Transcriptome Landscape of Human Folliculogenesis Reveals Oocyte and Granulosa Cell Interactions.

The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remain unknown. Using RNA sequencing (RNA-seq) analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks, and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle, which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in vivo and restoring fully competent oocytes in vitro.

The road to maturation: somatic cell interaction and self-organization of the mammalian oocyte.

Mammalian oocytes go through a long and complex developmental process while acquiring the competencies that are required for fertilization and embryogenesis. Recent advances in molecular genetics and quantitative live imaging reveal new insights into the molecular basis of the communication between the oocyte and ovarian somatic cells as well as the dynamic cytoskeleton-based events that drive each step along the pathway to maturity. Whereas self-organization of microtubules and motor proteins direct meiotic spindle assembly for achieving genome reduction, actin filaments are instrumental for spindle positioning and the establishment of oocyte polarity needed for extrusion of polar bodies. Meiotic chromatin provides key instructive signals while being 'chauffeured' by both cytoskeletal systems.

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.

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.

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.

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.

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.

[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.

Post-transcriptional silencing of Bos taurus prion family genes and its impact on granulosa cell steroidogenesis.

Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a downregulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology.

FTO protects human granulosa cells from chemotherapy-induced cytotoxicity.

BACKGROUND: Premature ovarian failure (POF) is a serious problem for young women who receive chemotherapy, and its pathophysiological basis is the dysfunction of granulosa cells. According to previous reports, menstrual-derived stem cells (MenSCs) can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF. Fat mass- and obesity-associated (FTO) was reported to be associated with oocyte development and maturation. FTO was decreased in POF and may be a biomarker for the occurrence of POF. Knockdown of FTO in granulosa cells promoted cell apoptosis and inhibited proliferation. But the relationship between FTO and ovarian repair was still unclear. This study was aimed at investigating the FTO expression level and the role of FTO in the MenSCs recovering the function of injured granulosa cells. METHOD: First, cisplatin was used to establish a granulosa cell injury model. Then, the MenSCs and injured granulosa cell coculture model and POF mouse model were established in this study to explore the role of FTO. Furthermore, gain- and loss-of-function studies, small interfering RNA transfection, and meclofenamic acid (MA), a highly selective inhibitor of FTO, studies were also conducted to clarify the regulatory mechanism of FTO in granulosa cells. RESULTS: MenSCs coculture could improve the function of injured granulosa cells by increasing the expression of FTO. MenSCs transplantation restored the expression of FTO in the ovaries of POF mice. Overexpression of FTO restored the injured cell proliferation and decreased apoptosis by regulating the expression of BNIP3. Down-regulation of FTO got the opposite results. CONCLUSIONS: In the treatment of MenSCs, FTO has a protective effect, which could improve the viability of granulosa cells after cisplatin treatment by decreasing the expression of BNIP3. Meanwhile, FTO may provide new insight into therapeutic targets for the chemotherapy-induced POF.

Genomic exploration of the targets of FOXL2 and ESR2 unveils their implication in cell migration, invasion, and adhesion.

FOXL2 and ESR2 are key transcriptional regulators in ovarian granulosa cells. To explore their transcriptional roles and their interplay, we have depleted Foxl2 and Esr2 in mouse primary granulosa cells to assess their ability to bind their targets and/or to modulate gene expression and cellular functions. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. A parallel ChIP-seq analysis showed that FOXL2 mainly binds to sites located in intergenic regions quite far from its targets. A bioinformatic analysis demonstrated that FOXL2-activated genes were enriched in peaks associated with the H3K27ac mark, whereas FOXL2-repressed genes were not, suggesting that FOXL2 can activate transcription through binding to enhancer sites. We also identified about 500 deregulated genes upon Esr2 silencing, of which one third are also targets of FOXL2. We provide evidence showing that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ESR2 targets are involved in cell motility and, consistently, granulosa cells depleted for either Foxl2 or Esr2 exhibit decreased migration, invasion and adhesion. This effect is paralleled by the depletion of their target Phactr1, involved in actin cytoskeleton dynamics. Our analysis expands the number of direct and indirect transcriptional targets of both FOXL2 and ESR2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134W FOXL2 pathogenic variant.

