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Ovarian tissue cryopreservation addresses critical challenges in fertility preservation for prepubertal female cancer patients, such as the lack of viable eggs and hormonal deficiencies. However, mitigating follicle and granulosa cell damage during freeze-thaw cycles remains an urgent issue. Luteinizing hormone (LH), upon binding to luteinizing hormone receptors (LHR) on granulosa cells, enhances estrogen synthesis and secretion, contributing to the growth of granulosa cells and follicles. This study examined mouse ovarian follicles and granulosa cells to identify optimal LH treatments using morphological assessments and LIVE/DEAD assays. The study found significant increases in the expression of Leucine-rich G-protein-coupled receptor 5 (Lgr5) and Forkhead box L2 (Foxl2) in mural and cumulus granulosa cells under LH influence, alongside marked reductions in active caspase-3 expression. Double immunofluorescence of Ki67 with Foxl2 and Lgr5 revealed ongoing proliferative activity in granulosa cells post freeze-thaw. In addition, LH treatment significantly boosted the expression of transforming growth factor (TGF-β) and its superfamily members in both granulosa cells and oocytes. These findings suggest that LH addition during cryopreservation can diminish damage to follicles and granulosa cells, offering new strategies to enhance the efficacy of mammalian ovarian cryopreservation. [ABSTRACT FROM AUTHOR] more...

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. • Detecting MSX2 expression in yak reproductive organs. • MSX2 is crucial for steroidogenesis in yak cumulus granulosa cells. [ABSTRACT FROM AUTHOR] more...

Growth differentiation factor 9 (GDF9), an oocyte-secreted factor, plays a vital role in porcine oocyte development. However, its function during oocyte in vitro maturation (IVM) remains unclear. In this study, we achieved GDF9 editing in approximately 59 % of cultured oocytes by cytoplasmic injection of a pre-assembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex into porcine oocytes at the germinal vesicle (GV) stage. GDF9 editing caused significant damage to porcine oocytes during IVM. Additionally, GDF9 editing impaired mitochondrial function, increased reactive oxygen species (ROS) accumulation, and decreased glutathione (GSH) levels. The impaired IVM of GDF9-edited porcine oocytes was primarily driven by active cAMP-PKA signaling, which inhibited MOS expression, leading to the activation of the WEE1B/MYT1 kinase and inactivation of CDC25B phosphatase. This cascade resulted in the inactivation of CDK1, thereby preventing the activation of maturation-promoting factor (MPF) and inhibiting first polar body (PB1) extrusion. Our findings enhance the understanding of GDF9's regulatory role in porcine oocyte IVM and provide a theoretical foundation for improving porcine reproductive performance. • Cytoplasmic injection of ctRNP can efficiently disrupt GDF9 in porcine oocytes, resulted in reduced oocyte maturation by impairing oocyte maturation promoting factor expression. • Disrupted expression of GDF9 in porcine oocytes impaired SMAD2/3 signaling in the cumulus granulosa cells. • Disrupted expression of GDF9 in porcine oocytes impaired cytoplasmic maturation by impairing mitochondrial function. [ABSTRACT FROM AUTHOR] more...

The maturation of oocytes has an important impact on the subsequent development of the embryo. However, during the in vitro maturation (IVM) of oocytes, oocytes are separated from the follicular environment, resulting in a low maturation rate of oocytes in vitro. In order to improve maturation rate of IVM of porcine oocytes, this study was conducted to investigate using sodium alginate (SA) to encapsulate porcine mural granulosa cells (MGs) to develop an SA three-dimensional (3D) co-culture system for IVM of porcine oocytes. And, gene expression, reactive oxygen species (ROS), ATP level, mitochondrial membrane potential, parthenogenetic activation development results of cultured oocytes, and as well as ROS and glutathione (GSH) levels in cumulus granulosa cells (CGs) were detected. Our results showed that the maturation rate of the SA 3D co-culture group was 85.41 %, that of the negative control (NC) group was 79.24 %, and that of the MGs co-culture group was 81.62 %. In SA 3D co-culture group, mitochondrial membrane potential level of oocytes was 1.6, ROS level was 19 and the ATP level was 1.7. While in NC group, mitochondrial membrane potential level of oocytes was 1.2, the ROS level was 52, and the ATP level was 0.4. The ROS level in the CGs of SA 3D co-culture group decreased by 1.5 times, and the glutathione content increased by 2.3 times. In the SA 3D co-culture group, GDF9 gene expression level was 2.0, and BMP15 gene expression level was 1.2. While in NC group, GDF9 gene expression level was 0.7, and BMP15 gene expression level was 0.6. The blastocyst rate in the SA 3D co-culture group was 41.4 %, and that in the NC group was 36.6 %. In conclusion, encapsulating MGs in SA gel and co-culturing them with porcine oocytes in 3D during IVM can improve the developmental potential of oocytes. This result will provide an important reference for improving the methods of in vitro maturation of oocytes. • The 3D co-culture system was established using sodium alginate encapsulated mural granulosa cells. • The 3D co-culture system improves the oocyte maturation rate. • The 3D co-culture system improves the development potential of oocytes. [ABSTRACT FROM AUTHOR] more...

This study investigated the potential role and underlying mechanisms of oleanolic acid (OA), a pentacyclic triterpene with antioxidant and anti-inflammatory properties, in porcine oocytes during in vitro maturation (IVM). The results showed that supplementation with 5 μM OA during IVM resulted in a greater percentage of mature oocytes, parthenogenetically activated embryos and somatic cell nuclear-transferred embryos. This was evidenced by significant increases in the rate of first polar body expulsion, the expansion of cumulus granulosa cells and the total cell number in blastocysts. Further analysis revealed that OA promoted fatty acid accumulation and upregulated the mRNA expression of genes involved in fatty acid β-oxidation. OA significantly increased the intracellular mitochondrial membrane potential and ATP levels and effectively inhibited BAX/BCL2 and Cleaved Caspase3 protein expression. Notably, OA increased the protein levels of intracellular Nrf2 and HO-1, and the GSH levels and the activities of the antioxidant enzymes SOD and catalase (CAT), while reducing ROS levels. Mechanistically, OA activated the Nrf2/HO-1 signalling pathway, which is crucial for regulating the expression of antioxidant-related targets in IVM porcine oocytes. Our findings indicated that OA improved antioxidant capacity by activating the Nrf2/HO-1 signalling pathway, thereby promoting porcine oocyte maturation. • Supplementation with OA during IVM promotes porcine oocyte maturation and fosters embryonic development in vitro. • OA sustains lipid metabolism in porcine oocytes. • OA elevates mitochondrial function and antioxidant capacity in porcine oocytes. • OA activated the Nrf2/HO-1 signalling pathway in porcine oocytes. [ABSTRACT FROM AUTHOR] more...

Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHRN316S mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHRN316S mice exhibited reduced numbers of corpora lutea and ovulation. The LHRN316S mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHRN316S female mice with ICR wild type male mice revealed that the infertility rate of LHRN316S mice was 21.4% (3/14). Litter sizes from LHRN316S mice were smaller than those from control wild type female mice. The oocytes from LHRN316S mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHRN316S mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment. [ABSTRACT FROM AUTHOR] more...

Background: Advanced maternal age is associated with decreased oocyte quantity and quality and in vitro fertilization (IVF) success rates. This study aimed to investigate whether melatonin supplementation can improve IVF outcomes in women of advanced maternal age by modulating cuproptosis and ferroptosis. Methods: This prospective cohort study included 161 women aged 35–45 years undergoing IVF-frozen embryo transfer cycles. Participants were assigned to either melatonin (n = 86, 2 mg daily for ≥ 8 weeks) or control (n = 75) groups. Cumulus cells were analyzed for cuproptosis and ferroptosis-related gene expression. Additional experiments were conducted on the HGL5 human granulosa cell line to assess mitochondrial function and metabolic reprogramming. Results: Melatonin supplementation significantly improved IVF outcomes in women aged ≥ 38 years, increasing clinical pregnancy rates (46.0% vs. 20.3%, P < 0.01), ongoing pregnancy rates (36.5% vs. 15.3%, P < 0.01), and live birth rates (33.3% vs. 15.3%, P < 0.05). In cumulus cells from patients, gene expression analysis revealed that melatonin modulated cuproptosis and ferroptosis-related genes, including ATP7B and GPX4, with more pronounced effects in the ≥ 38 years group. This suggests melatonin enhances cellular resilience against oxidative stress and metal-induced toxicity in the ovarian microenvironment. In vitro studies using HGL5 cells showed melatonin reduced oxidative stress markers, improved mitochondrial function, restored expression of glycolysis and TCA cycle-related genes and modulated cuproptosis and ferroptosis-related gene expression. These findings provide mechanistic insight into melatonin's protective effects against regulated cell death in ovarian cells, potentially explaining the improved IVF outcomes observed. Conclusions: Melatonin supplementation significantly improved IVF outcomes in women of advanced maternal age, particularly those ≥ 38 years old, likely by modulating cuproptosis and ferroptosis and enhancing mitochondrial function in cumulus and granulosa cells. These results suggest that melatonin could be a promising adjuvant therapy for improving IVF success rates in older women. [ABSTRACT FROM AUTHOR] more...

