Granulosa Cell Differentiation Research Articles

P62 promotes FSH-induced antral follicle formation by directing degradation of ubiquitinated WT1.

In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitin‒proteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.

Differential analysis of ovarian tissue between high and low-yielded laying hens in the late laying stage and the effect of LECT2 gene on follicular granulosa cells proliferation.

The ovaries of high-yield laying hens exhibited signs of aging beyond 400 days of age, subsequently resulting in a decline in both egg production and egg quality. Oxidative stress, characterized by an increase in the production of reactive oxygen species (ROS), stands as one of the principal processes contributing to ovarian aging. Elevated ROS levels are implicated in the induction of apoptosis in granulosa cells (GCs), provoking mitochondrial impairment, and diminishing the capacity of the antioxidant defense system. This investigation stratified laying hens into two distinct groups, predicated upon their egg production levels: high-yield hens (HH) and low-yield hens (LL). The study focused on evaluating oxidative stress markers and identifying differentially expressed genes between these two groups. The findings revealed that the LL group exhibited follicular atresia, mitochondrial disruptions, and apoptotic occurrences in ovarian GCs. Notably, ROS levels, Malondialdehyde (MDA) concentrations, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations in ovarian tissue and follicular GCs were substantially higher in the HH group. Furthermore, the RNA-sequencing results unveiled differential expression of the LECT2 gene between the HH and LL groups. Consequently, an overexpression vector for the LECT2 gene was successfully constructed and introduced into GCs. The quantitative polymerase chain reaction (QPCR) analysis exhibited significant downregulation (p<0.01) of key apoptotic genes such as Caspase-3 and C-myc and significant upregulation (p<0.01) of BCL2 following the overexpression of the LECT2 gene in GCs. In conclusion, oxidative stress emerges as a pivotal factor influencing the laying traits of both high and low-yield laying hens. The accumulation of reactive oxygen species (ROS) within the ovaries precipitates apoptosis in GCs, subsequently leading to follicular atresia and a reduction in egg production. Furthermore, we employed RNA sequencing technology to examine the ovarian matrix tissue in high and low laying hens during the late phase of egg laying. Our analysis revealed a substantial upregulation of the LECT2 gene in the ovarian matrix tissue of high laying hens. This observation implies that the LECT2 gene exerts a pivotal influence on driving the proliferation and differentiation of follicular GCs, thereby exerting a crucial regulatory role in follicular development.

Regulation of Follicular Development in Chickens: WIF1 Modulates Granulosa Cell Proliferation and Progesterone Synthesis via Wnt/β-Catenin Signaling Pathway.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated β-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |log2⁡(FoldChange)| > 1 and p_adj < 0.05). Cytokine-cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (log2⁡(FoldChange) = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

MiRNA profiling of chicken follicles during follicular development

MicroRNAs (miRNAs) play a crucial role as transcription regulators in various aspects of follicular development, including steroidogenesis, ovulation, apoptosis, and gene regulation in poultry. However, there is a paucity of studies examining the specific impact of miRNAs on ovarian granulosa cells (GCs) across multiple grades in laying hens. Consequently, this study aims to investigate the roles of miRNAs in chicken GCs. By constructing miRNA expression profiles of GCs at 10 different time points, encompassing 4 pre-hierarchical, 5 preovulatory, and 1 postovulatory follicles stage, we identified highly expressed miRNAs involved in GC differentiation (miR-148a-3p, miR-143-3p), apoptosis (let7 family, miR-363-3p, miR-30c-5p, etc.), and autophagy (miR-128-3p, miR-21-5p). Furthermore, we discovered 48 developmentally dynamic miRNAs (DDMs) that target 295 dynamic differentially expressed genes (DDGs) associated with follicular development and selection (such as oocyte meiosis, progesterone-mediated oocyte maturation, Wnt signaling pathway, TGF-β signaling pathway) as well as follicular regression (including autophagy and cellular senescence). These findings contribute to a more comprehensive understanding of the intricate mechanisms underlying follicle recruitment, selection, and degeneration, aiming to enhance poultry’s reproductive capacity.

Lnc RNA LOC102163816 Promotes Proliferation of Porcine Follicular Granulosa Cells Via miR-455-3p/PTK2B/PI3K/AKT Pathway.

