Granulosa Research Articles

Multi-Omics Reveals the Role of Arachidonic Acid Metabolism in the Gut-Follicle Axis for the Antral Follicular Development of Holstein Cows.

In vitro embryonic technology is crucial for improving farm animal reproduction but is hampered by the poor quality of oocytes and insufficient development potential. This study investigated the relationships among changes in the gut microbiota and metabolism, serum features, and the follicular fluid metabolome atlas. Correlation network maps were constructed to reveal how the metabolites affect follicular development by regulating gene expression in granulosa cells. The superovulation synchronization results showed that the number of follicle diameters from 4 to 8 mm, qualified oocyte number, cleavage, and blastocyst rates were improved in the dairy heifers (DH) compared with the non-lactating multiparous dairy cows (NDC) groups. The gut microbiota was decreased in Rikenellaceae_RC9_gut_group, Alistipes, and Bifidobacterium, but increased in Firmicutes, Cyanobacteria, Fibrobacterota, Desulfobacterota, and Verrucomicrobiota in the NDC group, which was highly associated with phospholipid-related metabolites of gut microbiota and serum. Metabolomic profiling of the gut microbiota, serum, and follicular fluid further demonstrated that the co-metabolites were phosphocholine and linoleic acid. Moreover, the expression of genes related to arachidonic acid metabolism in granulosa cells was significantly correlated with phosphocholine and linoleic acid. The results in granulosa cells showed that the levels of PLCB1 and COX2, participating in arachidonic acid metabolism, were increased in the DH group, which improved the concentrations of PGD2 and PGF2α in the follicular fluid. Finally, the expression levels of apoptosis-related proteins, cytokines, and steroidogenesis-related genes in granulosa cells and the concentrations of steroid hormones in follicular fluid were determinants of follicular development. According to our results, gut microbiota-related phosphocholine and linoleic acid participate in arachidonic acid metabolism in granulosa cells through the gut-follicle axis, which regulates follicular development. These findings hold promise for enhancing follicular development and optimizing oocyte quality in subfertile dairy cows.

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

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

ScRNA-seq revealing the molecular atlas of the goat follicular microenvironment over the time course of ovulation

Ovulation is paramount for female animal fertility, necessitating a thorough understanding of its process and molecular underpinnings. This study aimed to delineate the goat ovary temporal dynamics of ovulation. Utilizing single-cell sequencing, we analyzed follicular fluid samples obtained at 0, 6, 12, 18, and 24 h post-hCG administration, identifying 4 cell types and 6 myeloid cell subtypes. We elucidated gene expression and functional changes in granulosa cells over the time course of ovulation. Notably, our study detected and confirmed immune cell infiltration at 6 h post-LH peak. Additionally, we observed significant cellular interactions between granulosa cells and macrophages, with granulosa cells expressing IL1RAP, COL4A1, COL4A2 implicated in immune cell regulation, while macrophages expressed EREG to facilitate oocyte maturation. Collectively, our investigation has established a comprehensive single-cell transcriptome atlas of the ovulating goat ovary for advancing exploration into ovulation mechanisms and developing therapies for ovulatory disorders.

The DNA double-strand break repair proteins γH2AX, RAD51, BRCA1, RPA70, KU80, and XRCC4 exhibit follicle-specific expression differences in the postnatal mouse ovaries from early to older ages.

Ovarian aging is closely related to a decrease in follicular reserve and oocyte quality. The precise molecular mechanisms underlying these reductions have yet to be fully elucidated. Herein, we examine spatiotemporal distribution of key proteins responsible for DNA double-strand break (DSB) repair in ovaries from early to older ages. Functional studies have shown that the γH2AX, RAD51, BRCA1, and RPA70 proteins play indispensable roles in HR-based repair pathway, while the KU80 and XRCC4 proteins are essential for successfully operating cNHEJ pathway. Female Balb/C mice were divided into five groups as follows: Prepuberty (3weeks old; n = 6), puberty (7weeks old; n = 7), postpuberty (18weeks old; n = 7), early aged (52weeks old; n = 7), and late aged (60weeks old; n = 7). The expression of DSB repair proteins, cellular senescence (β-GAL) and apoptosis (cCASP3) markers was evaluated in the ovaries using immunohistochemistry. β-GAL and cCASP3 levels progressively increased from prepuberty to aged groups (P < 0.05). Notably, γH2AX levels varied in preantral and antral follicles among the groups (P < 0.05). In aged groups, RAD51, BRCA1, KU80, and XRCC4 levels increased (P < 0.05), while RPA70 levels decreased (P < 0.05) compared to the other groups. The observed alterations were primarily attributed to altered expression in oocytes and granulosa cells of the follicles and other ovarian cells. As a result, the findings indicate that these DSB repair proteins may play a role in the repair processes and even other related cellular events in ovarian cells from early to older ages.