The growth of the ovarian antral follicle is a complex process that is difficult to study, especially in human and nonhuman primates. Understanding the antral stage of development is key to new approaches to regulating reproduction. This study analyzed cohorts of three sizes of developing antral follicles obtained from adult rhesus macaque females using RNA sequencing of oocytes and cumulus and granulosa cells. The overall objective of this study was to identify key developmental changes in gene expression in oocytes, granulosa, and cumulus cells, as nonhuman primate antral stage follicles transition through progressively larger sizes in the absence of exogenous hormonal stimulation. Only a relatively small number of genes displayed altered mRNA expression levels in any of the three cell types during this period. Most of the identified differentially expressed genes (DEGs) decreased in the granulosa cells or increased in the cumulus cells. Although the number of DEGs observed was small, these DEGs indicate predicted effects on distinct upstream regulators in the cumulus and granulosa cells. This study is particularly important because it shows for the first time the gene expression changes during antral follicle growth in a medically relevant model. NEW & NOTEWORTHY: Changes in gene expression in oocytes, granulosa, and cumulus cells were determined in nonhuman primate antral stage ovarian follicles transitioning through progressively larger sizes without exogenous hormonal stimulation. Only a small number of genes displayed altered mRNA expression levels in any of the three cell types. Most of the differentially expressed genes (DEGs) decreased in granulosa cells or increased in cumulus cells. These results identified upstream regulators of antral follicle development. [ABSTRACT FROM AUTHOR] more...

Granulosa cells (GCs) in secondary follicles differentiate into cumulus cells (CCs) and mural granulosa cells (MGCs) in the antral follicle. Only CCs maintain direct connections with oocytes through transzonal projections (TZPs) and support oocyte growth. Here, we examined whether granulosa cells (GCs) from secondary follicles and MGCs from early and late antral follicles were able to reconstruct complexes with TZP-free denuded oocytes (DOs) and regenerate TZPs. Furthermore, to confirm that the regenerated TZPs were functional, the development of the reconstructed complexes and oocyte growth in the complexes were evaluated. After coculture, GCs and MGCs from early antral follicles reconstructed the complexes with DOs and regenerated TZPs. Furthermore, the oocytes in the integrally reconstructed complexes grew fully and acquired meiotic competence, suggesting that the regenerated TZPs were functional. In contrast, MGCs from the late antral follicles lost their ability to elongate TZPs. As the ability to regenerate TZPs differed among cells, we analyzed the transcriptomes of GCs, CCs, and MGCs collected from follicles of different sizes. The characteristics of TZP generation coincided with the transcriptome changes in two directions: from GCs to CCs and MGCs. In conclusion, until the early antral follicle stage, bovine GCs, CCs, and MGCs have common characteristics to elongate TZPs and form antrum-like structures that support oocyte growth in vitro. Furthermore, as the follicle develops, MGCs lose the ability to elongate TZPs. [ABSTRACT FROM AUTHOR] more...

Mature, vital, and motile spermatozoa are essential for reaching the oocyte and binding to hyaluronic acid (HA) in the cumulus oophorus matrix. This study aims to determine the relationship between sperm-migration ability and HA-binding potential, as well as the relationship between sperm concentration and motility. Semen samples were collected from 702 men aged 20–56 years (median 34.8). We evaluated the sperm concentration and motility from basic semen analysis, the swim-up test (expressed as millions per mL and the migration efficiency percentage), and the hyaluronan-binding assay (HBA). A moderate positive correlation was found between the migration test results and HBA (R = 0.48). The highest correlation was observed between the concentration of motile spermatozoa and the migration test results (R = 0.85) and HBA (R = 0.4). The sperm migration efficiency strongly correlated with progressive motility (R = 0.6). Although significantly higher sperm migration was observed in patients with normal HBA results, the results of the functional tests were found to differ in some cases. For infertility treatment, the current diagnostic algorithm should be enhanced with more comprehensive seminological methods that assess the sperm-migration ability and HA-binding potential. We also recommend incorporating the swim-up method into the diagnostic protocol before planning assisted reproductive technology (ART) treatment. [ABSTRACT FROM AUTHOR] more...

The oocyte quality decreases during ovarian tissue transport to the laboratories of in vitro embryo production. To provide additional information on how the conditions of transporting sheep ovaries impact the ovarian tissue and oocytes’ ability to develop into blastocyst stages, we have studied new transport media Ankara University Zootekni (AUZ1, AUZ2) supplemented with antioxidants (melatonin, Vit E, and Vit A), buffer solution, and energy substrates, and compared them with the traditional transport media: Phosphate-Buffered Saline (PBS), and Charles Rosenkrans 1 (CR1), Normal Saline (NS) at different temperatures (-6 to 30 °C). We also studied and compared how well different transport media preserve the ovarian tissue's structural integrity while transporting sheep ovaries at 4°C. Our findings indicated that various temperatures and transport media play critical roles in embryo development. The embryo development rates showed that when sheep ovaries are transported in AUZ1, they produce oocytes with a higher embryo development rate than other transport media at any temperature. In addition, histology examination revealed that the transport of sheep ovarian tissue in any medium at a temperature of 4 °C did not negatively impact the viability and histomorphology of the primordial, primary, and secondary follicles. In contrast to other transport media, the AUZ1 medium maintained the normal morphology of antral follicles, Graafian follicles, and the cumulus oophorus of sheep ovarian tissue. In conclusion, adding melatonin, buffer solution, and energy substrates to the transportation medium of ovarian tissues has a beneficial and positive role in maintaining ovarian tissue and increasing the rates of embryonic development. [ABSTRACT FROM AUTHOR] more...

Calcium (Ca2+) is a second messenger for many signal pathways, and changes in intracellular Ca2+ concentration ([Ca2+]i) are an important signaling mechanism in the oocyte maturation, activation, fertilization, function regulation of granulosa and cumulus cells and offspring development. Ca2+ oscillations occur during oocyte maturation and fertilization, which are maintained by Ca2+ stores and extracellular Ca2+ ([Ca2+]e). Abnormalities in Ca2+ signaling can affect the release of the first polar body, the first meiotic division, and chromosome and spindle morphology. Well-studied aspects of Ca2+ signaling in the oocyte are oocyte activation and fertilization. Oocyte activation, driven by sperm-specific phospholipase PLCz, is initiated by concerted intracellular patterns of Ca2+ release, termed Ca2+ oscillations. Ca2+ oscillations persist for a long time during fertilization and are coordinately engaged by a variety of Ca2+ channels, pumps, regulatory proteins and their partners. Calcium signaling also regulates granulosa and cumulus cells’ function, which further affects oocyte maturation and fertilization outcome. Clinically, there are several physical and chemical options for treating fertilization failure through oocyte activation. Additionally, various exogenous compounds or drugs can cause ovarian dysfunction and female infertility by inducing abnormal Ca2+ signaling or Ca2+ dyshomeostasis in oocytes and granulosa cells. Therefore, the reproductive health risks caused by adverse stresses should arouse our attention. This review will systematically summarize the latest research progress on the aforementioned aspects and propose further research directions on calcium signaling in female reproduction. [ABSTRACT FROM AUTHOR] more...

Copyright of Journal of Northwest A & F University - Natural Science Edition is the property of Editorial Department of Journal of Northwest A&F University (Natural Science Edition) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) more...

