Expression of thyroid antigens in the female reproductive system.

Thyroid autoimmunity (TAI) has been linked to fertility disorders and pregnancy complications, even in euthyroid women. However, the exact pathophysiological mechanism underlying this association is not fully understood. This study seeks to investigate the expression of thyroid antigens within the human female reproductive system, potentially identifying targets for thyroid antibodies. Human biopsies of endometrium and follicular granulosa cells were collected and thyroperoxidase (TPO) and thyroglobulin (TG) expression was evaluated in these tissues by immunohistochemistry. Results showed, for the first time, the expression of TG protein and confirmed the presence of thyroid TPO in human endometrium and granulosa cells. Results suggest that TPO antibodies (TPOAbs) and TG antibodies (TGAbs) could interact with TPO and TG expressed in the reproductive system in patients with positive thyroid antibodies, thereby disrupting the function of TPO and TG and generating an inflammatory response, leading to fertility disorders and pregnancy complications.

L'auto-immunité thyroïdienne (AIT) est associée à des troubles de la fertilité et à des complications de grossesse, même chez les femmes euthyroïdiennes. Cependant, le mécanisme physiopathologique sous-jacent à cette association n'est pas entièrement élucidé. Cette étude vise à examiner l'expression des antigènes thyroïdiens dans le système reproducteur féminin humain, afin d'identifier des cibles potentielles pour les anticorps antithyroïdiens. Des biopsies d'endomètre et de cellules de granulosa ont été analysées pour l'expression de la thyroperoxydase (TPO) et de la thyroglobuline (TG) par immunohistochimie. Les résultats montrent, pour la première fois, l'expression de la TG et confirment la présence de la TPO dans l'endomètre et les cellules de granulosa humaines. Ces résultats suggèrent que les anticorps anti-TPO et anti-TG pourraient interagir avec la TPO et TG exprimés au niveau du système reproducteur des patientes présentant des anticorps thyroïdiens positifs, perturbant ainsi leur fonction et entraînant une réponse inflammatoire pouvant conduire à des troubles de la fertilité et des complications de grossesse.

tsRNA-00764 Regulates Estrogen and Progesterone Synthesis and Lipid Deposition by Targeting PPAR-γ in Duck Granulosa Cells.

Transfer RNA-derived small RNAs (tsRNAs) are novel regulatory small non-coding RNAs that have been found to modulate many life activities in recent years. However, the exact functions of tsRNAs in follicle development remain unclear. Follicle development is a remarkably complex process that follows a strict hierarchy and is strongly associated with reproductive performance in ducks. The process of converting small yellow follicles into hierarchal follicles is known as follicle selection, which directly determines the number of mature follicles. We performed small RNA sequencing during follicle selection in ducks and identified tsRNA-00764 as the target of interest based on tsRNA expression profiles in this study. Bioinformatics analyses and luciferase reporter assays further revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) was the target gene of tsRNA-00764. Moreover, tsRNA-00764 knockdown promoted estrogen and progesterone synthesis and lipid deposition in duck granulosa cells, while a PPAR-γ inhibitor reversed the above phenomenon. Taken together, these results demonstrate that tsRNA-00764, differentially expressed in pre-hierarchal and hierarchy follicles, modulates estrogen and progesterone synthesis and lipid deposition by targeting PPAR-γ in duck granulosa cells, serving as a potential novel mechanism of follicle selection. Overall, our findings provide a theoretical foundation for further exploration of the molecular mechanisms underlying follicle development and production performance in ducks.

miR-221-5p_R-4 regulates internalized trehalose-induced autophagy by targeting NRBF2 in porcine granulosa cells.