The proliferation and differentiation of granulosa cells (GCs) is a crucial process in follicular development. However, the molecular regulatory mechanism of follicular proliferation and differentiation of GCs needs further research. Studies have reported that follicular fluid exosomes are involved in regulation of proliferation of GCs, but the specific mechanism is unclear. This study demonstrated that LOC102163816 is upregulated in porcine GCs treated with follicular fluid exosomes. Further study defined LOC102163816 to be a novel long noncoding RNA that is highly homologous to human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and enriched in porcine follicular fluid exosomes. We have speculated that LOC102163816 might have a cell-proliferative effect similar to that of MALAT1. We found that overexpression of LOC102163816 promoted transition from the G1 phase to the S phase of the cell cycle, thereby promoting proliferation of GCs. To explore the specific mechanism underlying this promotion of proliferation, miRNA sequencing was performed after overexpression of LOC102163816. Our results showed that LOC102163816 sponged miR-455-3p, promoting expression of protein tyrosine kinase 2 beta (PTK2B), thereby activating the PI3K/AKT signaling pathway to regulate proliferation of porcine follicular GCs. These findings provide useful insights into follicular development.

Resveratrol-βcd inhibited premature ovarian insufficiency progression by regulating granulosa cell autophagy

BackgroundThe ovarian environment of premature ovarian insufficiency (POI) patients exhibits immune dysregulation, which leads to excessive secretion of numerous proinflammatory cytokines that affect ovarian function. An abnormal level of macrophage polarization directly or indirectly inhibits the differentiation of ovarian granulosa cells and steroid hormone production, ultimately leading to POI. Resveratrol, as a health supplement, has been widely recognized for its safety. There is a substantial amount of evidence indicating that resveratrol and its analogs possess significant immune-regulatory functions. It has also been reported that resveratrol can effectively inhibit the progression of POI. However, the underlying immunological and molecular mechanisms through which resveratrol inhibits the progression of POI are still unclear.ResultsOur preliminary reports have shown that resveratrol-βcd, the beta-cyclodextrin complex of resveratrol, significantly enhances the stability of resveratrol. Resveratrol-βcd could regulate the dysfunctional immune status of macrophages and T cells in the tumor microenvironment. In this study, we treated busulfan and cyclophosphamide (B/C)-treated mice, which were used as a POI model, with resveratrol-βcd. After resveratrol-βcd treatment, the levels of IL-6 in the ovaries were significantly increased, and the progression of POI was suppressed. IL-6 activated granulosa cells (GCs) through soluble IL-6R (sIL-6R), promoting autophagy in GCs. Resveratrol-βcd and IL-6 had a synergistic effect on enhancing autophagy in GCs and promoting E2 secretion.ConclusionsWe partially elucidated the immune mechanism by which resveratrol inhibits the progression of POI and the autophagy-regulating function of GCs. This provides a theoretical basis for using resveratrol to prevent POI in future studies and clinical guidance.

Aquisição da dependência de gonadotrofinas pelos folículos antrais iniciais e os desafios para promover o seu crescimento in vitro

Abstract This review aims to discuss the main factors involved in the development of early antral follicles until gonadotropin dependence. This follicular phase is characterized by intense proliferation of granulosa cells, formation of a fluid-filled cavity, morphological differentiation of cumulus cells, mural granulosa cells and recruitment of theca cells. The interaction between oocyte, granulosa and theca cells is crucial for follicular growth and hormone production. Growth factors produced by the oocyte, such as growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), regulate granulosa cell proliferation and differentiation and antral cavity development, as well as stimulate the production of follicle-stimulating hormone (FSH) receptors in granulosa cells. In response to FSH, granulosa cells secrete C-type natriuretic peptide (CNP), which acts through its receptor to increase cyclic guanosine monophosphate (cGMP) production and consequently follicular development. Granulosa cells also produce insulin-like growth factor-1 (IGF-1) and increase aromatase enzyme activity, which results in greater sensitivity to gonadotropins and follicular steroidogenesis. The absence of IGF-1 signaling causes cessation of follicular growth at the early antral stage. Many other local factors are involved in the regulation of follicular development. Therefore, this review brings relevant data for a better understanding of the mechanisms involved in the control of early antral follicle growth, emphasizing the role of endocrine and paracrine factors, the oocyte-granulosa cell interaction and the processes of follicular atresia. The challenges for the establishment of efficient culture systems for in vitro growth of early antral follicles are also discussed.

