Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

Biomarkers Assessing the Role of Cumulus Cells on IVF Outcomes: A Systematic Review.

PURPOSE: Although significant improvements in assisted reproductive technology (ART) outcomes have been accomplished, a critical question remains: which embryo is most likely to result in a pregnancy? Embryo selection is currently based on morphological and genetic criteria; however, these criteria do not fully predict good-quality embryos and additional objective criteria are needed. The cumulus cells are critical for oocyte and embryo development. This systematic review assessed biomarkers in cumulus-oocyte complexes and their association with successful IVF outcomes. METHODS: A comprehensive search was conducted using PubMed, Embase, Scopus, and Web of Science from inception until November 2022. Only English-language publications were included. Inclusion criteria consisted of papers that evaluated genetic biomarkers associated with the cumulus cells (CCs) in humans and the following three outcomes of interest: oocyte quality, embryo quality, and clinical outcomes, including fertilization, implantation, pregnancy, and live birth rates. RESULTS: The search revealed 446 studies of which 42 met eligibility criteria. Nineteen studies correlated genetic and biochemical biomarkers in CCs with oocyte quality. A positive correlation was reported between oocyte quality and increased mRNA expression in CCs of genes encoding for calcium homeostasis (CAMK1D), glucose metabolism (PFKP), extracellular matrix (HAS2, VCAN), TGF-ß family (GDF9, BMP15), and prostaglandin synthesis (PTGS2). Nineteen studies correlated genetic and biochemical biomarkers in CCs with embryo quality. A positive correlation was reported between embryo quality and increased mRNA expression in CCs of genes encoding for extracellular matrix (HAS2), prostaglandin synthesis (PTGS2), steroidogenesis (GREM1), and decreased expression of gene encoding for hormone receptor (AMHR2). Twenty-two studies assessed genetic and biochemical biomarkers in CCs with clinical outcomes. Increased expression of genes encoding for extracellular matrix (VCAN), and TGF-ß family (GDF9, BMP15) were positively correlated with pregnancy rate. CONCLUSION: Genetic biomarkers from cumulus cells were associated with oocyte quality (CAMK1D, PFKP, HAS2, VCAN, GDF-9, BMP-15, PTGS2), embryo quality (GREM1, PTGS2, HAS2), and pregnancy rate (GDF9, BMP15, VCAN). These results might help guide future studies directed at tests of cumulus cells to devise objective criteria to predict IVF outcomes.

Dynamic regulation of semaphorin 7A and adhesion receptors in ovarian follicle remodeling and ovulation.

The ovarian follicle is a complex structure that protects and helps in the maturation of the oocyte, and then releases it through the controlled molecular and structural remodeling process of ovulation. The progesterone receptor (PGR) has been shown to be essential in regulating ovulation-related gene expression changes. In this study, we found disrupted expression of the cellular adhesion receptor gene Sema7A in the granulosa cells of PGR-/- mice during ovulation. We subsequently found that expression of Sema7A in preovulatory follicles is promoted by gonadotropins and hypoxia, establishing an asymmetrical pattern with the SEMA7A protein enriched at the apex of large antral follicles. Sema7A expression was downregulated through a PGR-dependent mechanism in the periovulatory period, the abundance of SEMA7A protein was reduced, and the asymmetric pattern became more homogeneous after an ovulatory stimulus. Receptors for Sema7A can either repel or promote intercellular adhesion. During ovulation, striking inverse regulation of repulsive Plxnc1 and adhesive Itga5/Itgb1 receptors likely contributes to dramatic tissue remodeling. The adhesive receptor Itga5 was significantly increased in periovulatory granulosa cells and cumulus-oocyte complexes (COCs), and functional assays showed that periovulatory granulosa cells and COCs acquire increased adhesive phenotypes, while Sema7A repels granulosa cell contact. These findings suggest that the regulation of Sema7A and its associated receptors, along with the modulation of integrin α5, may be critical in establishing the multilaminar ovarian follicle structure and facilitating the remodeling and apical release of the cumulus-oocyte complex during ovulation.

Human follicular fluid elicits select dose- and age-dependent effects on mouse oocytes and cumulus-oocyte complexes in a heterologous in vitro maturation assay.