P10-12 The mixture of PFAS may affect female reproductive health by causing disruption of mitochondrial function in ovarian granulosa cells

P10-12 The mixture of PFAS may affect female reproductive health by causing disruption of mitochondrial function in ovarian granulosa cells

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

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

P11-01 BPS and BPF by acting as mitochondrial disrupting chemicals cause changes in energy metabolism in human ovarian granulosa cells

P11-01 BPS and BPF by acting as mitochondrial disrupting chemicals cause changes in energy metabolism in human ovarian granulosa cells

In vivo study: black cumin extract (Nigella sativa) on antral follicle diameter and granulosa cell layer thickness as an effort to prevent infertility

Background: Infertility is a health problem that affects approximately 10-15% of couples trying to achieve pregnancy worldwide. Various studies have shown that Nigella sativa (N. Sativa) has many health benefits, including reproductive health. However, studies examining the effectiveness of N. Sativa in preventing folliculogenesis disorders have never been reported.Purpose: This study aims to determine the benefits of administering N. Sativa in preventing folliculogenesis disorders as measured by the diameter of the antral follicles and the thickness of the granulosa cell layer.Methods: This research design uses an experiment with a randomized posttest-only controlled group design approach. There were 27 experimental animals (mice) divided into three groups, namely the negative control group (9 animals) and the positive control group (9 animals), as well as the treatment group (9 animals). The positive control group was exposed to two cigarettes of cigarette smoke per day. The treatment group was exposed to cigarette smoke and N. Sativa 300 mg/KgBW/day for 28 days. After that, the mice were sacrificed to take their ovaries. Then, the ovarian organ samples were stained with Hematoxylin-eosin and examined for the diameter of the antral follicles and the thickness of the granulosa cell layer in the antral follicles with photo slides at 400x magnification in 5 fields of view, statistical analysis using the Independent T-test.Results: There were significant differences in antral follicle diameter and granulosa cell thickness in mice exposed to cigarette smoke and N. Sativa. Administration of N. Sativa extract can increase granulosa cell thickness through estrogenic function. Administration of N. Sativa extract showed estrogenic activity by increasing serum estradiol levels. N. Sativa heterotrophic activity cannot be separated from the flavonoid and phenolic compounds contained in N. Sativa, which have been proven to have high estrogenic activity.Conclusion: N. Sativa extract is efficacious in improving the diameter of antral follicles and the thickness of granulosa cells caused by exposure to cigarettes.

The role of stathmin expression in the differential diagnosis, prognosis, and potential treatment of ovarian sex cord-stromal tumors

BackgroundStathmin, a cytosolic microtubule-destabilizing phosphoprotein involved in the regulation of mitosis, is widely expressed in various malignancies and acts as an adverse prognostic factor. Our research analyzed its immunohistochemical expression on a large cohort of ovarian sex cord-stromal tumors, evaluating its potential utility in differential diagnosis, prognosis, and therapeutic application.MethodsWe examined 390 cases of ovarian sex cord-stromal tumors including 281 adult granulosa cell tumors (AGCT), 5 juvenile granulosa cell tumors (JGCT), 33 Sertoli-Leydig cell tumors (SLCT), 50 fibromas/thecomas (F/T), 11 Leydig cell tumors/steroid cell tumors (LCT/SterCT), 5 sex-cord stromal tumors NOS (SCST-NOS), 3 Sertoli cell tumors (SCT), and 2 sclerosing stromal tumors (ScST). Immunohistochemical analysis was performed using TMAs.ResultsStrong expression (> 50%) was observed in all cases of AGCT, JGCT, SLCT, SCST-NOS, SCT and 1 ScST. The other case of ScST exhibited mild expression (5–10%). The negative cases included exclusively F/T and LCT/SterCT, with F/T showing 24% of negative cases and LCT/SterCT comprising 64% of negative cases.ConclusionThe results of our study indicate that stathmin is neither a prognostic marker nor suitable for the differential diagnosis of challenging cases of ovarian sex cord-stromal tumors. However, its predictive value may be theoretically significant, as a decrease in stathmin expression potentialy influences response to chemotherapy treatment.