Single-cell transcriptomic atlas of goat ovarian aging

BackgroundThe ovaries are one of the first organs that undergo degenerative changes earlier in the aging process, and ovarian aging is shown by a decrease in the number and quality of oocytes. However, little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging, especially in goats. Therefore, the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.ResultsFor the first time, we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn, young and aging goats, and identified nine ovarian cell types with distinct gene-expression signatures. Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes, such as Wnt beta-catenin signalling was enriched in germ cells, whereas ovarian steroidogenesis was enriched in granulosa cells (GCs). Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system, oxidative phosphorylation, and apoptosis. Subsequently, we identified a series of dynamic genes, such as AMH, CRABP2, THBS1 and TIMP1, which determined the fate of GCs. Additionally, FOXO1, SOX4, and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.ConclusionsThis study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell type-specific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

Cellular atlases of ovarian microenvironment alterations by diet and genetically-induced obesity.

Obesity, which can arise from genetic or environmental factors, has been shown to cause serious damages to the reproductive system. The ovary, as one of the primary regulators of female fertility, is a complex organ comprised of heterogeneous cell types that work together to maintain a normal ovarian microenvironment (OME). Despite its importance, the effect of obesity on the entire ovary remains poorly documented. In this study, we performed ovary single-cell and nanoscale spatial RNA sequencing to investigate how the OME changed under different kinds of obesity, including high-fat diet (HFD) induced obesity and Leptin ablation induced obesity (OB). Our results demonstrate that OB, but not HFD, dramatically altered the proportion of ovarian granulosa cells, theca-interstitial cells, luteal cells, and endothelial cells. Furthermore, based on the spatial dynamics of follicular development, we defined four subpopulations of granulosa cell and found that obesity drastically disrupted the differentiation of mural granulosa cells from small to large antral follicles. Functionally, HFD enhanced follicle-stimulating hormone (FSH) sensitivity and hormone conversion, while OB caused decreased sensitivity, inadequate steroid hormone conversion, and impaired follicular development. These differences can be explained by the differential expression pattern of the transcription factor Foxo1. Overall, our study provides a powerful and high-resolution resource for profiling obesity-induced OME and offers insights into the diverse effects of obesity on female reproductive disorders.

Expression and regulation of Noggin4 gene in chicken ovarian follicles and its role in the proliferation and differentiation of granulosa cells

As a member of Noggin family, Noggin4 is reported to play an important role in the formation of head structure during the embryo development of Xenopus laevis and chicken. We previously detected an increase of Noggin4 transcript in the granulosa cells of chicken hierarchal follicles (Post-GCs) compared to pre-hierarchal follicles (Pre-GCs) by ONT transcriptome sequencing. To further clarify the role of Noggin4 in chicken follicle selection, in this study, we investigated its expression, regulation and function in follicles and granulosa cells. The mRNA expression of chicken Noggin4 exhibited dynamic changes during follicle development. It was significantly higher in the small yellow follicles than in the small white, F6, F5 and F4 follicles, and also increased in Post-GCs than in Pre-GCs. The Noggin4 mRNA could be stimulated by follicle stimulating hormone (FSH) and bone morphogenetic protein 4 (BMP4) in both Pre-GCs and Post-GCs. However, the estrogen and progesterone could exert opposing transcriptional regulations on Noggin 4 mRNA in both Pre- and Post-GCs. In chicken Post-GCs, knockdown of Noggin4 by siRNA reduced the mRNA expression of steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), but increased that of Wnt family member 4 (Wnt4), while overexpression of Noggin4 significantly decreased the mRNA expression of Wnt4 but had no marked effects on that of STAR and CYP11A1. Moreover, Noggin4 significantly decreased the mRNA expression of BMP4 in both Pre-GCs and Post-GCs. Overexpression of Noggin4 inhibited the proliferation of both Pre-GCs and Post-GCs. These data collectively suggest an important role of Noggin4 in chicken follicle selection, especially on the proliferation of granulosa cells.

