed that the RBP4 g.36491960G>C mutation added a novel binding site for transcription factor DP-1 (TFDP1). RT–qPCR results showed that the expression of TFDP1 was significantly higher in the high-prolificacy group than in the low-prolificacy group, and the expression of RBP4 was higher in both the CC and GC genotypes than that in the GG genotype. TFDP1 overexpression significantly increased the expression of RBP4 mRNA (p < 0.05) and the expression of the cell proliferation factors cyclin-D1, cyclin-D2 and CDK4 (p < 0.05). The opposite trend was observed after interference with TFDP1. Both the EdU and CCK-8 results showed that TFDP1 expression could regulate the proliferation of goat ovarian granulosa cells. In summary, our results showed that RBP4 g.36491960G>C was significantly associated with fecundity traits in goats. The g.36491960G>C mutation enhanced the transcriptional activity of RBP4 and increased the expression of RBP4, thus improving the fertility of Yunshang black goats.

Withdrawal: "Long Non-coding RNA NEAT1 Drives the Development of Polycystic Ovary Syndrome via Sponging Multiple MicroRNAs" by Xia Sang and Yuzhen Zhang.

A line of evidences have validated that multiple microRNAs (including miR-16, miR-483 and miR-324-3p) were abnormally expressed in granulosa cells, which may be closely related with the pathogenesis of polycystic ovary syndrome (PCOS). Long-chain non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has been proved to participate in the progression of various human diseases via regulating microRNAs, but its function in PCOS are not yet clear. In this work, RT-PCR was performed to detect NEAT1 expression in PCOS tissues, and human ovarian granulosa cell line KGN were taken as the model to construct the cell line with high expression and low expression of NEAT1. CCK-8 and flow cytometry were carried out to monitor cells proliferation and apoptosis, respectively. Besides, we performed luciferase reporter gene assay and RNA binding protein immunoprecipitation (RIP) assay to verify the interaction between NEAT1, miR-16, miR-483 and miR-324-3p. We demonstrated that NEAT1 was highly expressed in PCOS tissues and cells. Over-expressed NEAT1 promoted the proliferation and inhibited the apoptosis. Moreover, NEAT1 was negatively correlated with the expression levels of miR-16, miR-483 and miR-324-3p. Dual luciferase analysis and RIP assay confirmed that NEAT1 can specifically bind to miR-16, miR-483 and miR-324-3p, by which NEAT1 can reduce their expression. In conclusion, NEAT1 can promote the proliferation of ovarian granulosa cells and arrest apoptosis via impeding expressions of miR-16, miR-483 and miR-324-3p. Our research would shed new light on the molecular mechanism of ovarian granulosa cell disorder. This article is protected by copyright. All rights reserved.