Photobiomodulation ameliorates ovarian aging by alleviating oxidative stress and inflammation damage and improving mitochondrial function

Ovarian aging is a serious clinical concern. Few safe and effective methods are currently available to improve ovarian functions. Photobiomodulation (PBM) is a safe and noninvasive physical therapy that can modulate a series of biological processes. Recently, several studies have noted its potential to improve the function of ovary and reproductive cells. However, the effects of PBM treatment on natural ovarian aging remain unclear. In this study, we used a naturally reproductive aging mouse model to observe the effect of PBM on ovarian function. Young and aged female ICR mice were treated with or without PBM for 2 months. PBM was performed using a semiconductor InGaAlP laser emitting at 650 nm (80 mW, 6.7 mW/cm2 for 5 or 10 min, resulting in a dose of 2 or 4 J/cm2, respectively). After treatment, the effects of PBM and its role in oxidative stress, inflammation, and mitochondrial function were investigated. We found that PBM (4 J/cm2) effectively recovered the levels of sex hormones, increased the number of primordial and growing follicles, improved angiogenesis, and decreased cell apoptosis in naturally aged mice. Moreover, PBM reduced oxidative stress, inhibited chronic ovarian inflammation, and improved mitochondrial function in aged ovaries. Similar protective effects of PBM were observed in a hydrogen peroxide-induced oxidative stress model of human granulosa cell line (KGN) in vitro. Increased cell viability, cell proliferation, hormone secretion, mitochondrial membrane potential, and adenosine triphosphate levels and decreased apoptosis and oxidative stress were detected in KGN cells after PBM treatment. Collectively, this study suggest that PBM treatment is beneficial for restoring ovarian function in naturally reproductive aging mice and has a significant protective effect against oxidative stress damage in KGN cells. The mechanisms underlying the benefits of PBM in ovarian aging include antioxidant stress, reduction of inflammation, and preservation of mitochondrial function. Therefore, this study emphasizes the potential of PBM as a therapeutic intervention to ameliorate ovarian aging.

Fluorescence in-situ hybridization assessment of spindle cell-rich testicular sex cord stromal tumors demonstrates multiple chromosomal gains across histologic subtypes

Spindle cell-rich testicular sex cord-stromal tumors (TSCSTs) comprise a group that includes mostly (but not exclusively): myoid gonadal stromal tumor (MGST), adult granulosa cell tumor (AGCT), and unclassified TSCST. These entities demonstrate histopathologic overlap, and prior genomic studies have failed to identify specific oncogenic drivers. Results of DNA sequencing suggest that different types of spindle cell-rich TSCSTs harbor a recurrent pattern of chromosomal gains. However, these results have not been validated by alternative methods and the extent of these changes within individual tumors remains unknown. We used a combination of commercially available fluorescence in-situ hybridization (FISH) probes (3q11.2, 6p24.3, 6q11.1, 6q23, 7q11.21-q11.22, 9p21.3, 11q13.3, 17p11.2) to enumerate a subset of chromosomes identified as altered (gained) in prior studies. We analyzed 10 cases (3 MGST, 4 unclassified TSCST, 3 AGCT), including 7 that had been previously sequenced. FISH demonstrated gains of chromosomes 3, 6, 7, 9, and 11 above the pre-established threshold (25%) in 50%, 80%, 70%, 20%, and 40% of cases, respectively, with gains of chromosome 17 being present in only 1 unclassified TSCST. The proportion of cells with chromosomal gains ranged from 26% to 60%. Tumors with available copy number data from prior genomic analyses showed a partial discordance between FISH and sequencing results. This study demonstrates that spindle-cell rich TSCSTs harbor a recurrent pattern of chromosomal gains, which are present in variable subsets of neoplastic cells. Further studies are needed to determine if these chromosomal changes represent a mechanism relevant for oncogenesis or a secondary event.

Sustained fertility from first-wave follicle oocytes that pause their growth.