MiR-10a-5p inhibits steroid hormone synthesis in porcine granulosa cells by targeting CREB1 and inhibiting cholesterol metabolism

During growth, proliferation, differentiation, atresia, ovulation, and luteinization, the morphology and function of granulosa cells (GCs) change. Estrogen and progesterone are steroid hormones secreted by GCs that regulate the ovulation cycle of sows and help maintain pregnancy. miR-10a-5p is highly expressed in GCs and can inhibit GC proliferation. However, the role of miR-10a-5p in the steroid hormone synthesis of porcine GCs is unclear. In this study, miR-10a-5p agomir or antagomir was transfected into GCs. Overexpression of miR-10a-5p in GCs inhibited steroid hormone secretion and significantly downregulated steroid hormone synthesis via 3β-hydroxy steroid dehydrogenase and cytochrome P450 family 19 subfamily A member 1. Interference with miR-10a-5p had the opposite effect. Bodipy and Oil Red O staining showed that overexpression of miR-10a-5p significantly reduced the formation of lipid droplets. Overexpression significantly inhibited the content of total cholesterol esters in GCs. The mRNA and protein levels of 3-hydroxy-3-methylglutaryl-CoA reductase and scavenger receptor class B member 1 decreased significantly, and the opposite effects were seen by interference with miR-10a-5p. Bioinformatic analysis of potential targets identified cAMP-responsive element binding protein 1 as a potential target and dual-luciferase reporter system analysis confirmed that miR-10a-5p directly targets the 3’ untranslated region. These findings suggest that miR-10a-5p inhibits the expression of 3β-hydroxy steroid dehydrogenase and cytochrome P450 family 19 subfamily A member 1 to inhibit the synthesis of steroid hormones in GCs. In addition, miR-10a-5p inhibits the cholesterol metabolism pathway of GCs to modulate steroid hormone synthesis.

Multi-omics reveal the metabolic patterns in mouse cumulus cells during oocyte maturation

Bi-directional communication between cumulus cells and the surrounded oocytes is important for the development and functions of both compartments. However, the metabolic framework in cumulus cells has not been systematically described. In the present study, cumulus cells from cumulus-oocyte complexes (COCs) at three key time points were isolated (arrested GV stage, post-hCG 0h; meiotic resumption GVBD stage, post-hCG 3h; and metaphase II stage, post-hCG 12h), and the temporal metabolomic and proteomic profiling were performed. Integrated multi-omics analysis reveals the global metabolic patterns in cumulus cells during mouse oocyte maturation. In particular, we found the active hyaluronic acid metabolism, steroid hormone synthesis, and prostaglandin E2 (PGE2) production in cumulus cells. Meanwhile, accompanying the oocyte maturation, a progressive increase in nucleotide and amino acid metabolism was detected in the surrounding cumulus cells. In sum, the data serve as a valuable resource for probing metabolism during terminal differentiation of ovarian granulosa cells, and provide the potential biomarkers for improving and predicting oocyte quality.

Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1–5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

The mechanisms that control the preantral to early antral follicle transition and the strategies to have efficient culture systems to promote their growth in vitro.

Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.

Fec, CA5A and CLSTN2 genes and their function during sheep ovulation: a review.

Objective: To describe the function of Fec, CA5A and CLSTN2 genes during ovulation in ewes.&#x0D; Design/methodology/approach: A search and analysis of scientific information related to Fec, CA5A and CLSTN2 genes in sheep was performed.&#x0D; Results: Fec, CA5A and CLSTN2 genes are involved at the ovarian level; ewes carrying the first gene were found to have increased ovulation rate, folliculogenesis and granulosa cell differentiation. CA5A stimulates an increased follicular rate and plays an important role in pre-implantation. While CLSTN2 has activity in ovarian development and growth; it also has the ability to interact with other genes involved in follicular maturation, granulosa cell differentiation and development of the ovarian follicle.&#x0D; Limitations on study/implications: Ewes carrying these genes increase the prolificacy rate in the flock.&#x0D; Findings/conclusions: The expression of these genes acts synergistically in the ovulatory process, enhancing the ovulatory response by contributing to endocrine, paracrine and molecular synchronisation, so that the maturation of the oocyte occurs, leading to ovulation.