Follicular fluid (FF) is a primary microenvironment of the oocyte within an antral follicle. Although several studies have defined the composition of human FF in normal physiology and determined how it is altered in disease states, the direct impacts of human FF on the oocyte are not well understood. The difficulty of obtaining suitable numbers of human oocytes for research makes addressing such a question challenging. Therefore, we used a heterologous model in which we cultured mouse oocytes in human FF. To determine whether FF has dose-dependent effects on gamete quality, we performed in vitro maturation of denuded oocytes from reproductively young mice (6-12 weeks) in 10%, 50%, or 100% FF from participants of mid-reproductive age (32-36 years). FF impacted meiotic competence in a dose-dependent manner, with concentrations >10% inhibiting meiotic progression and resulting in spindle and chromosome alignment defects. We previously demonstrated that human FF acquires a fibro-inflammatory cytokine signature with age. Thus, to determine whether exposure to an aging FF microenvironment contributes to the age-dependent decrease in gamete quality, we matured denuded oocytes and cumulus-oocyte complexes (COCs) in FF from reproductively young (28-30 years) and old (40-42 years) participants. FF decreased meiotic progression of COCs, but not oocytes, from reproductively young and old (9-12 months) mice in an age-dependent manner. Moreover, FF had modest age-dependent impacts on mitochondrial aggregation in denuded oocytes and cumulus layer expansion dynamics in COCs, which may influence fertilization or early embryo development. Overall, these findings demonstrate that acute human FF exposure can impact select markers of mouse oocyte quality in both dose- and age-dependent manners.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Does the addition of follicular fluid in the in vitro maturation medium increase the oocyte maturation and embryo production in alpacas?

In alpacas (Vicugna pacos), the high cost of in vitro embryo production is also a consequence of the use of several substances in the culture medium. In addition, embryo production rates in this species are still considered low. Thus, in attempt to reduce the cost and to improve the in vitro embryo production rates, this study evaluates the effect of adding follicular fluid (FF) in the in vitro maturation medium on oocyte maturation and subsequent embryo production. After ovary collection at the local slaughterhouse, the oocytes were recovered, selected, and allocated in experimental groups: standard maturation medium (G1) and simplified medium added by 10% FF (G2). The FF was acquired from follicles between 7- and 12-mm diameter. The cumulus cell expansion and the embryo production rates were analyzed by chi-square with p < 0.05. No differences (p > 0.05) were observed in maturation rate between G1 (66.36%) and G2 (63.12%) groups. Likewise, no significant difference (p > 0.05) was verified between G1 and G2 for morula (40.85 vs 38.45%), blastocyst (7.01 vs 6.93%), and total number of embryos (47.87 vs 45.38%). In conclusion, it was possible to simplify the medium used for in vitro maturation of alpaca oocytes resulting in embryo production rates similar to the standard medium.

Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying.

PURPOSE: Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration. METHODS: Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR. RESULTS: CRNPs effectively transported trehalose into COCs within 4 h of co-incubation at 38.5 °C followed by a cold-triggered release at 4 °C for 15 min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1 at dehydration levels reaching up to 63% of water loss. CONCLUSION: Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

Interaction between epidermal growth factor receptor and C-C motif chemokine receptor 2 in the ovulatory cascade.