Trehalose, as a food and feed additive, can regulate physiological and pathological processes by activating autophagy, yet the molecular mechanism of trehalose dominated a rise in autophagy has not been elucidated. This study investigated the mechanisms of trehalose-induced autophagy in porcine granulosa cells (PGCs). Trehalose was internalized into PGCs by endocytosis and caused a temporary change of the lysosome pH, ultimately inducing autophagy. Furthermore, miRNA-seq analysis of PGCs indicated that trehalose upregulated the expression of miR-221-5p_R-4, which can target nuclear receptor binding factor 2 (NRBF2) to induce autophagy of PGCs. In summary, this study reveals that miR-221-5p_R-4 targeting NRBF2 was involved in the autophagy of PGCs challenged by trehalose, which was taken up by PGCs through endocytosis.

Effects of Astaxanthin on the Physiological State of Porcine Ovarian Granulose Cells Cultured In Vitro.

The physiological state of Granulosa cells (GCs) is intricately linked to the growth and development of oocytes. Oxidative stress has been found to cause damage to GCs in vitro. Astaxanthin (AST), a well-known natural ketone-type carotenoid, has demonstrated strong antioxidant properties. This study investigates the impact of astaxanthin supplementation on the physiological state of porcine ovarian granulosa cells cultured in vitro. Variations in morphology, apoptosis, reactive oxygen species (ROS) levels, and the expression of apoptosis and anti-oxidation-related genes in porcine GCs from different passages were observed. Significant morphological changes, increases in apoptosis, and decreases in antioxidant capacity resulting from passage were observed. Subsequently, treatment with 5 µmol/L astaxanthin significantly enhanced cell viability, proliferation, antioxidant capacity and mitochondrial function while also regulating the estradiol (E2) and progesterone (P4) levels. Additionally, the gene expression of antioxidation, E2, and P4 synthesis markers was assessed, revealing reduced apoptosis and ROS levels in porcine GCs. In conclusion, supplementation with 5 µmol/L astaxanthin in vitro effectively enhances the physiological condition of porcine GCs and optimizes the culture system for these cells in vitro. Optimizing the culture system of porcine GCs in vitro can simulate the function of granulosa cells in vivo and provide a theoretical reference for further promoting follicular development, which is beneficial to improving sow fertility in actual production.

ER stress-induced LINC00173 promotes the apoptosis of ovarian granulosa cells by regulating the HRK/PI3K/AKT pathway in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and metabolic abnormality disease that mainly affects women of reproductive age. LINC00173, a novel long noncoding RNA (lncRNA), has emerged as an important factor in the development of PCOS. However, the role of LINC00173 in PCOS development and its specific upstream and downstream mechanisms remain to be further clarified. Here, we found that LINC00173 was significantly upregulated in granulosa cells (GCs) of PCOS patients, and played a crucial role in promoting apoptosis of GCs. Mechanistically, we observed the activation of endoplasmic reticulum (ER) stress in the GCs of PCOS patients, and the ER stress sensor ATF4 could directly induce LINC00173 expression by binding to its promoter. LINC00173 further upregulated the expression of Harakiri (HRK) and subsequently inhibited downstream PI3K/AKT pathway. In conclusions, our study uncovered that ER stress-induced upregulation of LINC00173 leads to increased HRK expression and inhibition of the PI3K/AKT pathway, thereby promoting the progression of PCOS. These findings provide a new therapeutic strategy for the treatment of PCOS.

Investigation of FF-MAS oxysterole's role in follicular development and its relation to hedgehog signal pathway.