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells.

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.

Effect of mitophagy in oocytes and granulosa cells on oocyte quality†.

Mitophagy is the process by which cells selectively remove supernumerary or damaged mitochondria through autophagy, and is crucial for mitochondrial homeostasis and cell survival. Mitochondria play vital roles in determining the developmental competence of oocytes. During the early stages of oogenesis, aberrant mitochondria can be removed by mitophagy. After oocyte formation, mitophagy is not actively initiated to clear damaged mitochondria despite the presence of mitophagy regulators in oocytes, which leads to the transmission of dysfunctional mitochondria from the oocyte to the embryo. However, granulosa cells around oocytes can improve mitochondrial function through mitophagy, thereby improving oocyte developmental capacity. Furthermore, this review discusses recent work on the substances and environmental conditions that affect mitophagy in oocytes and granulosa cells, thus providing new directions for improving oocyte quality during assisted reproductive technology treatment.

Melatonin alleviates deoxynivalenol-induced apoptosis of human granulosa cells by reducing mutually accentuated FOXO1 and ER stress‡.

Deoxynivalenol (DON) is one of the most prevalent Fusarium mycotoxins, which cause detrimental effects on human and animal reproductive systems by inducing oxidative stress. Increasing evidence has suggested the potential roles of melatonin in protecting granulosa cells from oxidative injury, but the underlying mechanisms remain largely elusive. Here, we demonstrated that suppression of FOXO1 and endoplasmic reticulum (ER) stress was engaged in melatonin-mediated protection against oxidative damage in human granulosa cells upon DON exposure in vitro. DON induced excess reactive oxygen species accumulation, cells viability loss, reduced estradiol-17ß, and progesterone production in human granulosa cells, whereas melatonin ameliorated these phenotypes. Next, we found that the protective effect of melatonin against apoptosis was via reducing ER stress because the inhibition of ER stress displayed similar protective effects during DON treatment. Moreover, melatonin provided no additional protection when ER stress was inhibited. We further found that FOXO1 is a pivotal downstream effector of melatonin and ER stress in regulating DON-induced apoptosis in human granulosa cells. Blocking of FOXO1 reduced DON-induced cells death and FOXO1 activation could be suppressed by melatonin or ER stress inhibitor. However, melatonin failed to further restore cells viability in the presence of FOXO1 inhibitor. Collectively, our results reveal a new mechanism of melatonin in protecting against DON-induced apoptosis and dysfunction by suppressing ER stress and FOXO1 in human granulosa cells.

Leukaemia inhibitory factor modulates the differentiation of granulosa cells during sheep in vitro preantral to antral follicle development and improves oocyte meiotic competence.

In vitro follicle development from cryopreserved ovarian tissue could become an invaluable assisted reproduction technology for women with early ovarian failure. The challenge lies in producing, from small follicles present in the ovarian cortex, high-quality mature oocytes able to sustain embryo development. In vivo, an optimal combination of hormones and other factors coordinates the development of follicles and their enclosed oocyte. We have investigated the effect of the leukaemia inhibitory factor (LIF) cytokine, alone or in combination with FSH, on sheep in vitro follicle development from the preantral stage onwards. LIF did not alter follicle growth or antrum formation, but it modulated the differentiation of granulosa cells, as revealed by decreased production of anti-Müllerian hormone and abolished FSH-induced stimulation of oestradiol secretion. This modulatory role was also reflected in the abundance of mRNA from 35 genes, analysed by reverse-transcription coupled to microfluidic quantitative PCR. LIF stimulated or at least maintained the expression of genes involved in the dialogue between the oocyte and granulosa cells, through gap junctions (GJA4 encoding connexin 37) or paracrine signalling (Bone morphogenetic protein 15, KIT ligand and their receptors). Finally, the presence of both LIF and FSH during follicle growth strongly improved oocyte meiotic competence: most oocytes (56%) underwent subsequent nuclear maturation, a significant increase compared with their counterparts from follicles of similar size (550-900 µm) cultured with FSH only (28%) or developed in vivo (9%). Their ability to sustain embryo development remains to be evaluated. Combined supplementation with FSH and LIF certainly merits investigation with human follicles.

