Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Mitochondrial Dysfunction in Advanced Maternal Aged Cumulus Cells: A Possible Link to ATP Synthase Impairment?

Age-related changes in the mitochondrial status of human cumulus cells (hCCs) impact oocyte quality; however, the relationship between hCC mitochondrial (dys)function and reproductive aging remains poorly understood. This study aimed to establish the interplay between hCC mitochondrial dysfunction and women's reproductive potential. In this investigation, 266 women were enrolled and categorized into two groups based on their age: a young group (<35 years old) and an advanced maternal age (AMA) group (≥35 years old). Comprehensive analysis of reproductive outcomes was conducted in our population. Various mitochondrial-related parameters were analyzed across distinct subsets. Specifically, mitochondrial membrane potential (∆Ψm) and mitochondrial mass were examined in 53 samples, mtDNA content in 25 samples, protein levels in 23 samples, bioenergetic profiles using an XF24 Extracellular Flux Analyzer in 6 samples, and levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in 39 and 43 samples, respectively. In our study, the reproductive potential of AMA women sharply decreased, as expected. Additionally, an impairment in the mitochondrial function of hCCs in older women was observed; however, no differences were found in terms of mitochondrial content. Regarding oxidative phosphorylation, metabolic profiling of hCCs from AMA women indicated a decrease in respiratory capacity, which was correlated with an age-dependent decrease in the ATP synthase (ATP5A1) protein level. However, intracellular ROS and ATP levels did not differ between groups. In conclusion, our study indicates that age-related dysfunction in hCCs is associated with impaired mitochondrial function, and, although further studies are required, ATP synthase could be relevant in this impairment.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

FGF2, LIF, and IGF-1 supplementation improves mouse oocyte in vitro maturation via increased glucose metabolism†.

Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.

Long pentraxin 3 and vitamin D receptor mRNA expression pattern of cumulus granulosa cells isolated from PCOS oocytes at different stages of nuclear maturation.

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.

The follicle-stimulating hormone triggers rapid changes in mitochondrial structure and function in porcine cumulus cells.

Oocyte maturation is a key process during which the female germ cell undergoes resumption of meiosis and completes its preparation for embryonic development including cytoplasmic and epigenetic maturation. The cumulus cells directly surrounding the oocyte are involved in this process by transferring essential metabolites, such as pyruvate, to the oocyte. This process is controlled by cyclic adenosine monophosphate (cAMP)-dependent mechanisms recruited downstream of follicle-stimulating hormone (FSH) signaling in cumulus cells. As mitochondria have a critical but poorly understood contribution to this process, we defined the effects of FSH and high cAMP concentrations on mitochondrial dynamics and function in porcine cumulus cells. During in vitro maturation (IVM) of cumulus-oocyte complexes (COCs), we observed an FSH-dependent mitochondrial elongation shortly after stimulation that led to mitochondrial fragmentation 24 h later. Importantly, mitochondrial elongation was accompanied by decreased mitochondrial activity and a switch to glycolysis. During a pre-IVM culture step increasing intracellular cAMP, mitochondrial fragmentation was prevented. Altogether, the results demonstrate that FSH triggers rapid changes in mitochondrial structure and function in COCs involving cAMP.

Global crotonylome identifies EP300-regulated ANXA2 crotonylation in cumulus cells as a regulator of oocyte maturation.

Lysine crotonylation (Kcr), a newly discovered post-translational modification, played a crucial role in physiology and disease progression. However, the roles of crotonylation in oocyte meiotic resumption remain elusive. As abnormal cumulus cell development will cause oocyte maturation arrest and female infertility, we report that cumulus cells surrounding human meiotic arrested oocytes showed significantly lower crotonylation, which was associated with decreased EP300 expression and blocked cumulus cell expansion. In cultured human cumulus cells, exogenous crotonylation or EP300 activator promoted cell proliferation and reduced cell apoptosis, whereas EP300 knockdown induced the opposite effect. Transcriptome profiling analysis in human cumulus cells indicated that functions of crotonylation were associated with activation of epidermal growth factor receptor (EGFR) pathway. Importantly, we characterized the Kcr proteomics landscape in cumulus cells by LC-MS/MS analysis, and identified that annexin A2 (ANXA2) was crotonylated in cumulus cells in an EP300-dependent manner. Crotonylation of ANXA2 enhanced the ANXA2-EGFR binding, and then activated the EGFR pathway to affect cumulus cell proliferation and apoptosis. Using mouse oocytes IVM model and EP300 knockout mice, we further confirmed that crotonylation alteration in cumulus cells affected the oocyte maturation. Together, our results indicated that EP300-mediated crotonylation is important for cumulus cells functions and oocyte maturation.