The Hedgehog signaling pathway plays a crucial role in folliculogenesis; however, the association between FF-MAS oxysterol activity in folliculogenesis and the Hedgehog signaling pathway has not been revealed. The evaluation of FF-MAS activity in polycystic ovary syndrome (PCOS) with folliculogenesis disorder might provide a new approach to tackle follicular and oocyte maturation failure. The question is: does FF-MAS oxysterol affect granulosa cell (GC) proliferation? If so, is this effect facilitated through the Hedgehog pathway? To answer these questions, GCs were isolated from follicle fluids obtained from patients undergoing oocyte retrieval during in vitro fertilization (IVF) treatment. After the isolated GCs were incubated in different cell culture media, the levels of Hedgehog pathway components (SMO, Gli1) were measured by using immunohistochemical methods, cytoELISA, and qRT-PCR. Meanwhile, cell proliferation rates were determined. Significant increases (p < 0.001) in SMO and Gli1 expressions and cell proliferation were observed in the FF-MAS-treated subgroups of both PCOS and male factor participants compared to the FF-MAS deficient subgroup. Remarkably, FF-MAS positively affected the pathway components despite the pathway inhibitor cyclopamine. Although the increase in Hedgehog pathway components was slightly higher in the male factor group (MF), it was not statistically significant. In our study, we demonstrated for the first time the molecular effect of FF-MAS on human GCs in folliculogenesis. Since FF-MAS is already used in assisted reproductive techniques in animals and is known to be synthesized in the human body, it could be considered a new approach in human IVF treatments.

Taurine Protects against Silica Nanoparticle-Induced Apoptosis and Inflammatory Response via Inhibition of Oxidative Stress in Porcine Ovarian Granulosa Cells.

Silica nanoparticles (SNPs) induce reproductive toxicity through ROS production, which significantly limits their application. The protective effects of taurine (Tau) against SNP-induced reproductive toxicity remain unexplored. So this study aims to investigate the impact of Tau on SNP-induced porcine ovarian granulosa cell toxicity. In vitro, granulosa cells were exposed to SNPs combined with Tau. The localization of SNPs was determined by TEM. Cell viability was examined by CCK-8 assay. ROS levels were measured by CLSM and FCM. SOD and CAT levels were evaluated using ELISA and qPCR. Cell apoptosis was detected by FCM, and pro-inflammatory cytokine transcription levels were measured by qPCR. The results showed that SNPs significantly decreased cell viability, while increased cell apoptosis and ROS levels. Moreover, SOD and CAT were decreased, while IFN-α, IFN-ß, IL-1ß, and IL-6 were increased after SNP exposures. Tau significantly decreased intracellular ROS, while it increased SOD and CAT compared to SNPs alone. Additionally, Tau exhibited anti-inflammatory effects and inhibited cell apoptosis. On the whole, these findings suggest that Tau mitigates SNP-induced cytotoxicity by reducing oxidative stress, inflammatory response, and cell apoptosis. Tau may be an effective strategy to alleviate SNP-induced toxicity and holds promising application prospects in the animal husbandry and veterinary industry.

Advances in the Effects of Heat Stress on Ovarian Granulosa Cells: Unveiling Novel Ferroptosis Pathways.

Heat stress has been one of the key research areas for researchers due to the wide-ranging effects and complex mechanisms of action of its stress product reactive oxygen species (ROS). The aim of this paper is to comprehensively review and summarize the effects of heat stress on ovarian granulosa cells and their mechanism of action. We systematically reviewed the effects of heat stress on ovarian granulosa cells, including intracellular steroid hormone changes, oxidative stress, apoptosis, and mitochondrial function. Meanwhile, this paper discusses in detail several major mechanisms by which heat stress induces apoptosis in ovarian granulosa cells, such as through the activation of apoptosis-related genes, induction of endoplasmic reticulum stress, and the mitochondrial pathway. In addition, we analyzed the mechanism of ferroptosis in ovarian granulosa cells under heat stress conditions, summarized the potential association between heat stress and ferroptosis in light of the existing literature, and explored the key factors in the mechanism of action of heat stress, such as the signaling pathways of Nrf2/Keap1, HSPs, and JNK, and analyzed their possible roles in the process of ferroptosis. Finally, this paper provides an outlook on the future research direction, describing the possible interaction between heat stress and ferroptosis, with a view to providing a theoretical basis for further understanding and revealing the complex mechanism of ferroptosis occurrence in ovarian granulosa cells under heat stress.

Effect of Gossypol on Gene Expression in Swine Granulosa Cells.