Role of lncRNAs in the pathogenic mechanism of human decreased ovarian reserve.

Decreased ovarian reserve (DOR) is defined as a decrease in the quality and quantity of oocytes, which reduces ovarian endocrine function and female fertility. The impaired follicular development and accelerated follicle atresia lead to a decrease in the number of follicles, while the decline of oocyte quality is related to the disorder of DNA damage-repair, oxidative stress, and the dysfunction of mitochondria. Although the mechanism of DOR is still unclear, recent studies have found that long non-coding RNA (lncRNA) as a group of functional RNA molecules participate in the regulation of ovarian function, especially in the differentiation, proliferation and apoptosis of granulosa cells in the ovary. LncRNAs participate in the occurrence of DOR by affecting follicular development and atresia, the synthesis and secretion of ovarian hormones. This review summarizes current research on lncRNAs associated with DOR and reveals the potential underlying mechanisms. The present study suggests that lncRNAs could be considered as prognostic markers and treatment targets for DOR.

Identification of genes involved in chicken follicle selection by ONT sequencing on granulosa cells.

In chickens, follicle selection is an important process affecting laying traits, which is characterized by the differentiation of granulosa cells and the synthesis of progesterone by granulosa cells from hierarchical follicles. By using Oxford Nanopore Technologies (ONT) approach, we compared the transcriptomes of granulosa cells between pre-hierarchical (Pre-GCs) and hierarchical follicles (Post-GCs) to identify genes underlying chicken follicle selection. A total of 2,436 differentially expressed genes (DEGs), 3,852 differentially expressed transcripts (DETs) and 925 differentially expressed lncRNA transcripts were identified between chicken Pre-GCs and Post-GCs. For all of the significant DETs, the alternative 3'splice sites (A3) accounted for a maximum of 23.74% of all alternative splicing events. Three DETs of the 7-dehydrocholesterol reductase gene (DHCR7) named as T1, T3, and T4, differing in 5'untranslated regions (UTRs), increased in Post-GCs with different folds (T1: 1.83, T3: 2.42, T4: 5.06). The expression of the three DHCR7 transcripts was upregulated by estrogen in a dose-dependent manner, while was downregulated by bone morphogenetic protein 15 (BMP15) and transforming growth factor-beta 1 (TGF-β1). Follicle-stimulating hormone (FSH) and bone morphogenetic protein 4 (BMP4) promoted the expression of the three DHCR7 transcripts in Pre-GCs at lower concentrations, while repressed their expression at higher concentrations. The data from this study may provide a reference for better understanding of the genetic mechanisms underlying follicle selection in chicken and other poultry species.

Dual role of NR4A1 in porcine ovarian granulosa cell differentiation and granulosa-lutein cell regression in vitro

This study aimed to investigate the role of NR4A1 in forskolin (FSK)-induced granulosa cell (GC) differentiation and PGF2α-induced granulosa-lutein cell (GLC) regression. For experiment 1, primary porcine GCs were pre-cultured for 6 d before induced-differentiation by FSK with or without siNR4A1, and changes in GC proliferation, lipid droplets (LDs), and P4 level were detected. For experiment 2, the GLC model was established by FSK as in experiment 1, and then PGF2α was utilized to induce GLC regression with or without siNR4A1, changes in P4 secretion, apoptosis proteins, and associated signaling pathway members were detected. Results showed that in experiment 1, FSK up-regulated NR4A1 expression during GC differentiation and decreased GC proliferation activity, which was reversed by siNR4A1. siNR4A1 inhibited the FSK-induced decreases in Cyclin B1/D1 and CDK1/2 mRNA abundances, and increases in P21/P27 mRNA abundances, and FSK-induced LD accumulation. FSK up-regulated P4 secretion and StAR, CYP11A1 and HSD3B expression, decreased CYP19A1 expression, which were reversed by siNR4A1 except for StAR expression. In experiment 2, PGF2α induced NR4A1 expression and reduced GLC viability, which were reversed by siNR4A1. Compared with PGF2α group, the levels of P4 secretion and StAR expression were higher in PGF2α+siNR4A1 group, while CYP11A1 and HSD3B expressions held at low levels. siNR4A1 inhibited PGF2α-induced expression of apoptosis proteins (caspase3, Bax, Fas, TNFa), ATF3, and phosphorylated MAPKs (ERK1/2, P38, JNK). In summary, NR4A1 is involved in regulating porcine GC differentiation and GLC regression as well as the changes in cell proliferation, apoptosis, steroidogenesis, and MAPK pathways, which provide a theoretical basis for further understanding of the mechanism of porcine luteal formation and regression.