Ovulation results from the cyclical recruitment of non-renewing, quiescent oocytes for growth. Therefore, the primordial follicles that are established during development from an oocyte encapsulated by granulosa cells are thought to comprise the lifelong ovarian reserve 1-4 . However, using oocyte lineage tracing in mice, we observed that a subset of oocytes recruited for growth in the first juvenile wave remain paused for many months before continuing growth, ovulation, fertilization and development into healthy offspring. This small subset of genetically-labeled fetal oocytes, labeled with Sycp3-CreERT2, is distinguished by earlier entry and slower dynamics of meiotic prophase I. While labeled oocytes were initially found in both primordial follicles and growing follicles of the first wave, they disappeared from primordial follicles by puberty. Unexpectedly, these first-wave labeled growing oocytes persisted throughout reproductive lifespan and contributed to offspring at a steady rate beyond 12 months of age, suggesting that follicles can pause mid-growth for extended periods then successfully resume. These results challenge the conclusion from lineage tracing of granulosa cells that first-wave follicles make a limited contribution to fertility 5 and furthermore suggest that growth-paused oocytes comprise a second and previously unrecognized ovarian reserve.

Integrative analysis of transcriptome and metabolome reveals the heterogeneity of ovarian follicles between high-altitude Yak (Bos grunniens) and Low-altitude cattle (Bos taurus)

Differences including reproductive efficiency have been proved existence between high-altitude yak (Bos grunniens) and low-altitude cattle (Bos taurus), however, the discrepancies of ovarian follicles across them are still elusive. In the present study, RNA-seq results exhibited an obvious separation of yak and cattle granulosa cells (GCs), and 2329 differently expressed genes (DEGs) was obtained, with 1297 upregulated and 1032 downregulated DEGs in yaks. Advanced analysis revealed that DEGs were mainly enriched in cellular structure, DNA packaging and remodeling (GO and GSEA), hypoxia adaptability, energy metabolism (KEGG and GSEA), and epigenetic modification of histones (Reactome and GSEA). Metabolite profiles of yak and cattle follicular fluid (FF) were depicted by LC-MS/MS non-target metabolomics, and 350 different metabolites (DMs) were acquired, with 242 upregulated and 108 downregulated DMs in yaks. The KEGG pathway enrichment results demonstrated that DMs were primarily enriched in metabolism including lipid, carbohydrate and nucleic acid metabolism. Finally, twenty-nine pathways were significant enriched via integrative analysis, most of which were associated with metabolism including carbohydrate metabolism (e.g. Glycolysis / Gluconeogenesis) and lipid metabolism (e.g. Sphingolipid metabolism). Of note, the key hypoxia adaptation pathway, HIF-1 signaling pathway, was predominant enriched. Overall, we ascertained that the heterogeneity in ovarian follicles of yaks and cattle was existence, which were mainly reflected on hypoxia adaptation and energy metabolism. These results implied that the above processes were evolved specifically for the normal development of yak follicles under plateau atmosphere.

Effects of selection for litter size variability on ovarian folliculogenesis, ovarian cell proliferation, apoptosis, and production of regulatory peptides in rabbits

The present study aimed to identify the novel mechanisms regulating rabbit fecundity. For this purpose, the association between litter size variability, fecundity, ovarian morphology, and markers of proliferation, apoptosis, steroidogenesis, and the presence of regulatory proteins were examined in ovarian cells from two rabbit lines divergently selected for low (LL) and high (HL) variability in litter size throughout sixteen generations. Ovaries and uteri were isolated from multiparous non-lactating female rabbits from each line at sixteen generations of selection. One part of the ovary was subjected to histomorphometric analysis of folliculogenesis. From the rest of ovary, ovarian granulosa cells (OGCs) were isolated, cultured, and cell viability, proliferation (accumulation of PCNA and of cyclin B1, and BrdU-positive cells), and apoptosis (accumulation of caspase 3, bax and DNA fragmentation) were evaluated by the Trypan blue exclusion test and BrdU, quantitative immunocytochemistry, and cell death detection assays. Furthermore, OGCs were subjected to proteomic analysis by using the nano HPLC-Chip-MS/MS method. The release of progesterone and oestradiol was measured by ELISA. The LL had more than one kit per litter than the HL (7.6 kits vs. 6.5 kits, p ≤ 0.05). No differences were found in the diameter of primordial and primary ovarian follicles, theca, and granulosa thickness, but the diameter of oocytes in the primary and secondary follicles was higher in the LL than in the HL (88.49 µm in the LL vs. 77.86 µm in the HL for oocytes of primary follicles, p ≤ 0.05; 122.10 µm in the LL vs. 109.87 µm in the HL for oocytes of secondary follicles, p ≤ 0.05). Preovulatory follicles were presented only in the ovaries of the LL. The LL had higher incorporation of BrdU and reduced accumulation of bax within OGCs (0.92% in the LL vs. 0.44% in the HL for incorporation of BrdU, p ≤ 0.05; 41% in the LL vs. 48% in the HL for accumulation of bax, p ≤ 0.05). Ovarian fragments from the LL produced less progesterone and oestradiol than those of the HL (12 ng/mg tissue/day in the LL vs, 45 ng/mg tissue/day in the HL for progesterone, p ≤ 0.05; 11 ng/mg tissue/day in the LL vs. 30 ng/mg tissue/day in the HL for oestradiol, p ≤ 0.05). Besides, the OGCs from the LL produced a higher number of specific regulatory proteins involved in cell differentiation, proliferation, and adhesion than the HL (50 vs 38, p ≤ 0.05). In conclusion, higher prolificacy in the LL line would be caused by: (1) the selection of growing primordial ovarian follicles; (2) better transformation to preovulatory follicles; (3) increased cytoplasmic maturation of oocytes; (4) increased DNA synthesis and decreased cytoplasmic apoptosis in OGCs; (5) changes in ovarian steroidogenesis; and (6) changes in the number of peptides involved in cell differentiation, proliferation, and adhesion. HIGHLIGHTS Assessing fecundity in two divergently rabbit lines for variability in litter size. Fecundity and its variability are related to the diameter and quality of oocytes. Selection and better growth of preovulatory follicles have been related to fecundity.