Gossypol (GP), a polyphenolic compound in cottonseed, has notable effects on female reproduction and the respiratory system in pigs. This study aimed to discern the alterations in gene expression within swine granulosa cells (GCs) when treated with two concentrations of GP (6.25 and 12.5 µM) for 72 h, in vitro. The analysis revealed significant changes in the expression of numerous genes in the GP-treated groups. A Gene Ontology analysis highlighted that the differentially expressed genes (DEGs) primarily pertained to processes such as the mitotic cell cycle, chromosome organization, centromeric region, and protein binding. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated distinct impacts on various pathways in response to different GP concentrations. Specifically, in the GP6.25 group, pathways related to the cycle oocyte meiosis, progesterone-mediated oocyte maturation, and p53 signaling were prominently affected. Meanwhile, in the GP12.5 group, pathways associated with PI3K-Akt signaling, focal adhesion, HIF-1 signaling, cell cycle, and ECM-receptor interaction showed significant alterations. Notably, genes linked to female reproductive function (CDK1, CCNB1, CPEB1, MMP3), cellular component organization (BIRC5, CYP1A1, TGFB3, COL1A2), and oxidation-reduction processes (PRDX6, MGST1, SOD3) exhibited differential expression in GP-treated groups. These findings offer valuable insights into the changes in GC gene expression in pigs exposed to GP.

Exploration of transcriptional regulation network between buffalo oocytes and granulosa cells and its impact on different diameter follicles.

BACKGROUND: Buffalo is a globally important livestock species, but its reproductive performance is relatively low than cattles. At present, dominant follicle development specific process and mechanistic role of follicular growth related genes in water buffaloes are not well understood. Therefore, we comprehensively performed transcriptomics of granulosa cells and oocytes from different-sized follicles in water buffalo to identify key candidate genes that influence follicle development and diameter, and further explored the potential regulatory mechanisms of granulosa cells and oocytes in the process of water buffalo follicle development. RESULTS: In this study, we found918 granulosa cell transcripts and 1401 oocyte transcripts were correlated in follicles of different diameters, and the expression differences were significant. Subsequent enrichment analysis of the co-expressed differentially expressed transcripts identified several genes targeted by long non-coding RNAs (lncRNAs) and associated with follicular development. Notably, the upregulation of BUB1 regulated by MSTRG.41325.4 and interactive action of SMAD2 and SMAD7 might have key regulatory role in follicular development. Additionally, we also detected key differentially expressed genes that potentially influence follicular hormone metabolism and growth, like ID2, CHRD, TGIF2 and MAD2L1, and constructed an interaction network between lncRNA transcripts and mRNAs. CONCLUSIONS: In summary, this study preliminarily revealed the differences in gene expression patterns among buffalo follicles of different sizes and their potential molecular regulatory mechanisms. It provides a new perspective for exploring the mechanism of buffalo follicular dominance and improving buffalo reproductive performance.

[Comparative analysis of the impact of repetitive transcranial magnetic stimulation and burst transcranial magnetic stimulation at different frequencies on memory function and neuronal excitability of mice].

Transcranial magnetic stimulation (TMS) as a non-invasive neuroregulatory technique has been applied in the clinical treatment of neurological and psychiatric diseases. However, the stimulation effects and neural regulatory mechanisms of TMS with different frequencies and modes are not yet clear. This article explores the effects of different frequency repetitive transcranial magnetic stimulation (rTMS) and burst transcranial magnetic stimulation (bTMS) on memory function and neuronal excitability in mice from the perspective of neuroelectrophysiology. In this experiment, 42 Kunming mice aged 8 weeks were randomly divided into pseudo stimulation group and stimulation groups. The stimulation group included rTMS stimulation groups with different frequencies (1, 5, 10 Hz), and bTMS stimulation groups with different frequencies (1, 5, 10 Hz). Among them, the stimulation group received continuous stimulation for 14 days. After the stimulation, the mice underwent new object recognition and platform jumping experiment to test their memory ability. Subsequently, brain slice patch clamp experiment was conducted to analyze the excitability of granulosa cells in the dentate gyrus (DG) of mice. The results showed that compared with the pseudo stimulation group, high-frequency (5, 10 Hz) rTMS and bTMS could improve the memory ability and neuronal excitability of mice, while low-frequency (1 Hz) rTMS and bTMS have no significant effect. For the two stimulation modes at the same frequency, their effects on memory function and neuronal excitability of mice have no significant difference. The results of this study suggest that high-frequency TMS can improve memory function in mice by increasing the excitability of hippocampal DG granule neurons. This article provides experimental and theoretical basis for the mechanism research and clinical application of TMS in improving cognitive function.

Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells.

As a member of peroxisome proliferator-activated receptor (PPAR) family, PPARG has been reported to be involved in glucolipid metabolism in various species. However, the function of PPARG in estradiol (E2) synthesis, apoptosis, and proliferation in goat ovarian granulosa cells (GCs) is unclear. In this study, we found that goat PPARG was expressed in GCs of all grades of follicles, and localized in the cytoplasm and nucleus of GCs. Transfection of small interfering RNA-PPARG2 (si-PPARG2) decreased E2 synthesis and the abundances of HSD3B and CYP19A1 mRNA and protein. It also promoted cell apoptosis with significant increases in the ratio of BAX/BCL-2 and Caspase3 mRNA and protein. Meanwhile, cell cycle was inhibited by si-PPARG2 transfection, accompanied by decreased mRNA levels of CDK4, CKD6, MYC, CCND1, CCND2, PCNA, and CCNB, increased mRNA level of P53, and decreased protein levels of CDK4, MYC, and CCND1. Furthermore, PPARG interference affected the mRNA and protein abundances of CREB as well as the phosphorylation of CREB but not PKA. In conclusion, our data suggest that PPARG plays an important role in regulating E2 synthesis, cell apoptosis, and proliferation of goat GCs, including the CREB expression and phosphorylation. These results provide evidences for the in-depth study of PPARG in the regulation of goat GCs function.

CREBRF regulates apoptosis and estradiol via ISG15/ISGylation in pig granulosa cells.

Granulosa cells play a crucial role in the reproductive processes of female animals, as their proliferation, apoptosis, and hormonal secretion are vital for follicular development and ovulation. Although the role and mechanisms of CREBRF in the reproductive system have been partly reported, its functions in ovarian granulosa cells have not been fully explored. In this study, the results indicated that the expression of CREBRF in the ovaries at 30 days after birth was significantly higher than that during puberty and sexual maturity. Studies on the function of CREBRF found that CREBRF could enhance the synthesis of estradiol and had no effect on progesterone synthesis in pig granulosa cells. At the same time, CREBRF could suppress apoptosis through the Bax/caspase3/caspase9 pathway and modulation of ISG15/ISGylation in pig granulosa cells. During this process, the expression of many genes changed in granulosa cells. Several genes (CMPK2, MX1, MX2, ZBP1, PML, CHAC1, and BAX) which were promoted apoptosis, were upregulated after CREBRF knockdown with siRNA. ISG15-protein conjugation genes (HERC5, UBA7, UBE2L6, ISG15) were also were upregulated. On the contrary, the expression of anti-apoptotic (RFK, SNAP23) genes decreased. In conclusion, CREBRF could enhance the synthesis of estradiol and acted as anti-apoptosis role in pig granulosa cells. This discovery can provide novel insights for further elucidating the molecular mechanisms of granulosa cells in the ovary and potentially identifies CREBRF as a molecular target for improving fertility.

Molecular characterization of MSX2 gene and its role in regulating steroidogenesis in yak (Bos grunniens) cumulus granulosa cells.