The epidermal growth factor receptor (EGFR) signaling pathway is one of the main pathways responsible for propagating the luteinizing hormone (LH) signal throughout the cumulus cells and the oocyte. Recently, we have proposed the C-C motif chemokine receptor 2 (CCR2) and its main ligand (monocyte chemoattractant protein-1, MCP1) as novel mediators of the ovulatory cascade. Our previous results demonstrate that the gonadotropins (GNT), amphiregulin (AREG), and prostaglandin E2 (PGE2) stimulation of periovulatory gene mRNA levels occurs, at least in part, through the CCR2/MCP1 pathway, proposing the CCR2 receptor as a novel mediator of the ovulatory cascade in a feline model. For that purpose, feline cumulus-oocyte complexes (COCs) were cultured in the presence or absence of an EGFR inhibitor, recombinant chemokine MCP1, and gonadotropins [as an inducer of cumulus-oocyte expansion (C-OE), and oocyte maturation] to further assess the mRNA expression of periovulatory key genes, C-OE, oocyte nuclear maturation, and steroid hormone production. We observed that MCP1 was able to revert the inhibition of AREG mRNA expression by an EGFR inhibitor within the feline COC. In accordance, the confocal analysis showed that the GNT-stimulated hyaluronic acid (HA) synthesis, blocked by the EGFR inhibitor, was recovered by the addition of recombinant MCP1 in the C-OE culture media. Also, MCP1 was able to revert the inhibition of progesterone (P4) production by EGFR inhibitor in the C-OE culture media. Regarding oocyte nuclear maturation, recombinant MCP1 could also revert the inhibition triggered by the EGFR inhibitor, leading to a recovery in the percentage of metaphase II (MII)-stage oocytes. In conclusion, our results confirm the chemokine receptor CCR2 as a novel intermediate in the ovulatory cascade and demonstrate that the EGFR/AREG and the CCR2/MCP1 signaling pathways play critical roles in regulating feline C-OE and oocyte nuclear maturation, with CCR2/MCP1 signaling pathway being downstream EGFR/AREG pathway within the ovulatory cascade.

Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos.

Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes' thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability.

Minimized microbial contamination rate in conventional IVF cycles after modifications of cumulus-oocyte complex handling.

RESEARCH QUESTION: Could the microbial contamination rate of oocytes and embryos in conventional IVF cycles be further reduced by modifying the handling procedures of cumulus-oocyte complexes (COC)? DESIGN: Two modifications were applied to COC handling procedures. First, a mark was made on the outer wall of the 10-cm Petri dish to indicate the site from which the follicular fluid was poured out during the oocyte retrieval process (modified handling 1) since May 2018. Second, a modified way of pipetting during denuding (modified handling 2) has been adopted since June 2019. The microbial contamination rates before and after each modification were compared. The clinic outcomes of patients with a history or at a high risk of microbial contamination were reported after incorporating the two modifications of COC handling. RESULTS: After the first modification was implemented, the contamination rate was remarkably reduced from 0.37% (9/2436) to 0.18% (2/1089). After adding the second modification, no new contamination occurred in the subsequent 3178 conventional IVF cycles (P = 0.001). Moreover, no contamination was noted in patients with a history of microbial contamination or persistent candidal vaginitis during conventional IVF after modifications of COC handling. CONCLUSIONS: Modifying the handling procedures of COC can minimize the microbial contamination rate in conventional IVF cycles. Contamination risk directly derived from the urogenital tracts might be less likely than what we thought to be with current IVF techniques.

Integrative analysis of transcriptomic and metabolomic profiles reveals abnormal phosphatidylinositol metabolism in follicles from endometriosis-associated infertility patients.

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.

Expression of the melatonergic system during meiotic maturation of cynomolgus monkey cumulus-oocyte complexes.

BACKGROUND: Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non-human primate cumulus-oocyte complexes (COCs). METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect transcripts and proteins of the rate-limiting enzyme in melatonin synthesis (arylalkylamine N-acetyltransferase, AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NRH: quinone oxidoreductase 2, NQO2) in cynomolgus monkey COCs. RESULTS: RT-PCR analyses revealed the presence of AANAT, MT1, MT2, and NQO2 transcripts in granulosa cells, germinal vesicle (GV)- and metaphase II (MII)-stage cumulus cells, and oocytes. Immunocytochemistry revealed the presence of AANAT, MT1, MT2, and NQO2 proteins in GV- and MII-stage COCs. CONCLUSIONS: Our results provide the first evidence for the existence of the rate-limiting enzyme required for melatonin synthesis, melatonin membrane receptors, and a melatonin binding site in non-human primate COCs.

Oocyte in vitro maturation: physiological basis and application to clinical practice.