The study describes the morphometric characteristics of the ovaries, follicles, and oocytes of an adult female gray wolf. Macroscopic examination revealed ellipsoidal ovaries, which measured 9x18 mm and weighed 844 mg and 752 mg for the left and right ovaries, respectively. Two large follicles (4 mm) were prominent on the surface of the left ovary. The mean diameter of the collected primary oocytes (n=22) was 116.8±12.35 μm (including zona pellucida). Microscopic examination revealed dark, finely granulated, homogenous cytoplasm surrounded by a clearly visible zona pellucida and 2-5 layers of cumulus cells. Limited scientific studies are available due to challenges in obtaining biological material. The present analysis offers preliminary insights into the morphological features of the gray wolf oocytes and is informative for future research aimed at species' conservation. [ABSTRACT FROM AUTHOR] more...

Cellular metabolism, apoptosis, fertilization, and proliferation of granulosa cells belong to a battery of processes where microRNAs can be detected and associated with infertility. The aim of the present review is to focus on mammalian oocyte maturation events and the association between oocyte growth and miRNA expression. PubMed/Medline, Google Scholar and Scopus databases were searched, and 33 studies were included. Regarding the correlation among miRNA expression and the regulation of granulosa cells and cumulus cells, the most important miRNAs were let-7b, let-7c and miR-21. Additionally, the loss of Dicer, an enzyme involved in miRNA biogenesis, is probably a crucial factor in oogenesis, oocyte maturation and embryogenesis. Furthermore, miRNAs interfere with different cellular mechanisms like apoptosis, steroidogenesis, genome integrity, angiogenesis, antioxidative response and, consequently, oocyte maturation. Hence, it is of major importance to clarify the role and mechanism of each miRNA as understanding its action may develop new tools and establish new diagnostic and treatment approaches for infertility and ovarian disorders. [ABSTRACT FROM AUTHOR] more...

Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment. [ABSTRACT FROM AUTHOR] more...

(1) Background: In this paper we aim to study the relationship between the expression levels of molecules involved in apoptotic/survival pathways, considered as molecular markers of oocyte competence (i.e., AKT, p-AKT, ERK1/2, and p-ERK1/2) in mural granulosa cells (MGCs) and the administration of r-FSH alone or combined with exogenous r-LH, in ovarian stimulation protocol. Moreover, we aim to evaluate oocyte competence by comparing normally cleaved embryos that were transferred in the uterus, with embryos that were arrested during in vitro culture. (2) Methods: The study included 34 normo-responder women undergoing ICSI procedures. All subjects were divided into two groups. Group A consisted of 18 women stimulated with r-FSH and used as a control group; Group B consisted of 14 women stimulated with r-FSH combined with r-LH. The MGCs were obtained from individual follicles. Immunoblot analyses were carried out to analyze the AKT, p-AKT, ERK1/2, and p-ERK1/2 levels in MGCs and to correlate them with the ovarian stimulation protocol. Furthermore, the oocyte competence was evaluated, for each follicle, according to the development of the embryo during in vitro culture and the pregnancy outcome. (3) Results: We found no significant difference in the levels of molecules in isolated MGCs between groups A and B. These results, in light of our previous research, suggest for the first time, to our knowledge, that cumulus cells and mural granulosa cells in the same follicle show different expression levels of molecules involved in the apoptotic mechanism. (4) Conclusions: Our results could clarify some controversial data in the literature where cumulative cell pools of cumulus and granulosa were analyzed, described as ovarian follicle cells, and used as markers of oocyte competence. In this paper, we found evidence that cumulus and granulosa cells need to be analyzed separately. [ABSTRACT FROM AUTHOR] more...

This article, titled "Editorial: Cytoprotective role of mitochondria in reproduction," explores the important role of mitochondria in reproductive biology. Mitochondria, traditionally known as the powerhouse of the cell, have been found to play a multifaceted role in reproduction. They regulate germ cell fate, ensure cellular integrity, and protect reproductive tissues from age-related degeneration. The article discusses the potential therapeutic strategies for reproductive diseases by targeting mitochondrial pathways. It also highlights studies on mitochondrial function in conditions such as primary ovarian insufficiency, obesity-related fertility issues, embryo development, and preeclampsia. The research contributes to a better understanding of the cytoprotective mechanisms of mitochondria in reproduction and opens avenues for future medical interventions to improve human reproduction. [Extracted from the article] more...

Background: Quiescin sulfhydryl oxidase 2 (QSOX2) is a flavin adenine dinucleotide-dependent sulfhydryl oxidase that is known to be involved in protein folding, cell growth regulation, and redox state modification through oxidative activities. Earlier studies demonstrated the tissue and cellular localization of QSOX2 in the male reproductive tract, as well as the highly-regulated mechanism of QSOX2 protein synthesis and expression through the coordinated action of testosterone and epididymal-enriched amino acid, glutamate. However, the presence and the functions of QSOX2 in female reproduction are unknown. In this study, we applied the Cre-loxP gene manipulation system to generate the heterozygous and homozygous Qsox2 knockout mice and examined its effects on ovarian function. Results: We demonstrated that QSOX2 was detected in the follicle-supporting cells (granulosa and cumulus cells) of ovarian follicles of all stages but was absent in the corpus luteum, suggesting its supportive role in folliculogenesis. In comparison with reproductive organogenesis in wild-type mice, there was no difference in testicular and epididymal structure in male Qsox2 knockout; however, Qsox2 knockout disrupted the regular ovulation process in female mice as a drastic decrease in the formation of the corpus luteum was detected, and no pregnancy was achieved when mating males with homozygous Qsox2 knockout females. RNAseq analyses further revealed that Qsox2 knockout altered critical signaling pathways and genes that are responsible for maintaining ovarian functions. Conclusion: Our data demonstrated for the first time that Qsox2 is critical for ovarian function in mice. [ABSTRACT FROM AUTHOR] more...

In the field of human in vitro fertilization (IVF), selecting the best oocyte for freezing or embryo for transfer remains an important focus of clinical practice. Although several techniques are and have been used for this goal, results have generally not been favorable and/or are invasive such that damage to some embryos occurs, resulting in a reduced number of healthy births. Therefore, the search continues for non-invasive oocyte and embryo quality markers that signal the development of high-quality embryos. Multiple studies indicate the important positive effects of retinoic acid (RA) on oocyte maturation and function. We previously showed that a high follicular fluid (FF) RA concentration at the time of oocyte retrieval in IVF protocols was associated with oocytes, giving rise to the highest quality embryos, and that cumulus granulosa cells (CGCs) are the primary source of follicle RA synthesis. Data also demonstrated that connexin-43 (Cx43), the main connexin that forms gap junctions in CGCs, is regulated by RA and that RA induces a rapid increase in gap junction communication. Here, we hypothesize that CGC RA plays a causal role in oocyte competency through its action on Cx43 and, as such, may serve as a biomarker of oocyte competence. Multiple studies have demonstrated the requirement for Cx43 in CGCs for the normal progression of folliculogenesis, and that the increased expression of this connexin is linked to the improved developmental competence of the oocyte. The data have shown that RA can up-regulate gap junction intercellular communication (GJIC) in the cumulus–oocyte complex via a non-genomic mechanism that results in the dephosphorylation of Cx43 and enhanced GJIC. Recognizing the positive role played by gap junctions in CGCs in oocyte development and the regulation of Cx43 by RA, the findings have highlighted the possibility that CGC RA levels may serve as a non-invasive indicator for selecting high-quality oocytes for IVF procedures. In addition, the data suggest that the manipulation of Cx43 with retinoid compounds could provide new pharmacological approaches to improve IVF outcomes in cases of failed implantation, recurrent miscarriage, or in certain diseases that are characterized by reduced fecundity, such as endometriosis. [ABSTRACT FROM AUTHOR] more...