Regulation of granulosa cell functions through NRP-1 mediated internalization of follicular fluid non-exosomal miR-210.

Crosstalk between follicular fluid (FF) and granulosa cells (GCs) plays a vital role in the regulation of folliculogenesis, ensuring regular reproductive cycle in mammals. This crosstalk is primarily mediated by hormones and signaling molecules, such as cytokines and chemokines. Recently, extracellular microRNAs (miRNAs) have gained a lot of attention in cell-to-cell communication. Extracellular miRNA transportation occurs through exosomes, a kind of micro-vesicles produced from almost all cells. However, the mode of non-exosomal miRNA internalization is not much studied. In the present study, we explored the role of neuropilin-1 (NRP-1) as a receptor in internalizing FF non-exosomal miRNAs in GCs. We first confirmed the expression of NRP-1 in GCs during follicular development followed by its role in the internalization of miR-210, a non-exosomal miRNA. This study showed that incubation of GCs with a non-exosomal fraction of FF increased the content of miR-210 in GCs as compared to their control. To illustrate the role of NRP-1 as a receptor, NRP-1 was knockdown using siRNA. Silencing experimental results showed a significant decrease in uptake of miR-210 in NRP-1 knockdown GCs. Furthermore, downstream expression analysis of miR-210 target genes (CYP19A1, PCNA, and EFNA3) also confirmed the NRP-1 mediated miR-210 internalization. Results of the present study clearly demonstrated that FF non-exosomal miR-210 can be internalized through the NRP-1 receptor. Furthermore, differential expression of NRP-1 in GCs suggests its role in follicular development. Overall, these findings suggest that FF non-exosomal miRNA plays an important role in GC functions and female reproduction.

Irisin deletion induces a decrease in growth and fertility in mice.

BACKGROUND: Irisin, which is cleaved from fibronectin type III domain-containing protein 5 (Fndc5), plays an important role in energy homeostasis. The link between energy metabolism and reproduction is well known. However, the biological actions of irisin in reproduction remain largely unexplored. METHODS: In this study, we generated Fndc5 gene mutation to create irisin deficient mice. Female wild-type (WT) and Fndc5 mutant mice were fed with standard chow for 48 weeks. Firstly, the survival rate, body weight and fertility were described in mice. Secondly, the levels of steroid hormones in serum were measured by ELISA, and the estrus cycle and the appearance of follicles were determined by vaginal smears and ovarian continuous sections. Thirdly, mRNA-sequencing analysis was used to compare gene expression between the ovaries of Fndc5 mutant mice and those of WT mice. Finally, the effects of exogenous irisin on steroid hormone production was investigated in KGN cells. RESULTS: The mice lacking irisin presented increased mortality, reduced body weight and poor fertility. Analysis of sex hormones showed decreased levels of estradiol, follicle-stimulating hormone and luteinizing hormone, and elevated progesterone levels in Fndc5 mutant mice. Irisin deficiency in mice was associated with irregular estrus, reduced ratio of antral follicles. The expressions of Akr1c18, Mamld1, and Cyp19a1, which are involved in the synthesis of steroid hormones, were reduced in the ovaries of mutant mice. Exogenous irisin could promote the expression of Akr1c18, Mamld1, and Cyp19a1 in KGN cells, stimulating estradiol production and inhibiting progesterone secretion. CONCLUSIONS: Irisin deficiency was related to disordered endocrinology metabolism in mice. The irisin deficient mice showed poor growth and development, and decreased fertility. Irisin likely have effects on the expressions of Akr1c18, Mamld1 and Cyp19a1 in ovary, regulating the steroid hormone production. This study provides novel insights into the potential role of irisin in mammalian growth and reproduction.

The LINC00477/miR-128 axis promotes the progression of polycystic ovary syndrome by regulating ovarian granulosa cell proliferation and apoptosis.