A Circular RNA Derived from the Pumilio 1 Gene Could Regulate PTEN in Human Cumulus Cells.

CircRNAs are a class of non-coding RNAs able to regulate gene expression at multiple levels. Their involvement in physiological processes, as well as their altered regulation in different human diseases, both tumoral and non-tumoral, is well documented. However, little is known about their involvement in female reproduction. This study aims to identify circRNAs potentially involved in reproductive women's health. Candidate circRNAs expressed in ovary and sponging miRNAs, already known to be expressed in the ovary, were selected by a computational approach. Using real time PCR, we verified their expression and identified circPUM1 as the most interesting candidate circRNA for further analyses. We assessed the expression of circPUM1 and its linear counterpart in all the follicle compartments and, using a computational and experimental approach, identified circPUM1 direct and indirect targets, miRNAs and mRNAs, respectively, in cumulus cells. We found that both circPUM1 and its mRNA host gene are co-expressed in all the follicle compartments and proposed circPUM1 as a potential regulator of PTEN, finding a strong positive correlation between circPUM1 and PTEN mRNA. These results suggest a possible regulation of PTEN by circPUM1 in cumulus cells and point out the important role of circRNA inside the pathways related to follicle growth and oocyte maturation.

Antioxidant sericin averts the disruption of oocyte-follicular cell communication triggered by oxidative stress.

Antioxidants are free radical scavengers that increase oocyte quality and improve female fertility by suppressing oxidative stress. However, the related mechanisms remain unclear. The present study was designed to examine whether a reduction of oxidative stress from using the antioxidant sericin led to expanded cumulus cell (CC)-oocyte communication and oocyte developmental acquisition in a bovine model. We found that cumulus-oocyte complexes (COCs) matured in the presence of sericin showed a significantly increased oocyte meiotic maturation rate (P < 0.01) and accelerated subsequent blastocyst formation, as more blastocysts were found at the hatched stage (P < 0.05) compared to that in the control group. In contrast to the control group, sericin suppressed H2O2 levels in COCs, resulting in a markedly enhanced CC-oocyte gap junction communication index and number of transzonal projections, which were preserved until 18 h of oocyte maturation. These findings indicate that sericin reduces disruption of oocyte-follicular cell communication induced by oxidative stress. Sericin consistently increased intra-oocyte glutathione (GSH) levels and reduced oocyte H2O2 levels (P < 0.05), both of which were ablated when GSH synthesis was inhibited by buthionine sulfoximide (an inhibitor of GSH synthesis). Furthermore, the inhibition of GSH synthesis counteracted the positive effects of sericin on subsequent embryo developmental competence (P < 0.01). Intra-oocyte GSH levels were positively associated with blastocyst development and quality. These outcomes demonstrate new perspectives for the improvement of oocyte quality in assisted reproductive technology and may contribute to developing treatment strategies for infertility and cancer.

Lipopolysaccharide impairs the in vitro growth, steroidogenesis, and maturation of oocyte-cumulus-granulosa cell complexes derived from bovine early antral follicles.

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria causes uterine or mammary inflammation, resulting in low fertility. The present study aimed to investigate the effect of LPS on the in vitro growth (IVG), steroidogenesis, and maturation of oocyte-cumulus-granulosa cell complexes (OCGCs) derived from bovine early antral follicles. OCGCs were isolated from bovine early antral follicles (0.5-1 mm in diameter) and cultured in vitro for 12 days using media containing 0 (control), 0.01, or 1 µg/mL of LPS. The viability, cavity formation, and oocyte diameter of the OCGCs, as well as the concentrations of estradiol (E2) and progesterone (P4) in the IVG culture media, were determined. After IVG culture, oocytes collected from viable OCGCs were matured in vitro (IVM) in a medium without LPS. The nuclear maturation rate and the mitochondrial membrane potential of oocytes were determined. Bovine oocytes and cumulus-granulosa complexes derived from early antral follicles expressed genes encoding LPS receptor complex, such as toll-like receptor 4 (TLR4). Immunohistochemistry analysis further localized TLR4 expression predominantly in follicular granulosa and theca cells of early antral follicles. The viability of OCGCs and cavity formation in OCGCs were lower in the 0.01 and 1 µg/mL LPS groups than in the control group. No significant difference in oocyte diameter was observed between the treatment groups throughout the culture period. Moreover, E2 production was suppressed in the 0.01 and 1 µg/mL LPS groups from Days 4-8, whereas P4 production increased in the 1 µg/mL LPS group from Days 0-8. The nuclear maturation rate after IVM was lower in the 0.01 and 1 µg/mL LPS groups than in the control group. The mitochondrial membrane potential of post-IVM oocytes was lower in the 0.01 and 1 µg/mL LPS groups than in the control group. Taken together, these results indicate that LPS inhibited the growth and steroidogenesis of OCGCs and the meiosis and mitochondrial function of oocytes derived from early antral follicles. This study suggests that the detrimental effects of LPS on developing oocytes may contribute to long-term decreased fertility in postpartum dairy cows.