Polycystic ovarian syndrome (PCOS) is a commonly occurring reproductive disorder among the reproductive-aged women. Its global occurrence varies based on diagnostic guidelines, ethnicities, and locations of concern. Insulin resistance (IR) is commonly observed around 65–70% of women diagnosed with PCOS, representing a prevalent association. Consequently, the study was designed with an objective of illustrating the effect of insulin on mural and cumulus granulosa cells (GCs) of PCOS patients in comparison to normal ovulating women. This study is a case–control design, wherein a total of 80 participants were recruited meeting criterion of inclusion and exclusion, divided into 8 groups with each group consisting of 10 samples. The process involves the isolation and culturing of mural granulosa cells (MGC) and cumulus granulosa cells (CGC) with and without exposure to insulin. The proteins released by untreated GCs and insulin-treated GCs were extracted, and complex protein mixtures were digested with trypsin, followed by tandem mass spectrometry analysis and data processing using bioinformatics. We found 595 proteins in both control and PCOS samples, of which 310 were contributed by MGCs and 285 by CGCs. The PCOS MGCs expressed 20%, both the normal MGCs and CGCs have equal representation of 16% by each, whereas the PCOS CGCs proteins contributed 15% of the total of the proteomic expression. However, the poor expression observed with the Insulin exposure, the Insulin treated PCOS CGCs contributes 13%, PCOS MGCs contributes 8%. The normal MGCs upon the Insulin treatment give 8% then and there only 4% of proteins expressed by normal CGCs after Insulin treatment. The Venn analysis widened on their precise expression topographies. The examination of strings exhibited important protein–protein interaction pathways. This is a pioneering investigation aimed to establish the link between hyperinsulinemia in localized follicular GCs and PCOS mechanisms by comparing them to control group. The examination of various attributes, mechanisms, and traits shown by genes and proteins in individuals with PCOS compared to control populations, alongside the investigation of the dynamics of these genes and proteins following exposure to insulin, holds promise for the formulation of novel hypotheses and strategies in the identification of new biomarkers. [ABSTRACT FROM AUTHOR] more...

Type 2 taste receptors (TAS2Rs), traditionally known for their role in bitter taste perception, are present in diverse reproductive tissues of both sexes. This review explores our current understanding of TAS2R functions with a particular focus on reproductive health. In males, TAS2Rs are believed to play potential roles in processes such as sperm chemotaxis and male fertility. Genetic insights from mouse models and human polymorphism studies provide some evidence for their contribution to male infertility. In female reproduction, it is speculated that TAS2Rs influence the ovarian milieu, shaping the functions of granulosa and cumulus cells and their interactions with oocytes. In the uterus, TAS2Rs contribute to uterine relaxation and hold potential as therapeutic targets for preventing preterm birth. In the placenta, they are proposed to function as vigilant sentinels, responding to infection and potentially modulating mechanisms of fetal protection. In the cervix and vagina, their analogous functions to those in other extraoral tissues suggest a potential role in infection defense. In addition, TAS2Rs exhibit altered expression patterns that profoundly affect cancer cell proliferation and apoptosis in reproductive cancers. Notably, TAS2R agonists show promise in inducing apoptosis and overcoming chemoresistance in these malignancies. Despite these advances, challenges remain, including a lack of genetic and functional studies. The application of techniques such as single‐cell RNA sequencing and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated endonuclease 9 gene editing could provide deeper insights into TAS2Rs in reproduction, paving the way for novel therapeutic strategies for reproductive disorders. [ABSTRACT FROM AUTHOR] more...

Purpose: LH induces the expression of EGF‐like factors and their shedding enzyme (ADAM17) in granulosa cells (GCs), which is essential for ovulation via activation of the ErbB–ERK1/2 pathway in cumulus cells (CCs). Neurotensin (NTS) is reported as a novel regulator of ovulation, whereas the NTS‐induced maturation mechanism in oocytes remains unclear. In this study, we focused on the role of NTS in the expression of EGF‐like factors and ErbBs, and ADAM17 activity, during oocyte maturation and ovulation in mice. Methods: The expression and localization in GC and CC were examined. Next, hCG and NTS receptor 1 antagonist (SR) were injected into eCG‐primed mice, and the effects of SR on ERK1/2 phosphorylation were investigated. Finally, we explored the effects of SR on the expression of EGF‐like factors and ErbBs, and ADAM17 activity in GC and CC. Results: NTS was significantly upregulated in GC and CC following hCG injection. SR injection suppressed oocyte maturation and ERK1/2 phosphorylation. SR also downregulated part of the expression of EGF‐like factors and their receptors, and ADAM17 activity. Conclusions: NTS induces oocyte maturation through the sustainable activation of the ERK1/2 signaling pathway by upregulating part of the EGF‐like factor‐induced pathway during oocyte maturation in mice. [ABSTRACT FROM AUTHOR] more...

Purpose: We investigated the interactions between mural granulosa cells (MGCs) and cumulus granulosa cells (CGCs) during ovulation after the LH surge. Methods: We performed clustering, pseudotime, and interactome analyses utilizing reported single‐cell RNA sequencing data of mouse ovary at 6 h after eCG‐hCG injection. Results: Clustering analysis classified granulosa cells into two distinct populations, MGCs and CGCs. Pseudotime analysis divided granulosa cells into before and after the LH surge, and further divided them into two branches, the ovulatory MGCs and the ovulatory CGCs. Interactome analysis was performed to identify the interactions between MGCs and CGCs. Twenty‐six interactions were acting from CGCs toward MGCs, involving ovulation and steroidogenesis. Thirty‐six interactions were acting from MGCs toward CGCs, involving hyaluronan synthesis. There were 25 bidirectional interactions, involving the EGFR pathway. In addition, we found three novel interactions: Ephrins–Ephs pathway and Wnt–Lrp6 pathway from CGCs to MGCs, associated with steroidogenesis and lipid transport, respectively, and TGF‐β–TGFBR1 pathway from MGCs to CGCs, associated with hyaluronan synthesis. Conclusions: MGCs and CGCs interact with each other in the preovulatory follicle after the LH surge, and their interactions have roles in corpus luteum formation, oocyte maturation, and follicle rupture. [ABSTRACT FROM AUTHOR] more...

Background: A fine-tuned pro-inflammatory and anti-inflammatory balance in the follicular unit is essential for cumulus expansion and successful ovulation. While the long pentraxin 3 (PTX3) gene is required for the expansion of cumulus cells (CCs), ovulation, resumption of meiosis and fertilization, the vitamin D receptor gene (VDR-X2) is required for intra-follicle redox balance. This study was planned to determine the expression pattern of VDR-X2 and PTX3 mRNA in CCs isolated from germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes of PCOS patients with ovulatory dysfunction. Methods: The relative expression of CC-PTX3 and CC-VDR-X2 mRNA were evaluated using qRT-PCR in a total of 79 CC samples collected from individual cumulus-oocyte complex of 40 infertile patients (20 PCOS and 20 non-PCOS normal responders) who underwent ovarian stimulation with the GnRH antagonist protocol. Results: Relative PTX3 mRNA expressions of CCMI-control and CCMII-control showed 3- and 9-fold significant upregulation compared to CCGV-control, respectively. The relative PTX3 mRNA expression of CCMII-control increased approximately three fold compared to CCMI-control. Compared to CCGV-pcos, a 3-fold increase was noted in the relative PTX3 mRNA expression of CCMI-pcos and an approximately 4-fold increase in the PTX3 mRNA expression of CCMII-pcos. Relative PTX3 mRNA expression values of CCMII-pcos and CCMI-pcos were similar. A 6-fold upregulation of relative PTX3 mRNA and a 4-fold upregulation of VDR-X2 mRNA were detected in CCMII-control compared to CCMII-pcos. CC-VDR-X2 expression patterns of the PCOS and control groups overlapped with the CC-PTX3 pattern. Fertilization rates of the PCOS group exhibiting failed transcript expression were similar to normal responders. Conclusion: The fact that relative CC-PTX3 and CC-VDR mRNA expression does not increase during the transition from MI to MII stage in PCOS as in normal responders suggests that PTX3 and VDR expression may be defective in cumulus cells of PCOS patients with ovulatory dysfunction. [ABSTRACT FROM AUTHOR] more...

BACKGROUND: The granulosa cell state greatly affects the quality of oocytes. As the increased proportion of aged women in assisted reproduction, granulosa cells from patients of different ages are examined at transcript levels in order to better assess oocyte quality. OBJECTIVE: To investigate the expression changes of mRNA and long non-coding RNA in granulosa cells from the young and aged patients. METHODS: Waste cumulus granulosa cells obtained during the assisted reproductive cycle. Young group was in 25-35 years old, and the aged group was in 38- 45 years old. Granulosa cells collected from the young and aged females were analyzed using RNA sequencing, three replications for each group. RESULTS AND CONCLUSION: (1) The RNA sequencing analysis results showed that the average sequencing volume of samples was more than 14 G; the data quality Q30 after quality control was more than 93%; the average mapping rate of data was 98.4%, which indicates that the data had good quality. (2) The results of principal component analysis and correlation analysis showed that the samples of the aged group and the young group could be clearly distinguished. (3) The difference analysis results showed that compared with the young group, a total of 410 differentially expressed mRNAs were detected in the aged group (167 up-regulated and 243 down-regulated). GO analysis results showed that the down-regulated genes were mainly enriched in regulatedexocytosis and the emetabolicprocess. GSEA analysis results showed that secretion-related pathways in the aged group were down-regulated. (4) Compared with the young group, 662 differentially expressed long non-coding RNAs were detected in the aged group, and 1 772 protein-coding genes were directly regulated by these long non-coding RNAs, among which 59 genes overlaped with differentially expressed genes. The results showed that the expression of secretion-related genes and pathways in granulosa cells of the aged group was down-regulated, thus affecting oocyte quality. At the same time, these down-regulated genes were regulated by long non-coding RNA. Therefore, the expression of long non-coding RNA might be related to age. [ABSTRACT FROM AUTHOR] more...