BACKGROUND: Long noncoding RNAs (lncRNAs) participate in the pathogenesis of various human diseases. This study aims to investigate the roles of lncRNA LINC00477 in polycystic ovary syndrome (PCOS), especially the impacts of LINC00477 on the proliferation and migration of human granulosa cells and the related mechanisms. METHODS: qRT-PCR analysis was performed to examine the expression pattern of LINC00477 in serum samples of PCOS patients as well as PCOS animal models. The effect of LINC00477 on the viability and apoptosis of ovarian granulosa cells was detected by MTT and flow cytometry assays. The correlation between LINC00477 and miR-128 was verified by bioinformatics analysis and dual-luciferase reporter and RNA pull-down assays. Finally, rescue assays were performed to analyze the effects of the LINC00477-miR-128 axis on the biological behaviors of granulosa cells. RESULTS: LINC00477 was significantly upregulated in the serum of PCOS patients as well as PCOS mouse models. LINC00477 overexpression inhibited the proliferation and promoted the apoptosis of granulosa cells, whereas knockdown of LINC00477 yielded the opposite effects. Moreover, miR-128 mimics partially abrogated the effect of LINC00477 on granulosa cells. CONCLUSION: LINC00477 may function as a ceRNA to inhibit proliferation and apoptosis of granulosa cells by modulating miR-128 expression.

Metabolic exchanges between the oocyte and its environment: focus on lipids.

Finely regulated fatty acid (FA) metabolism within ovarian follicles is crucial to follicular development and influences the quality of the enclosed oocyte, which relies on the surrounding intra-follicular environment for its growth and maturation. A growing number of studies have examined the association between the lipid composition of follicular compartments and oocyte quality. In this review, we focus on lipids, their possible exchanges between compartments within the ovarian follicle and their involvement in different pathways during oocyte final growth and maturation. Lipidomics provides a detailed snapshot of the global lipid profiles and identified lipids, clearly discriminating the cells or fluid from follicles at distinct physiological stages. Follicular fluid appears as a main mediator of lipid exchanges between follicular somatic cells and the oocyte, through vesicle-mediated and non-vesicular transport of esterified and free FA. A variety of expression data allowed the identification of common and cell-type-specific actors of lipid metabolism in theca cells, granulosa cells, cumulus cells and oocytes, including key regulators of FA uptake, FA transport, lipid transformation, lipoprotein synthesis and protein palmitoylation. They act in harmony to accompany follicular development, and maintain intra-follicular homeostasis to allow the oocyte to accumulate energy and membrane lipids for subsequent meiotic divisions and first embryo cleavages.

miR-18b regulates the function of rabbit ovary granulosa cells.

MicroRNAs (miRNAs) have been determined to participate in the process of oestradiol production. Generally, there are two pathways by which oestradiol levels change, one being the state of cells (i.e. the status of enzymes involved in the synthesis of hormones such as oestradiol) and the other being the number of cells that secrete oestradiol. It is known that oestrogens are the main steroids produced by granulosa cells (GCs) of mature ovarian follicles. In this study we explored the function of miR-18b in rabbit GCs by overexpressing or inhibiting its activity. We found that miR-18b silencing promoted the secretion of oestradiol by significantly affecting the expression of steroidogenesis-related genes. Thus, miR-18b may act as a negative regulator of the production of enzymes related to oestradiol synthesis and affect oestradiol production. Furthermore, the effects of miR-18b on the proliferation, cell cycle and apoptosis of GCs were investigated using a cell counting kit (CCK-8) proliferation assay, detection of annexin V-fluorescein isothiocyanate apoptosis, flow cytometry and quantitative polymerase chain reaction. The results showed that miR-18b upregulated GC apoptosis (miR-18b overexpression decreases cell growth and stimulates apoptosis). These findings suggest that miR-18b and the oestrogen receptor 1 (ESR1) gene may be attractive targets to further explore the molecular regulation of GCs. The miR-18b may also explain, in part, the abnormal folliculogenesis in mammals caused by conditions such as polycystic ovary syndrome, primary ovarian insufficiency, and others.