Cumulus granulosa cells (CGCs) are somatic cells surrounding the oocyte that play an important role in oocyte growth, meiotic maturation, ovulation, and fertilization in mammals. Therefore, revealing the molecular mechanisms related to the development and function of CGCs is essential for further understanding the regulatory network in female reproduction. MSX2 belongs to the highly conserved msh homeobox gene family and plays diverse roles in different biological processes. This study cloned the coding sequence (CDS) of the yak MSX2 gene and detected the abundance and localization of MSX2 in the major female reproductive organs. The results indicated that the CDS of this gene included 747 base pairs and encoded 248 amino acids. The abundance of MSX2 mRNA was highly expressed in the luteal phase of the yak ovary during the estrous cycle, and MSX2 protein was widely expressed in different female reproductive organs, including the ovary, corpus luteum, uterus, and oviduct. Repressing MSX2 abundance in yak CGCs declined the cell viability and defective steroidogenesis. Several genes abundances related to cell proliferation, apoptosis, and sterogenesis also changed after MSX2 knockdown. MSX2 overexpression had the opposite effect on cell viability in yak CGCs. These results reveal the specific mechanism by which MSX2 regulates the development and function of yak CGCs and give novel and valuable insights into the mechanisms involved in yak reproduction.

In vitro growth of secondary follicles from cryopreserved-thawed ovarian cortex.

STUDY QUESTION: Can secondary follicles be obtained from cultured cryopreserved-thawed human ovarian cortical tissue? SUMMARY ANSWER: We obtained high-quality secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue from cis female donors (cOVA), but not from trans masculine donors (tOVA) in the same culture conditions. WHAT IS KNOWN ALREADY: The in vitro growth of oocytes present in unilaminar follicles into metaphase II stage (MII) oocytes has been previously achieved starting from freshly obtained ovarian cortical tissue from adult cis female donors. This involved a multi-step culture protocol and the first step included the transition from unilaminar follicles to multilayered secondary follicles. Given that the ovarian cortex (from both cis female and trans masculine donors) used for fertility preservation is cryopreserved, it is crucial to investigate the potential of unilaminar follicles from cryopreserved-thawed ovarian cortex to grow in culture. STUDY DESIGN, SIZE, DURATION: Cryopreserved-thawed ovarian cortical tissue from adult trans masculine donors (n = 3) and adult cis female donors (n = 3) was used for in vitro culture following the first culture step described in two published culture protocols (7-8 days and 21 days) and compared to freshly isolated ovarian cortex from trans masculine donors (n = 3) and to ovarian cortex prior to culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortical tissue was obtained from adult trans masculine donors undergoing gender-affirming surgery while using testosterone, and from adult cis female donors undergoing oophorectomy for fertility preservation purposes before chemotherapy. The ovarian cortex was fixed either prior (day 0) or after the culture period. Follicular survival, growth, and morphology were assessed through histology and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: We quantified the different stages of follicular development (primordial, primary, secondary, and atretic) after culture and observed an increase in the percentage of secondary follicles as well as an increase in COLIV deposition in the stromal compartment regardless of the culture media used. The quality of the secondary follicles obtained from cOVA was comparable to those prior to culture. However, in the same culture conditions, the secondary follicles from tOVA (fresh and cryo) showed low-quality secondary follicles, containing oocytes with small diameter, granulosa cells that expressed abnormal levels of KRT19 and steroidogenic-marker STAR and lacked ACTA2+ theca cells, when compared to tOVA secondary follicles prior to culture. LIMITATIONS, REASONS FOR CAUTION: The number of different donors used was limited. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed that cryopreserved-thawed cOVA can be used to generate high-quality secondary follicles after culture and those can now be further tested to evaluate their potential to generate functional MII oocytes that could be used in the clinic. However, using the same culture protocol on tOVA (fresh and cryo) did not yield high-quality secondary follicles, suggesting that either the testosterone treatment affects follicular quality or adapted culture protocols are necessary to obtain high-quality secondary follicles from tOVA. Importantly, caution must be taken when using tOVA to optimize folliculogenesis in vitro. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the European Research Council Consolidator Grant OVOGROWTH (ERC-CoG-2016-725722 to J.S.D.V. and S.M.C.D.S.L.), the Novo Nordisk Foundation (reNEW NNF21CC0073729 to H.C., F.W., J.S.D.V., S.M.C.D.S.L.), and China Scholarship Council (CSC 202008320362 and CSC 202008450034 to H.C. and F.W.), respectively. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Seasonal environmental fluctuations alter the transcriptome dynamics of oocytes and granulosa cells in beef cows.