To acquire the capacity to fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical reactions in the female reproductive tract, which are collectively called capacitation. The capacitated spermatozoa subsequently interact with the oocyte zona-pellucida and undergo the acrosome reaction, which enables the penetration of the oocyte and subsequent fertilization. However, the spontaneous acrosome reaction (sAR) can occur prematurely in the sperm before reaching the oocyte cumulus oophorus, thereby jeopardizing fertilization. One of the main processes in capacitation involves actin polymerization, and the resulting F-actin is subsequently dispersed prior to the acrosome reaction. Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR. In the present review, we describe the protective mechanisms that regulate sperm capacitation and prevent sAR. [ABSTRACT FROM AUTHOR] more...

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan. [ABSTRACT FROM AUTHOR] more...

Objective: To investigate the expression and localization of Vasorin (Vasn) in human female reproductive system. Methods: The presence of Vasorin was evaluated by RT-PCR and immunoblotting analyses in patient-derived endometrial, myometrial and granulosa cells (GCs) primary cultures. Immunostaining analyses were performed to detect Vasn localization in primary cultures and in ovarian and uterine tissues. Results: Vasn mRNA was detected in patient-derived endometrial, myometrial and GCs primary cultures without significant differences at the transcript level. Otherwise, immunoblotting analysis showed that Vasn protein levels were significantly higher in GCs than proliferative endometrial stromal cells (ESCs) and myometrial cells. Immunohistochemistry performed in ovarian tissues revealed that Vasn was expressed in the GCs of ovarian follicles at different stages of development with a higher immunostaining signal in mature ovarian follicles such as the antral follicle or on the surface of cumulus oophorus cells than in early-stage follicles. The immunostaining of uterine tissues showed that Vasn was expressed in the proliferative stroma endometrium while it was significantly less expressed in the secretory endometrium. Conversely, no protein immunoreactivity was revealed in health myometrial tissue. Conclusions: Our results revealed the presence of Vasn in the ovary and the endometrium. The pattern of Vasn expression and distribution suggests that this protein may have a role in the regulation of processes such as folliculogenesis, oocyte maturation, and endometrial proliferation. [ABSTRACT FROM AUTHOR] more...

The oocyte cumulus complex is mainly composed of an oocyte, the perivitelline space, zona pellucida and numerous granulosa cells. The cumulus granulosa cells (cGCs) provide a particularly important microenvironment for oocyte development, regulating its growth, maturation and meiosis. In this study, we studied the internal structures and cell-to-cell connections of mouse cGCs using focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed three-dimensional models to display characteristic connections between the oocyte and cGCs, and to illustrate various main organelles in cGCs together with their interaction relationship. A special form of cilium identified in granulosa cell was never reported in previous literature. [ABSTRACT FROM AUTHOR] more...

In the literature, there is a well-known correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice, and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC) compared with the routinely used swim-up method (SU). To determine the effectiveness of this method, biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality, were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared with both SU-selected and unselected (U) sperm. Furthermore, using this modified swim-up procedure, significantly reduced sperm DNA damage (p < 0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice. [ABSTRACT FROM AUTHOR] more...

Simple Summary: Oocytes are the foundation of life in mammals. They develop in small sacs called follicles in the ovaries. Each follicle holds one oocyte and numerous accompanying cells, and the final stage of oocyte development happens in a follicle called the antral follicle. The successful growth and maturation of oocytes depend on complex interactions with neighboring cells, mainly granulosa cells. Understanding the differences among these granulosa cells within the antral follicle and how they communicate with each other is essential for comprehending how mammals reproduce and grow. In this study, we collected cells from these follicles in pigs and utilized single-cell RNA sequencing to profile the transcriptomic features of these cells. The study discovered diverse subpopulations of granulosa cells within antral follicles, and they were found to communicate extensively. These findings provide us with valuable insights into how follicles develop and oocytes mature in pigs. This could potentially help improve pig reproduction efficacy and advance our knowledge in human reproductive medicine. The antral follicle stage is a critical period in mammalian oocyte maturation, marked by complex interactions between oocyte development and neighboring granulosa cells. Understanding the heterogeneity and communication patterns of granulosa cells within antral follicles is crucial for deciphering their roles in follicle development and oocyte maturation. Here, we employed single-cell RNA-sequencing to explore the molecular and cellular characteristics of porcine antral follicles. Our analysis revealed distinct subpopulations within mural and cumulus granulosa cells, indicating diverse cellular states and functions within the follicles. Functional enrichment analysis unveiled the involvement of specific subpopulations in steroid biosynthesis, cumulus expansion, and cellular communication. Moreover, comparing mature and less mature follicles highlighted differences in cell distribution and functions, indicating developmental-specific variations. Our findings shed light on the intricate cellular heterogeneity and communication network within porcine antral follicles, providing valuable insights into the regulation of follicle development and oocyte maturation in pigs. These results hold promise for improving pig reproductive efficiency and advancing human reproductive medicine. [ABSTRACT FROM AUTHOR] more...

This study aimed to investigate the effect of supplementation of chitosan nanoparticles (CSNPs) on the capacitation of bovine spermatozoa during the in vitro fertilization process. Hyperactivated motility (HAM) and acrosome reaction (AR) of sperm cells as well as in vitro fertilization and cleavage rates are the main parameters used to estimate the effect of CSNPs on bovine spermatozoa's fertilizing ability. In this study, three different concentrations of CSNPs (10, 20 and 100 μg/mL) were prepared and characterized. Motile spermatozoa were separated from frozen–thawed semen by a swim‐up technique and capacitated in Sperm‐TALP medium supplemented with heparin only without CSNPs treatment (positive control), heparin + 10 μg/mL CSNPs, heparin + 20 μg/mL CSNPs, heparin + 100 μg/mL CSNPs and the last one served as a negative control tube which supplemented with 10 μg/mL CSNPs without adding heparin. Sperm cells were incubated for 90 min at 39°C in a 5% CO2 incubator and evaluated every 30 min at intervals. Cumulus oophorus complex (COCs) were matured in a 5% CO2 incubator at 39°C and inseminated in vitro with frozen–thawed bull sperm of the above concentrations. The inseminated oocytes were incubated at 39°C in a 5% CO2 incubator for 24 h and then examined for evidence of fertilization. The results of this investigation showed that HAM and AR were best affected by CSNPs at a concentration of 20 μg/mL during an incubation time of 60 min. As time went on, the overall proportion of spermatozoa with progressive motility (PM) decreased across all groups, and a substantially lower value was found at the dose mentioned above. Additionally, the impact of sperm treated with CSNPs on fertilization rate was assessed. The outcomes demonstrated that in comparison to the other concentrations (10 and 100 μg/mL), the positive control and the negative control, the proportion of fertilized oocytes was significantly higher in the CSNPs concentration (20 μg/mL). In conclusion, it could be inferred from this investigation that CSNPs support sperm functions during IVF and can be used for biomedical interventions in bovine spermatozoa. Additionally, a high IVF rate was achieved by using sperm treated with CSNPs as CSNPs enhance sperm capacitation and acrosome reaction. [ABSTRACT FROM AUTHOR] more...