The Differentiation Fate of Granulosa Cells and the Regulatory Mechanism in Ovary.

Granulosa cells (GCs) are important drives of the reproductive process, not only the supporting cells for nutrition, but also cells with endocrine functions. Their differentiation and development parallel the entire menstruation period and even during pregnancy, making it tightly linked to the fate of the follicle. To elucidate the underlying mechanism is of great significance for related researches. The life course of GCs is briefly divided into five stages, from epithelial cells to pre-granulosa cells, GCs, mural and cumulus cells, lutein cells, and eventually disappear. A wide variety of genes and transcription factors participate in the regulation of different stages, and more importantly, various hormones secreted by the pituitary gland and GCs themselves play a leading role. These endogenous and exogenous signalling molecules interact to form a cross-linked communication network, promoting the development of GCs. Together with oocytes, theca cells and other functional cells in the ovary, GCs drive one of the most vital biological processes in women.

SIK2 and SIK3 Differentially Regulate Mouse Granulosa Cell Response to Exogenous Gonadotropins In Vivo.

Salt-inducible kinases (SIKs), a family of serine/threonine kinases, were found to be critical determinants of female fertility. SIK2 silencing results in increased ovulatory response to gonadotropins. In contrast, SIK3 knockout results in infertility, gonadotropin insensitivity, and ovaries devoid of antral and preovulatory follicles. This study hypothesizes that SIK2 and SIK3 differentially regulate follicle growth and fertility via contrasting actions in the granulosa cells (GCs), the somatic cells of the follicle. Therefore, SIK2 or SIK3 GC-specific knockdown (SIK2GCKD and SIK3GCKD, respectively) mice were generated by crossing SIK floxed mice with Cyp19a1pII-Cre mice. Fertility studies revealed that pup accumulation over 6 months and the average litter size of SIK2GCKD mice were similar to controls, although in SIK3GCKD mice were significantly lower compared to controls. Compared to controls, gonadotropin stimulation of prepubertal SIK2GCKD mice resulted in significantly higher serum estradiol levels, whereas SIK3GCKD mice produced significantly less estradiol. Cyp11a1, Cyp19a1, and StAR were significantly increased in the GCs of gonadotropin-stimulated SIK2GCKD mice. However, Cyp11a1 and StAR remained significantly lower than controls in SIK3GCKD mice. Interestingly, Cyp19a1 stimulation in SIK3GCKD was not statistically different compared to controls. Superovulation resulted in SIK2GCKD mice ovulating significantly more oocytes, whereas SIK3GCKD mice ovulated significantly fewer oocytes than controls. Remarkably, SIK3GCKD superovulated ovaries contained significantly more preantral follicles than controls. SIK3GCKD ovaries contained significantly more apoptotic cells and fewer proliferating cells than controls. These data point to the differential regulation of GC function and follicle development by SIK2 and SIK3 and supports the therapeutic potential of targeting these kinases for treating infertility or developing new contraceptives.