BACKGROUND: Examining the mechanistic cellular responses to heat stress could aid in addressing the increasing prevalence of decreased fertility due to elevated ambient temperatures. Here, we aimed to study the differential responses of oocytes and granulosa cells to thermal fluctuations due to seasonal differences. Dry beef cows (n = 10) were housed together, synchronized and subjected to a stimulation protocol to induce follicular growth before ovum pick-up (OPU). Two OPU's were conducted (summer and winter) to collect cumulus-oocyte-complexes (COCs) and granulosa cells. In addition, rectal temperatures and circulating blood samples were collected during OPU. Oocytes were separated from the adherent cumulus cells, and granulosa cells were isolated from the collected OPU fluid. RNA was extracted from pools of oocytes and granulosa cells, followed by library preparation and RNA-sequencing. Blood samples were further processed for the isolation of plasma and leukocytes. The transcript abundance of HSP70 and HSP90 in leukocytes was evaluated using RT-qPCR, and plasma cortisol levels were evaluated by immunoassay. Environmental data were collected daily for three weeks before each OPU session. Data were analyzed using MIXED, Glimmix or GENMOD procedures of SAS, according to each variable distribution. RESULTS: Air temperatures (27.5 °C vs. 11.5 °C), average max air temperatures (33.7 °C vs. 16.9 °C), and temperature-humidity indexes, THI (79.16 vs. 53.39) were shown to contrast significantly comparing both the summer and winter seasons, respectively. Rectal temperatures (Summer: 39.2 ± 0.2 °C; Winter: 38.8 ± 0.2 °C) and leukocyte HSP70 transcript abundance (Summer: 4.18 ± 0.47 arbitrary units; Winter: 2.69 ± 0.66 arbitrary units) were shown to increase in the summer compared to the winter. No visual differences persisted in HSP90 transcript abundance in leukocytes and plasma cortisol concentrations during seasonal changes. Additionally, during the summer, 446 and 940 transcripts were up and downregulated in oocytes, while 1083 and 1126 transcripts were up and downregulated in the corresponding granulosa cells, respectively (Fold Change ≤ -2 or ≥ 2 and FDR ≤ 0.05). Downregulated transcripts in the oocytes were found to be involved in ECM-receptor interaction and focal adhesion pathways, while the upregulated transcripts were involved in protein digestion and absorption, ABC transporters, and oocyte meiosis pathways. Downregulated transcripts in the granulosa cells were shown to be involved in cell adhesion molecules, chemokine signaling, and cytokine-cytokine receptor interaction pathways, while those upregulated transcripts were involved in protein processing and metabolic pathways. CONCLUSION: In conclusion, seasonal changes dramatically alter the gene expression profiles of oocytes and granulosa cells in beef cows, which may in part explain the seasonal discrepancies in pregnancy success rates during diverging climatic weather conditions.

Nur77 ameliorates cyclophosphamide-induced ovarian insufficiency in mice by inhibiting oxidative damage and cell senescence.