Cumulus granulosa cells (CGCs), an important type of ovarian somatic cells, carries out various functions related to oogenesis, follicular development, and steroidogenesis. Studying the biological mechanisms involved in the development and function of CGCs makes a great contribution to understanding the reproductive regulation in female animals. Stanniocalcin-1 (STC1) is an important Ca2+-regulated glycoprotein hormone that exhibits high expression levels in ovaries. In this study, we cloned the coding sequence of the yak STC1 , predicted the structure of STC1 protein, detected the expression and localization of STC1 in yak ovaries, and analyzed the functions of STC1 in yak CGCs. The CDS (coding sequence) region of yak STC1 gene was found to be 744 bp and encoded 247 amino acids. Homology comparison revealed that STC1 protein was highly conserved among mammals. The STC1 mRNA displayed dynamic expression profiles in different stages of yak ovaries, and the highest expression was found in the follicular phase. Regarding localization, STC1 protein was widely distributed in various kinds of yak ovarian cells, including oocytes, mural granulosa cells, CGCs, and thecal cells. Repressing the expression of STC1 resulted in defective proliferation and survival of yak CGCs. In addition, knockdown the expression of STC1 repressed the secretion of progesterone and promoted the secretion of estrogen. Overexpression of STC1 partially rescued the proliferation of CGCs and resulted in opposite effects on the secretion of progesterone and estrogen. Several apoptosis and steroidogenesis-related genes, including BAX , BCL2 , HSD3B1 , HSD17B1, CYP11A1 and CYP17A1 showed altered expressions after repressing or increasing the expression of STC1 in yak CGCs. To the best of our knowledge, this study is the first to focus on the role of STC1 in yak CGCs, and the outcomes offer fresh insights into the mechanism governing yak reproduction. • Cloning the coding sequence of the yak STC1 gene and predicting the structure of STC1 protein. • Identifying the expression and localization of STC1 protein in yak ovaries. • STC1 is essential for the survival and steroidogenesis in yak cumulus granulosa cells. [ABSTRACT FROM AUTHOR] more...

Endometriosis is a common gynecological disorder that causes female infertility. Our recent research found that excessive oxidative stress in ovaries of endometriosis patients induced senescence of cumulus granulosa cells. Here, we analyzed the transcriptomic and metabolomics profiles of follicles in a mouse model of endometriosis and in patients with endometriosis and investigated the potential function of changed metabolites in granulosa cells. RNA‐sequencing indicated that both endometriosis lesions and oxidative stress in mice induced abnormalities of reactive oxidative stress, steroid hormone biosynthesis, and lipid metabolism. The mouse model and women with endometriosis showed altered lipid metabolism. Nontargeted metabolite profiling of follicular fluid from endometriosis and male‐factor infertility patients by liquid chromatography mass spectrometry identified 55 upregulated and 67 downregulated metabolites. These differential metabolites were mainly involved in steroid hormone biosynthesis and glycerophospholipid metabolism. Phosphatidylinositol (PI 16:0/18:2) was significantly elevated in follicular fluid from endometriosis patients compared with controls (p < 0.05), while lysophosphatidylinositol (LPI 18:2, 20:2, 18:1, 20:3 and 18:3) was reduced (p < 0.05). Upregulated PI and downregulated LPI correlated with oocyte retrieval number and mature oocyte number. LPI inhibited cellular reactive oxidative stress induced by hemin in granulosa cells. Cell proliferation inhibition, senescence, and apoptosis induced by hemin were partially reversed by LPI. Moreover, LPI administration rescued hemin blocking of cumulus‐oocyte complex expansion and stimulated expression of ovulation‐related genes. Transcriptomic Switching mechanism at 5' end of the RNA transcript sequencing and western blot revealed that LPI effects on granulosa cells were associated with its regulation of MAPK‐ERK1/2 signaling, which was suppressed in the presence of hemin. In conclusion, our results revealed the dysregulation of lipid metabolism in endometriotic follicles. LPI may represent a novel agent for in vitro follicular culture that reverses the excessive oxidative stress from endometriotic lesions. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR] more...

Primary culture of granulosa cells is a prerequisite for a complete study of the normal functioning of the ovary and its pathologies. In this work, we selected the optimal protocol for obtaining a primary culture of mouse granulosa cells in the most functionally active state and revealed the effects of androstenedione and serotonin on the expression of ovarian markers that reflect the functional status of granulosa cells. The morpho-functional analysis of the ovary after PMSG stimulation revealed that 48 hours after PMSG stimulation is the optimal time for obtaining granulosa cells in the most active functional state. Using the set of 14 ovarian functional state marker genes we reveal that androstenedione inhibits cumulus and immature granulosa markers but stimulates genes characteristic of the mature state of granulosa. At the same time, granulosa cells express serotonergic receptors and transporter SERT. The ovarian marker genes expression analysis revealed that serotonin affects the expression of genes characterizing the differentiation of granulosa cells towards cumulus cells. Summarizing, we can conclude that serotonin and androstenedione have an antagonistic effect on the functional state of mouse granulosa cells in primary culture in vitro. [ABSTRACT FROM AUTHOR] more...

While morphokinetic evaluation of embryos has become the most commonly used technique in IVF to select embryos for transfer, studies have demonstrated that mitochondrial DNA (mtDNA) copy number is correlated with embryo viability and transfer outcomes. Correspondingly, this cohort study aims to evaluate the association between the mtDNA copy number in cumulus granulosa cells (CGCs) with embryo morphokinetic parameters and chromosomal status. Real-time PCR was employed to measure the mtDNA copy number of the 129 CGCs in samples obtained from 30 patients undergoing the IVF-IMSI program at Morula IVF Jakarta between July and October 2020. Bivariate and multiple analyses were utilized to determine its relationship with embryo morphokinetics, blastocyst yield, and chromosomal status. According to the analysis, there was a significant correlation between the mtDNA copy number and the blastocyst status after adjusting for the maternal age and sperm morphology (coefficient 0.832, p value = 0.032, RR value 2.299). Moreover, a significant link was observed between mtDNA copy number in CGC and early embryo developmental phase M1 (t2–t8), using the equation of M1 is 5.702–0.271 mtDNA copy number of CGCs + 0.017 maternal age + 0.013 sperm motility –0.115 sperm morphology (p value = 0.032). However, no correlation was found between the mtDNA copy number in CGCs with the other morphokinetic parameters (M2: tC–tEB, M3: t2–tEB, DC, RC, MN with p > 0.05), or the chromosomal status of the embryos (euploid: 139.44 ± 133.12, aneuploid: 142.40 ± 111.30, p = 0.806). In conclusion, our study suggests that mtDNA copy number in CGCs can serve as a useful biomarker for blastocyst status and early embryo developmental phase but not for chromosomal status. [ABSTRACT FROM AUTHOR] more...

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC. [ABSTRACT FROM AUTHOR] more...

Cumulus granulosa cells (CGCs) are a type of important ovarian somatic cells that carries out various functions related to oogenesis, follicular development and embryogenesis. The study on the development and function of CGCs facilitates the understanding of reproductive regulation in female animals. Sirtuin 7 (SIRT7) is a member of the sirtuin family of NAD+‐dependent deacetylases mediating numerous biological processes. In this study, we detected the localization of SIRT7 in yak ovaries as well as explored the function of SIRT7 in yak CGCs. The results revealed that the SIRT7 protein was mainly localized in the cytoplasm of oocytes, granulosa cells and theca cells. The knockdown of SIRT7 in yak CGCs repressed cell proliferation and impacted the expressions of several apoptosis‐related genes. Furthermore, oestrogen synthesis was also inhibited in SIRT7‐deficient yak CGCs. The expressions of several sterogenesis‐related genes decreased significantly following SIRT7 knockdown. In addition, the lack of SIRT7 in yak CGCs resulted in decreased levels of the TGFB/SMAD family members TGFB1, TGFBR1 SMAD2 and SMAD3. Moreover, the activation of the TGFB/SMAD pathway by adding TGFB/SMAD pathway activator SRI‐011381 partially rescued the level of oestrogen secreted by SIRT7‐deficient yak CGCs, as well as the expressions of steroidogenesis‐related genes NR5A1 and CYP19A1. This research is the first to focus on the role of SIRT7 in yak ovary, and the outcomes offer new insights into the mechanism governing yak reproduction. [ABSTRACT FROM AUTHOR] more...

Background: Development of ovarian follicles is regulated by a complex interaction of intra‐ and extra‐follicular signals. Oocyte‐derived paracrine factors (ODPFs) play a central role in this process in cooperation with other signals. Methods: This review provides an overview of the recent advances in our understanding of the paracrine regulation of antral follicle development in mammals. It specifically focuses on the regulation of granulosa cell development by ODPFs, along with other intrafollicular signals. Main Findings: Bi‐directional communication between oocytes and surrounding cumulus cells is a fundamental mechanism that determines cumulus cell differentiation. Along with estrogen, ODPFs promote the expression of forkhead box L2, a critical transcription factor required for mural granulosa cells. Follicle‐stimulating hormone (FSH) facilitates these processes by stimulating estrogen production in mural granulosa cells. Conclusion: Cooperative interactions among ODPFs, FSH, and estrogen are critical in determining the fate of cumulus and mural granulosa cells, as well as the development of oocytes. [ABSTRACT FROM AUTHOR] more...