miR-302d Targeting of CDKN1A Regulates DNA Damage and Steroid Hormone Secretion in Bovine Cumulus Cells.

(1) Background: DNA damage in cumulus cells hinders oocyte maturation and affects steroid hormone secretion. It is crucial to identify the key factors that regulate cellular DNA damage and steroid hormone secretion. (2) Methods: Treatment of bovine cumulus cells with bleomycin to induce DNA damage. The effects of DNA damage on cell biology were determined by detecting changes in DNA damage degree, cell cycle, viability, apoptosis, and steroid hormones. It was verified that mir-302d targeted regulation of CDKN1A expression, and then affected DNA damage and steroid hormone secretion in cumulus cells. (3) Results: Bleomycin induced increased DNA damage, decreased G1-phase cells, increased S-phase cells, inhibited proliferation, promoted apoptosis, affected E2 and P4 secretion, increased CDKN1A expression, and decreased miR-302d expression. Knockdown of CDKN1A reduced DNA damage, increased G1-phase cells, decreased G2-phase cells, promoted proliferation, inhibited apoptosis, increased E2 and P4 secretion, and increased the expression of BRCA1, MRE11, ATM, CDK1, CDK2, CCNE2, STAR, CYP11A1, and HSD3B1. The expression of RAD51, CCND1, p53, and FAS was decreased. Overexpression of CDKN1A resulted in the opposite results. miR-302d targets CDKN1A expression to regulate DNA damage and then affects the cell cycle, proliferation, apoptosis, steroid hormone secretion, and the expression of related genes. (4) Conclusions: miR-302d and CDKN1A were candidate molecular markers for the diagnosis of DNA damage in bovine cumulus cells.

ID3 regulates progesterone synthesis in bovine cumulus cells through modulation of mitochondrial function.

DNA binding inhibitory factor 3 (ID3) has been shown to have a key role in maintaining proliferation and differentiation. It has been suggested that ID3 may also affect mammalian ovarian function. However, the specific roles and mechanisms are unclear. In this study, the expression level of ID3 in cumulus cells (CCs) was inhibited by siRNA, and the downstream regulatory network of ID3 was uncovered by high-throughput sequencing. The effects of ID3 inhibition on mitochondrial function, progesterone synthesis, and oocyte maturation were further explored. The GO and KEGG analysis results showed that after ID3 inhibition, differentially expressed genes, including StAR, CYP11A1, and HSD3B1, were involved in cholesterol-related processes and progesterone-mediated oocyte maturation. Apoptosis in CC was increased, while the phosphorylation level of ERK1/2 was inhibited. During this process, mitochondrial dynamics and function were disrupted. In addition, the first polar body extrusion rate, ATP production and antioxidation capacity were reduced, which suggested that ID3 inhibition led to poor oocyte maturation and quality. The results will provide a new basis for understanding the biological roles of ID3 as well as cumulus cells.

Molecular cloning and characterization of STC1 gene and its functional analyses in yak (Bos grunniens) cumulus granulosa cells.

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.

Regulation of proteolysis in bovine cumulus cells with possible inclusion of proton pump activators.

The aim of this study was to reveal the effects of V-ATPase proton pump activation on lysosomal acidity and protein degradation in cultured cumulus cells. Cumulus cells from bovine ovaries were cultured in the presence of 10 and 50 µM doses of V-ATPase proton pump activators PIP2, PMA and DOG for 12 and 24 h. At the end of the culture period, the level of protein degradation was evaluated through DQ-Red-BSA analysis and the lysosomes were detected through a fluorescent probe. In addition, total and phosphorylated MAPK1/3 and AKT protein levels of cumulus cells were determined through Western blotting. PIP2 and PMA were shown to increase protein degradation and lysosomal acidity in cultured bovine cumulus cells, whereas DOG did not have any significant effects on these cells. Total and phosphorylated MAPK and AKT protein levels were higher in PIP2 and PMA groups compared with the control and DOG. It was concluded that particular proton pump activators can enhance protein degradation and lysosomal acidification in cultured bovine cumulus cells without having detrimental effects on cell signalling members required for cell viability and proper functioning. Due to the cellular interactions, increasing the lysosomal activity in cumulus cells in the culture environment could also affect the removal of protein aggregates in the oocytes. This strategy could be effective for improving in vitro maturation of the oocytes by providing proteostasis.