Transcriptomic analysis of ovarian follicles uncovers the crucial genes relevant to follicle selection and preovulatory hierarchy in hens.

Follicle selection and preovulatory hierarchy of hen ovaries were important stages of follicle development and crucially determining egg-laying performance. The selected follicles with a higher expression level of follicle-stimulating hormone receptor (FSHR) mRNA that facilitates response to FSH, and rapidly develops into preovulatory follicles with distinctive characteristics of granulosa cells (GCs) proliferation and differentiation. Identification of the key genes involved in these developmental events is helpful for elucidation of the molecular mechanism underlying egg-laying traits in chicken and other domestic fowl. Herein, the comparative transcriptomic analysis of ovarian prehierarchical follicles before selection (BSF), follicles at selection stage (ASF), and hierarchical follicles (HF) were implemented in the Jilin Black chicken (JB) and Lohmann Brown layer (LB) with the divergences in their egg-laying performance by RNA-sequencing. The results showed that nine deferentially expressed genes (DEGs), including STMN4, FABP3, ROBO2, RSPO4, and DMRT1 were revealed between follicles BSF and ASF; and seventeen DEGs, such as SLC6A15, SLITRK3, PRKG2 and TMC3 were mined between ASF and HF. These two group DEGs being co-expressed between BSF and ASF, and between ASF and HF were compared and substantiated in the JB and LB layers, respectively. Furthermore, 10 signaling pathways, such as cAMP signaling, PPAR signaling pathway, AMPK(Adenosine 5'-monophosphate (AMP)-activated protein kinase) pathway, and estrogen signaling pathway were also identified. Moreover, the roles of two representative candidates ROBO2 and PRKG2 genes presented as downregulated mRNA expression pattern in the transcriptomic profiles were further verified in vitro. The results demonstrated that downregulation of ROBO2 or PRKG2 significantly increased the expression levels of FSHR mRNA and protein with the boosted expression of CCND1, STAR, and BCL-2, whereas remarkably inhibited the expression of Caspase-3, consequently, brought about the decrease of GC apoptosis in the ovarian follicles, but increase of GC proliferation and differentiation serving as the hallmarks for follicle selection. It indicated that ROBO2 and PRKG2 may play indispensable roles in follicle selection and preovulatory hierarchy of hen ovaries separately. Our findings provided a comparative transcriptomic evidence for clarifying the molecular mechanism of the follicle development underlying egg-laying traits in chicken.

MiR-145 regulates steroidogenesis in mouse primary granulosa cells by targeting Arpc5 and subsequent cytoskeleton remodeling.

MicroRNA (miR)-145 is enriched in the follicular granulosa cells (GCs) of 3-week-old mice. Downregulating miR-145 inhibits the proliferation and differentiation of GCs and induces evident changes in their cytoskeleton. In this study, we examined how miR-145 induces cytoskeletal changes in mouse GCs and its potential mechanism in regulating GC steroidogenesis. We found that actin related protein 2/3 complex subunit 5 (Arpc5) is a target of miR-145. The miR-145 antagomir increased ARPC5 expression but not β-ACTIN, β-TUBULIN, and PAXILLIN expression. Arpc5 overexpression inhibited GC proliferation, differentiation, and progesterone synthesis. Furthermore, the expression of progesterone synthesis-associated enzymes was downregulated in the Arpc5 overexpression group, and the GC cytoskeleton exhibited evident changes. We conclude that Arpc5, a new target of miR-145, regulates primary GC proliferation and progesterone production by regulating the cytoskeleton remodeling.