Objective: We aimed to evaluate the potential apoptotic effects of propofol, dexmedetomidine and medetomidine that were used during oocyte retrieval on cumulus cells in rat ovulation induction model. Methods: After the ovulation induction model was created, rats were received dexmedetomidine in Group D, propofol in Group P, medetomidine in Group M. Oocytes collection was performed 10 minutes after the administration of study drugs. For Caspase-3 immunohistochemical evaluation, the staining level was scored in five randomly selected areas and the areas with the highest score were determined. Immunohistochemical staining scoring for each section was performed using a scoring algorithm called H-score. Results: Caspase-3 expression in cumulus cells was found to be lowest in Group D and highest in Group P. The mean caspase-3 H-score was lower in Group D than that of Group P and M (p<0.001), and in Group M than that of Group P (p<0.001). Conclusion: Our results demonstrate that dexmedetomidine and medetomidine exhibit less apoptotic effects in terms of caspase-3 activity in oocyte cumulus cells than propofol in a rat ovulation induction model. [ABSTRACT FROM AUTHOR] more...

In the current study, 930 equine oocytes were collected, 765 (82.25%) of which were considered viable for in vitro maturation. All selected oocytes were matured for 27 h at 38.50 ℃ in an atmosphere of 5% CO2 in humidified air. 25 mm hepes-buffered TCM-199 supplemented with 2 mm sodium pyruvate, 1mm l-glutamine, penicillin (75 mg/ml), streptomycin (50 mg/ml) and 10% fetal calf serum was used for in vitro maturation. Following maturation, matured COCs were examined to investigate for first polar body formation. The morphocytometric assessment was performed using the motic image plus (MIP) software with an inverted microscope. Morphocytometric examination results showed highly significant differences (p<0.001) among groups of matured oocytes (zona pellucida thickness; <13 μm, cumulus oophorus thickness; < 10 μm and oocytes diameter; < 100 μm), where the rates were 63.39%, 33.59% and 30.58%, respectively. The results of morphocytometric evaluation based on zona pellucida thickness, cumulus oophorus thickness and oocyte diameter of the total 765 cultured oocytes showed that 197 oocytes (25.75%) were classified as excellent mature, 108 oocytes (14.11%) as mature good, 203 oocytes (26.53%) as immature and 257 oocytes (33.59%) were considered as degenerated. It is concluded thatthe oocytes rates differed according to the parameters (cumulus oophorus, oocytes diameter, zona pellucida). [ABSTRACT FROM AUTHOR] more...

Conventional sperm processing uses centrifugation has a negative effect on sperm parameters and DNA integrity. We designed and fabricated a novel microfluid device based on chemotaxis and thermotaxis, and compared it with the swim‐up method. Twenty normal samples with high DNA fragmentation were included. Each sample was divided into four groups: Group 1, control, Group 2: sperm selection by thermotaxis, Group 3: sperm selection by chemotaxis, and Group 4: sperm selection with thermotaxis and chemotaxis. We used cumulus cells in a microfluid device to create chemotaxis, and, two warm stages to form a temperature gradient for thermotaxis. The spermatozoa were assessed based on the concentration, motility, and fine morphology using Motile Sperm Organelle Morphology Examination, mitochondrial membrane potential (MMP), acrosome reaction (AR), and sperm DNA fragmentation. Concentration (22.40 ± 5.39 vs. 66.50 ± 19.21; p < 0.001) and DNA fragmentation (12.30 ± 3.96% vs. 17.95 ± 2.89%; p < 0.001) after selection in the chemotaxis and thermotaxis microfluid device were significantly lower than control group. The progressive motility (93.75 ± 4.39% vs. 75.55 ± 5.86%, p < 0.001), normal morphology (15.45 ± 2.50% vs. 10.35 ± 3.36, p < 0.001), MMP (97.65 ± 1.81% vs. 94 ± 3.89%, p = 0.02), and AR status (79.20 ± 5.28% vs. 31.20 ± 5.24%, p < 0.001) in the chemotaxis and thermotaxis microfluid device were significantly increased compared to control group. According to these findings, spermatozoa that have penetrated the cumulus oophorus have better morphology and motility, as well as acrosome reactivity and DNA integrity. [ABSTRACT FROM AUTHOR] more...

【Objective】The present paper aimed to study the localization and expression pattern of histone deacetylases（HDACS） family members HDAC3 and HDAC8 in growing follicles of Tan sheep, in order to provide reference for further research on the epigenetics mechanism of follicular development and oocyte maturation regulation in Tan sheep. 【Method】The ovarian tissues of healthy tan sheep were collected and the following growth follicles were mechanically separated: Small antral follicles（diameter 0.5-3.0 mm）, large antral follicles（diameter 3.0-5.0 mm） and preovulatory follicles（diameter>5.0 mm）. The localization and expression of HDAC3 and HDAC8 in different follicles were detected by immunohistochemistry and immunofluorescence respectively. At the same time, the expression of HDAC3 and HDAC8 in the follicles of Tan sheep was detected by Western blotting.【Result】HDAC3 was mainly located in the granular cells of primitive follicles, primary follicles and secondary follicles, granular cells of antral follicles and cumulus cells of Tan sheep, and its expression was up-regulated with follicular development. The localization of HDAC8 in growing follicles was different from that of HDAC3, which was not only distributed in the oocyte and granulosa cytoplasm of primordial follicles, primary follicles and secondary follicles, it was also localized in the cytoplasm of oocytes, granulosa cells and cumulus cells with antral follicles.【Conclusion】These results indicated that the localization and expression of HDAC3 and HDAC8 in growing follicles of Tan sheep were different, suggesting that HDAC3 and HDAC8 may be involved in the regulation of follicular development and oocyte maturation in different ways. [ABSTRACT FROM AUTHOR] more...

Polycystic ovarian syndrome (PCOS) is one of the most common endocrinological disorders affecting between 6 to 20% of reproductive aged women. However, the etiology of PCOS is still unclear. Epidermal growth factor receptor (EGFR) plays a critical role in the growth and development of ovarian follicles. In our previous study, we showed that the expression level of EGFR was significantly higher in the cumulus granulosa cells from women with PCOS than that of normal women, suggesting that EGFR may play a potential role in the pathogenesis of PCOS. The present study further evaluated the association between EGFR and PCOS through both in clinical observation and animal experiments. We firstly validated the differential expression of EGFR in cumulus granulosa cells between PCOS patients and normal subjects by qRT-PCR and immunofluorescence staining. Then we generated a mouse model (n=20) of PCOS by injecting dehydroepiandrosterone (DHEA). The PCOS mice were then injected with an E corpus GFR inhibitor (AG1478) (n=10), which significantly improved the sex hormone levels in the estrous cycle stage, and the serum levels of LH, FSH and testosterone were compared with the PCOS mice without EGFR inhibitor treatment (n=10). Decreasing the expression level of EGFR in the PCOS mice also improved the ovulatory function of their ovaries which was indicated by the multifarious follicle stage in these mice as compared with the PCOS mice without EGFR inhibitor treatment. Also, the number of corpopa lutea were higher in the control group and the EGFR inhibitor treated group than in the PCOS group. The sex hormone levels and reproductive function were not significantly different between the control mice and the PCOS mice treated with the EGFR inhibitor. Our results demonstrated that EGF/EGFRsignaling affected the proliferation of cumulus granulosa cells, oocyte maturation and meiosis, and played a potential role in the pathogenesis of PCOS. Therefore, the selective inhibition of EGFR may serve as a novel strategy for the clinical management of PCOS. [ABSTRACT FROM AUTHOR] more...