Casein kinase 1α mediates estradiol secretion via CYP19A1 expression in mouse ovarian granulosa cells

BackgroundCasein kinase 1α (CK1α), expressed in both ovarian germ and somatic cells, is involved in the initial meiosis and primordial follicle formation of mouse oocytes. Using in vitro and in vivo experiments in this study, we explored the function and mechanism of CK1α in estrogen synthesis in mice ovarian granulosa cells.MethodsA CK1α knockout (cKO) mouse model, targeted specifically to ovarian granulosa cells (GCs), was employed to establish the influence of CK1α on in vivo estrogen synthesis. The influence of CK1α deficiency on GCs was determined in vivo and in vitro by immunofluorescence analysis and Western blot assay. Transcriptome profiling, differentially expressed genes and gene functional enrichment analyses, and computation protein–protein docking, were further employed to assess the CK1α pathway. Furthermore, wild-type female mice were treated with the CK1α antagonist D4476 to elucidate the CK1α's role in estrogen regulation.ResultsOvarian GCs CK1α deficiency impaired fertility and superovulation of female mice; also, the average litter size and the estradiol (E2) level in the serum of cKO female mice were decreased by 57.3% and 87.4% vs. control mice, respectively.This deficiency disrupted the estrous cycle and enhanced the apoptosis in the GCs. We observed that CK1α mediated the secretion of estradiol in mouse ovarian GCs via the cytochrome P450 subfamily 19 member 1 (CYP19A1).ConclusionsThese findings improve the existing understanding of the regulation mechanism of female reproduction and estrogen synthesis.Trial registrationNot applicable.

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

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

Bisphenol A Disrupts Ribosome Function during Ovarian Development of Mice.

This study examines the impact of Bisphenol A (BPA), a prevalent environmental estrogenic toxicant, on the ovarian development of mice. Mice were exposed to varying BPA doses from in utero to postnatal stages, up to weaning (day 21, PND 21) and puberty (day 45, PND 45). The BPA content in the serum of the offspring mice on PND 45 was higher than that of the mice sacrificed at PND 21. However, the ovary organ index of the mice of PND 21 was significantly increased, and the ovarian structure was damaged when exposed to BPA. In contrast, the mice with PND 45 did not show apparent ovarian lesions. On the other hand, granulosa cell apoptosis was detected in both PND 21 and PND 45 mice ovaries, and ERβ was increased under the influence of BPA. Transcriptomic analysis revealed BPA's significant impact on ribosomal gene expression, marked downregulation of Rpl21 and Rpsa, and upregulation of Rps2 in both age groups. These transcriptomic alterations were further corroborated by real-time PCR, highlighting a dose-dependent effect of BPA on Rps2. Our findings confirm BPA's detrimental effects on ovarian health, with more pronounced damage in younger mice, suggesting heightened vulnerability in this group. The study underscores ribosomes as critical targets in BPA-induced ovarian developmental disruptions.

Phospholipid Phosphatase 3 (PLPP3) Induces Oxidative Stress to Accelerate Ovarian Aging in Pigs.

Ovarian aging results in reproductive disorders and infertility in mammals. Previous studies have reported that the ferroptosis and autophagy caused by oxidative stress may lead to ovarian aging, but the mechanisms remain unclear. In this study, we compared the morphological characteristics between the aged and young ovaries of pigs and found that the aged ovaries were larger in size and showed more corpora lutea. TUNEL assay further showed that the apoptosis level of granulosa cells (GCs) was relatively higher in the aged ovaries than those in young ovaries, as well as the expressions of autophagy-associated genes, e.g., p62, ATG7, ATG5, and BECN1, but that the expressions of oxidative stress and aging-associated genes, e.g., SOD1, SIRT1, and SIRT6, were significantly lower. Furthermore, the RNA-seq, Western blotting, and immunofluorescence suggested that phospholipid phosphatase 3 (PLPP3) protein was significantly upregulated in the aged ovaries. PLPP3 was likely to decrease the expressions of SIRT1 and SIRT6 to accelerate cellular senescence of porcine GCs, inhibit the expressions of SOD1, CAT, FSP1, FTH1, and SLC7A11 to exacerbate oxidative stress and ferroptosis, and arouse autophagy to retard the follicular development. In addition, two SNPs of PLPP3 promoter were significantly associated with the age at puberty. g.155798586 (T/T) and g.155798718 (C/C) notably facilitated the mRNA and protein level of PLPP3. In conclusion, PLPP3 might aggravate the oxidative stress of GCs to accelerate ovarian aging, and two molecular markers of PLPP3 were identified for ovarian aging in pigs. This work not only contributes to investigations on mechanisms for ovarian aging but also provides valuable molecular markers to postpone ovarian aging in populations.