Premature ovarian failure (POF) is among the primary causes of ovarian dysfunction that severely affects women's physical and mental health. The main purpose of this study was to explore the expression level of Nerve growth factor-induced protein B (Nur77/NR4A1) in cyclophosphamide (CTX)-induced POF. We then tested whether Nur77 can exert a protective effect after CTX treatment and investigated the mechanism of Nur77's role during ovarian injury. CTX promotes follicular atresia by inducing redox imbalance, apoptosis, and senescence, thereby causing direct toxicity to gonads. Additionally, CTX decreases ovarian reserve consumption by stimulating the excessive activation of primordial follicles. Nur77 can be stimulated by oxidative stress, DNA damage, metabolism, inflammation, etc. However, its relationship with POF remains unelucidated. We here found that Nur77 is expressed at low levels in POF ovaries. Therefore, Nur77 was identified as a regulator of ovarian injury and follicular development. According to the results, Nur77 overexpression alleviated redox imbalances, reduced cell senescence and apoptosis, and improved follicular reserve. Nur77 protects ovarian function by restoring disordered sex hormone levels and estrus cycles and promoting follicle growth and development at all levels. Moreover, the rapamycin protein kinase (AKT)/mammalian target of the rapamycin (mTOR) is a crucial regulator of the primordial follicle pool and follicular development. A relationship was observed between Nur77 and AKT through string and molecular docking. Experiments confirmed the involvement of the AKT/mTOR signaling pathway in the regulatory role of Nur77 in ovarian function. Thus, Nur77 is a critical target for POF prevention and treatment.

Regulation of Bone Morphogenetic Protein Receptor Type II Expression by FMR1/Fragile X Mental Retardation Protein in Human Granulosa Cells in the Context of Poor Ovarian Response.

Fragile X mental retardation protein (FMRP) is a translational repressor encoded by FMR1. It targets bone morphogenetic protein receptor type II (BMPR2), which regulates granulosa cell (GC) function and follicle development. However, whether this interaction affects folliculogenesis remains unclear. Therefore, this study investigated the potential effect of FMRP-BMPR2 dysregulation in ovarian reserves and infertility. COV434 cells and patient-derived GCs were used to evaluate FMRP and BMPR2 expression. Similarly, FMR1, BMPR2, LIMK1, and SMAD expression were evaluated in GCs with normal (NOR) and poor (POR) ovarian responses. FMRP and BMPR2 were expressed in both cell types. They were co-localized to the nuclear membrane of COV434 cells and cytoplasm of primary GCs. FMR1 silencing increased the mRNA and protein levels of BMPR2. However, the mRNA levels of FMR1 and BMPR2 were significantly lower in the POR group. FMR1 and BMPR2 levels were strongly positively correlated in the NOR group but weakly correlated in the POR group. Additionally, SMAD9 expression was significantly reduced in the POR group. This study highlights the crucial role of FMR1/FMRP in the regulation of BMPR2 expression and its impact on ovarian function. These findings indicate that the disruption of FMRP-BMPR2 interactions may cause poor ovarian responses and infertility.

Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination.

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. SIGNIFICANCE: The application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome.

The dual role of ATG7: Regulation of autophagy and apoptosis in porcine ovarian follicular granulosa cells.

The regulation of mammalian ovarian development involves the coordinated processes of autophagy and apoptosis. The autophagy-related gene ATG7 plays a pivotal role in mediating crosstalk between these pathways. Despite its recognized importance, the specific function of ATG7 in ovarian follicular granulosa cells remains poorly understood. This study aimed to explore the effects of ATG7 overexpression on apoptosis and autophagy in porcine ovarian follicular granulosa cells and thereby provide insights into the interplay between these fundamental cellular mechanisms. An ATG7 overexpression vector was introduced into cells, followed by assessment of cell proliferation using the CCK-8 assay, quantification of related gene expression via real-time quantitative PCR and western blotting, and evaluation of apoptosis using TUNEL staining. ATG7 exhibited a predominant cytoplasmic localization and additional nuclear expression in porcine ovarian follicular granulosa cells. The transfection efficiency of the vector was initially verified, indicating that its overexpression notably increased expression of ATG7 protein. Further investigations confirmed that overexpression of ATG7 inhibited cell proliferation, stimulated autophagy, and promoted apoptosis in these cells. In summary, overexpression of ATG7 influences the viability of porcine ovarian follicular granulosa cells by regulating the interplay between autophagy and apoptosis. This study not only broadens the understanding of functional regulation of autophagy and apoptosis by ATG7, but also sheds light on the intricate mechanisms governing ovarian follicular atresia.