The cumulus oophorus is a structure that surrounds the mammalian egg and plays a key role in fertilization. However, very little is known with regards to how secretions from the cumulus cells can specifically promote fertilization. We hypothesized that secretions from bovine cumulus cells, and the reduction of oxygen stress by metabolic change, would enhance the fertilization capacity of sperm during in vitro fertilization (IVF) procedures. To prove our hypothesis, sperm were pre-incubated in chemically defined capacitation media containing methyl-beta-cyclodextrin and used to inseminate cumulus cell oocyte complexes, or denuded oocytes, with some components. While sperm capacitation was induced in capacitation media, fertilization was impeded by the removal of cumulus cells from cumulus cell oocyte complexes. Secretions from cumulus cells promoted the formation of two pronuclei via a filter and the fertilization of denuded oocytes was dramatically enhanced with hyaluronate, low oxygen concentration, or progesterone in fertilization media (P < 0.05). This demonstrates that these factors-maintained sperm motility and capacitation or enhanced the hyper-activation of capacitated sperm (P < 0.05). We conclude that cumulus cells secrete progesterone, hyaluronate and undergo metabolic events to reduce oxidative stress in fertilization media. These phenomenons help to improve the fertilization capacity of sperm. We believe that this study makes a significant contribution to our understanding of the function of cumulus cells during fertilization in animals and humans. [ABSTRACT FROM AUTHOR] more...

Cumulus-oocyte complex (COC) expansion and oocyte maturation are crucial processes for embryo development and fertility across species. Although miR-29b has been detected in porcine ovarian granulosa cells, its specific role in regulating oocyte maturation remains largely unknown. In this study, using the pig as a model, we report that over-expression of miR-29b lead to a decrease of COC expansion area and inhibits oocyte maturation (P <0.05). This suppression correlated with a decrease expression of COC-expansion-associated genes, including SHAS2 , ADAMTS1 , ADAMTS2 , ADAMTS17 and PTX 3 in both mural granulosa cells (mGCs) and cumulus granulosa cells (cGCs). Further investigation revealed that miR-29b over-expression induces reactive oxygen species (ROS) accumulation in both mGCs and cGCs, conversely, knock-down of miR-29b reverses all these effects. Treatment with the antioxidant β-mercaptoethanol alleviates ROS accumulation, rescues COC expansion and restores oocyte polar body formation impaired by miR-29b mimics. Computational analysis predicted CYCS , the gene encoding cytochrome C, as a potential target of miR-29b. Subsequent examination demonstrated that miR-29b downregulates CYCS at both mRNA and protein levels. Dual-luciferase reporter assays further confirmed that miR-29b interacts with the 3'-untranslated region (3'UTR) of CYCS. Over-expression of CYCS decreases ROS accumulation and promotes COC expansion (P <0.05). These results indicate that miR-29b regulates COC expansion and oocyte maturation in vitro by inducing ROS, likely through targeting of CYCS. This study sheds light on the role of miR-29b in oocyte maturation and provides insight into the regulatory function of miRNAs in ovarian physiology. • MiR-29b reduces porcine COC expansion and inhibits oocyte maturation by inducing ROS.· • CYCS, a cytochrome C gene, is a direct miR-29b target, affecting ROS and maturation.· • Antioxidants restore COC expansion and oocyte maturation, countering miR-29b effects.· • Study reveals roles of miR-29b and CYCS in ovarian function with fertility implications. [ABSTRACT FROM AUTHOR] more...

Background: Growing oocytes acquire the ability to mature through two-way communication between gametes and surrounding somatic cumulus cells (CCs). Granulosa cells (GCs) support oocyte growth, regulate meiosis progression, and modulate global oocyte transcription activity. However, the proliferation and differentiation of the yak ovary in GCs and CCs remain unclear. To characterize the important roles of long non-coding RNA, (lncRNA), microRNA (miRNA), and messenger RNA (mRNA), whole-transcriptome analysis was performed. Real-time quantitative fluorescence PCR was performed to verify the selected RNA sequences. Results: Important gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways related to differentiation and oocyte development were identified for the target genes of differentially expressed lncRNAs, miRNAs, and mRNAs. In total,6223 mRNAs (2197 upregulated, 4026 downregulated), 643 lncRNAs (204 upregulated, 479 downregulated), and 559 miRNAs (311 upregulated, 248 downregulated) were significantly altered between the two groups. Target genes involved in cell adhesion, cell differentiation, regulation of developmental processes, cell proliferation, embryo development, signal transduction, apoptosis, and aromatic compound biosynthetic processes were significantly enriched. These RNAs were involved in ECM-receptor interaction, MAPK signaling, Hippo signaling, PI3K-Akt signaling, cell cycle, cell adhesion, leukocyte trans-endothelial migration, and actin cytoskeleton regulation. Conclusions: A comprehensive analysis of the co-expression network of competing endogenous RNAs (ceRNAs) will facilitate the understanding of the process of granulosa cell proliferation and differentiation and offer a theoretical basis for the development of oocytes. [ABSTRACT FROM AUTHOR] more...

In the bovine cumulus oophorus, 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1)-mediated cortisol production dramatically increases during the periovulatory period. This event is closely associated with increased progesterone (P4) production, implying a functional connection between these C21 steroids. In this study, we investigated the mutual regulation of P4 and cortisol production in the bovine cumulus oophorus. Bovine cumulus-oocyte complexes (COCs) were aspirated from follicles 2–5 mm in diameter and subjected to in vitro maturation (IVM) for 24 h in an M199 supplemented with fetal calf serum (FCS) and follicle-stimulating hormone (FSH). COCs were treated with trilostane (0, 0.1, 1, 10 mM), an inhibitor of P4 synthesis, RU486 (0, 0.1, 1, 10 mM), a receptor antagonist for the progesterone receptor (PR) and glucocorticoid receptor (GR), and various concentrations of a synthetic progestogen nomegestrol acetate (NA; 0, 0.001, 0.01, 0.1, 1, 10 mM) to examine effect of P4. The effects of cortisol (0, 0.1, 1, 10 mM) were also examined in the presence or absence of trilostane. Trilostane and RU486 suppressed cumulus expansion, cortisol production, and HSD11B1 but not hexose-6-phosphate dehydrogenase (H6PDH) expression. Concomitant treatment with NA reversed the effects of trilostane. Unlike NA, cortisol did not alter the antagonistic effects of trilostane on cumulus expansion and HSD11B1 expression. Cortisol did not affect P4 production or steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1), and HSD11B1 expression. Collectively, these results indicate that locally produced P4 is crucial in regulating the local glucocorticoid environment through PRtg in the maturing bovine cumulus oophorus. Cortisol, however, does not appear to regulate P4 or its production. • Maturing bovine cumulus oophorus cannot generate cortisol de novo, but they can generate cortisol from cortisone via HSD11B1. • Locally produced P4 potentiated HSD11B1-led glucocorticoid activation in the maturing bovine cumulus oophorus. • Cortisol does not regulate P4 production nor HSD11B1-led cortisol production in the maturing bovine cumulus oophorus. [ABSTRACT FROM AUTHOR] more...

Aims: Debate over the optimal timing of oocyte denudation following oocyte retrieval continues. Multinucleation has been associated with lower embryo quality and implantation rate. Defining oocyte characteristics might increase the chances of better embryo selection. This study aimed to investigate the laboratory and clinical outcomes of intracytoplasmic sperm injection when the oocytes were denuded immediately after oocyte retrieval. Methods: A prospective randomized sibling-oocyte study was performed. The participants were under 40 years of age with more than 5 oocytes. Patients with male factors were excluded. The study and control groups were formed by simple randomization. The study group underwent oocyte denudation immediately. In the control group, oocytes were routinely incubated in an equilibration solution for 2 h until the removal of the cumulus cells. Outcome measures were normal fertilization (two pronuclei, 2PN), multinucleation rate, the proportion of good quality embryos, blastocyst formation, and pregnancy rate. Results: A total of 792 oocytes were collected from 54 participants (mean age: 30.6±3.7 years). The fertilization rate was higher in the study group (n=209 in 376) compared with the controls (n=201 in 416) (55.6% vs. 48.3%, p=0.041). The multinucleation rate (6.6% vs. 4.3%, p=0.150), proportion of grade 1 embryos on day 3 (48.7% vs. 43%, p=0.112) and day 5 (27.7% vs. 23.8%, p=0.214), the proportion of embryos reaching blastocyst stage (34.0% vs. 28.1%, p=0.072), and the pregnancy rates (78.6% vs. 71.4%, p=1.000) were similar. Conclusions: Our results demonstrated that immediate removal of cumulus cells does not have any negative impact on intracytoplasmic sperm injection outcomes. [ABSTRACT FROM AUTHOR] more...