Oocyte secreted factors control genes regulating FSH signaling and the maturation cascade in cumulus cells: the oocyte is not in a hurry.

PURPOSE: To assess the effects of the oocyte on mRNA abundance of FSHR, AMH and major genes of the maturation cascade (AREG, EREG, ADAM17, EGFR, PTGS2, TNFAIP6, PTX3, and HAS2) in bovine cumulus cells. METHODS: (1) Intact cumulus-oocyte complexes, (2) microsurgically oocytectomized cumulus-oolema complexes (OOX), and (3) OOX + denuded oocytes (OOX+DO) were subjected to in vitro maturation (IVM) stimulated with FSH for 22 h or with AREG for 4 and 22 h. After IVM, cumulus cells were separated and relative mRNA abundance was measured by RT-qPCR. RESULTS: After 22 h of FSH-stimulated IVM, oocytectomy increased FSHR mRNA levels (p=0.005) while decreasing those of AMH (p=0.0004). In parallel, oocytectomy increased mRNA abundance of AREG, EREG, ADAM17, PTGS2, TNFAIP6, and PTX3, while decreasing that of HAS2 (p<0.02). All these effects were abrogated in OOX+DO. Oocytectomy also reduced EGFR mRNA levels (p=0.009), which was not reverted in OOX+DO. The stimulatory effect of oocytectomy on AREG mRNA abundance (p=0.01) and its neutralization in OOX+DO was again observed after 4 h of AREG-stimulated IVM. After 22 h of AREG-stimulated IVM, oocytectomy and addition of DOs to OOX caused the same effects on gene expression observed after 22 h of FSH-stimulated IVM, except for ADAM17 (p<0.025). CONCLUSION: These findings suggest that oocyte-secreted factors inhibit FSH signaling and the expression of major genes of the maturation cascade in cumulus cells. These may be important actions of the oocyte favoring its communication with cumulus cells and preventing premature activation of the maturation cascade.

Effect of progesterone on in vitro meiotic maturation of canine oocytes associated with Cx37 and Cx43 gene expression.

Progesterone (P4) concentrations in canines are exceptionally high in the periovulatory period. However, the mechanisms by which P4 modulates final oocyte development in dogs remain to be characterized. The aim of this study was to evaluate the effect of P4 on meiotic development related to the gene expression of connexin 37 (Cx37) and connexin 43 (Cx43) in the canine cumlus oocyte complexes (COCs). COCs were isolated from 120 canine ovaries after a routine ovariohysterectomy. In each experiment, groups of COCs retrieved from the antral follicles were subjected to in vitro maturation (IVM) for 72 h without (control) or with P4 (50 µg/mL and 100 µg/mL) or the P4 receptor antagonist, aglepristone (RU534 at 1 µM and 10 µM). Some of the COCs recovered (from each group) after 72 h of IVM were subjected to meiotic evaluation; the remaining COCs, and those not subjected to IVM, were used to analyze the gene expression of Cx37 and Cx43 by qPCR. The results were evaluated using ANOVA. The addition of P4 increased (P < 0.05) the meiotic development compared to that in the control or aglepristone groups. The highest (P < 0.05) percentage of oocytes in the MII stage was observed upon P4 supplementation. In contrast, the highest percentage (P < 0.05) of oocytes arrested in the GV stage and the lowest (P < 0.05) percentages in the MII stage were observed for COCs cultured with aglepristone. Although a significant decrease in the mRNA levels of both connexins was observed after culturing, no effect on Cx37 and Cx43 gene expression was observed when exogenous P4 was added compared to those of the control group. However, COCs cultured with aglepristone exhibited higher (P < 0.05) expression of Cx37 and Cx43 than COCs in the control IVM-group, regardless of the concentration. In conclusion, our results suggest that a high dosage of P4 during IVM enhances the nuclear maturation of canine oocytes without altering the gene expression levels of Cx37 and Cx43. However, the increase in their expression upon treatment with a P4 antagonist indicates an in vivo role for this hormone in the endogenous modulation of both Cx37 and Cx43.

TBX2 affects proliferation, apoptosis and cholesterol generation by regulating mitochondrial function and autophagy in bovine cumulus cell.

BACKGROUND: T-box transcription factor 2 (TBX2) is a member of T-box gene family whose members are highly conserved in evolution and encoding genes and are involved in the regulation of developmental processes. The encoding genes play an important role in growth and development. Although TBX2 has been widely studied in cancer cell growth and development, its biological functions in bovine cumulus cells remain unclear. OBJECTIVES: This study aimed to investigate the regulatory effects of TBX2 in bovine cumulus cells. METHODS: TBX2 gene was knockdown with siRNA to clarify the function in cellular physiological processes. Cell proliferation and cycle changes were determined by xCELLigence cell function analyzer and flow cytometry. Mitochondrial membrane potential and autophagy were detected by fluorescent dye staining and immunofluorescence techniques. Western blot and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the expression changes of proliferation and autophagy-related proteins. Aadenosine triphosphate (ATP) production, glucose metabolism, and cholesterol synthesis of cumulus cells were measured by optical density and chemiluminescence analysis. RESULTS: After inhibition of TBX2, the cell cycle was disrupted. The levels of apoptosis, ratio of light chain 3 beta II/I, and reactive oxygen species were increased. The proliferation, expansion ability, ATP production, and the amount of cholesterol secreted by cumulus cells were significantly decreased. CONCLUSIONS: TBX2 plays important roles in regulating the cells' proliferation, expansion, apoptosis, and autophagy; maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells.

Expression profile of key genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin or fetal calf serum.

Cumulus cells from cumulus-oocyte complexes (COC) matured in vitro in serum-free medium show high incidence of apoptosis and DNA double-strand breaks (DSB). This study aimed to characterize the transcript expression profile of selected genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin (BSA) or fetal calf serum (FCS). Briefly, bovine cumulus-oocyte complexes were in vitro matured with either, 0.4% BSA or 10% FCS for 3, 6, 12 or 24 h. The total RNA of cumulus cells was used for real-time PCR analysis. Transcript abundance of XRCC6, XRCC5, DNAPK, GAAD45B, TP53BP1, RAD50, RAD52, ATM and BRCA2 target genes changed as the IVM proceeded (P < 0.05). However, an interaction between protein source (FCS or BSA) and time was not detected (P ≥ 0.05). Cumulus cells from COCs matured with BSA presented higher mRNA expression of two genes compared to FCS group: TP53BP1 at 6 h and BRCA1 at 3, 6, 12 and 24 h (P < 0.05). In summary, our results showed for the first time the expression profile of the key genes involved in DSB repair mechanisms in cumulus cells obtained from bovine COCs matured with FCS or BSA. The higher mRNA expression of BRCA1 and TP53BP1 and lower mRNA expression of TNFAIP6 suggests an increase in apoptosis rate and DNA damage in cumulus cells cultured in BSA-supplemented medium and may explain, at least to some extent, the reduced developmental potential of bovine oocytes matured in serum-free medium.

Sirtuin 7 is essential for the survival and synthesis of oestrogen in yak (Bos grunniens) cumulus granulosa cells.

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.

Age-related changes in Folliculogenesis and potential modifiers to improve fertility outcomes - A narrative review.

Reproductive aging is characterized by a decline in oocyte quantity and quality, which is directly associated with a decline in reproductive potential, as well as poorer reproductive success and obstetrical outcomes. As women delay childbearing, understanding the mechanisms of ovarian aging and follicular depletion have become increasingly more relevant. Age-related meiotic errors in oocytes are well established. In addition, it is also important to understand how intraovarian regulators change with aging and how certain treatments can mitigate the impact of aging. Individual studies have demonstrated that reproductive pathways involving antimullerian hormone (AMH), vascular endothelial growth factor (VEGF), neurotropins, insulin-like growth factor 1 (IGF1), and mitochondrial function are pivotal for healthy oocyte and cumulus cell development and are altered with increasing age. We provide a comprehensive review of these individual studies and explain how these factors change in oocytes, cumulus cells, and follicular fluid. We also summarize how modifiers of folliculogenesis, such as vitamin D, coenzyme Q, and dehydroepiandrosterone (DHEA) may be used to potentially overcome age-related changes and enhance fertility outcomes of aged follicles, as evidenced by human and rodent studies.