Oocyte in vitro maturation (IVM) is an assisted reproductive technology with a long and sometimes checked history. It is a minimally invasive technique involving the deliberate collection of immature oocytes from patients that have received no or minimal ovarian stimulation and the culture of oocytes to maturity in vitro, before standard procedures thereafter. Now, IVM is classified as nonexperimental and is primarily indicated for patients with a high antral follicle count, especially patients with polycystic ovaries or polycystic ovary syndrome, as well as for fertility preservation in cancer patients. In the recent past, IVM practice has had a confusing array of clinical protocols and has been slow to adapt to new scientific insights; however, recently, significant advances have been made in IVM culture methods based on new knowledge from animal studies, combined with defining a simple patient treatment protocol. These improvements have led to significant recent progress in IVM practice to the extent that IVM is now routinely practiced in a growing number of centers with specialized expertise around the world.

Intercellular communication in the cumulus-oocyte complex during folliculogenesis: A review.

During folliculogenesis, the oocyte and surrounding cumulus cells form an ensemble called the cumulus-oocyte complex (COC). Due to their interdependence, research on the COC has been a hot issue in the past few decades. A growing body of literature has revealed that intercellular communication is critical in determining oocyte quality and ovulation. This review provides an update on the current knowledge of COC intercellular communication, morphology, and functions. Transzonal projections (TZPs) and gap junctions are the most described structures of the COC. They provide basic metabolic and nutrient support, and abundant molecules for signaling pathways and regulations. Oocyte-secreted factors (OSFs) such as growth differentiation factor 9 and bone morphogenetic protein 15 have been linked with follicular homeostasis, suggesting that the communications are bidirectional. Using advanced techniques, new evidence has highlighted the existence of other structures that participate in intercellular communication. Extracellular vesicles can carry transcripts and signaling molecules. Microvilli on the oocyte can induce the formation of TZPs and secrete OSFs. Cell membrane fusion between the oocyte and cumulus cells can lead to sharing of cytoplasm, in a way making the COC a true whole. These findings give us new insights into related reproductive diseases like polycystic ovary syndrome and primary ovarian insufficiency and how to improve the outcomes of assisted reproduction.

Industrialization possibilities of purified pig sperm hyaluronidase.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

Effect of follicular ablation and gonadotropin priming on the recovery and quality of oocytes in Boran cows.

The objective of the study was to evaluate the effect of follicle ablation, exogenous FSH application, and different coasting time prior to ovum pick-up (OPU) on the number of follicles suitable for aspiration, oocyte quality, and cleavage rate in Ethiopian Boran cows. The experiment was carried out in three parts, (I) cows were synchronized using 500 µg PGF2α given 11 days apart. Cows were then subjected to a biweekly ovum pickup session before ovulation (n = 5) or starting day 7 after ovulation (n = 4) for 3 weeks. (II) Cows were synchronized, and all visible follicles were ablated on the first days of overt estrus, and cows were grouped into those that received a divided dose of 350 IU FSH (n = 5) or 175 IU FSH (n = 5) over 3 days. In both groups, OPU was carried out weekly starting 48 h after the last FSH for 6 weeks. (III) Protocol was similar to part II, but in group with 350 IU FSH (n = 5), coasting period was increased to 72 h. The covariates of follicles and oocyte were not affected (P > 0.05) by corpus luteum presence at OPU. The mean number of medium (7.36 ± 0.57) and large (8.28 ± 0.96) follicles were significantly higher (P < 0.05) in the group that received divided 350 IU FSH. Similarly, the mean number of grade-1 (4.19 ± 0.24) and grade-2 (4.32 ± .27) oocytes, maturation rate (70.41%), and cleavage rate (47.5%) were significantly higher (P < 0.05) in the group that received 350 IU FSH. COC quality was significantly (P < 0.05) influenced by coasting period. However, both maturation and cleavage rates were not affected by the coasting period. This study demonstrated that follicular ablation and treatment with FSH improves follicular population and oocyte recovery rate in Boran cows.

Notch Signaling Induced by Endoplasmic Reticulum Stress Regulates Cumulus-Oocyte Complex Expansion in Polycystic Ovary Syndrome.

Endoplasmic reticulum (ER) stress activated in granulosa cells contributes to the pathophysiology of polycystic ovary syndrome (PCOS). In addition, recent studies have demonstrated that Notch signaling plays multiple roles in the ovary via cell-to-cell interactions. We hypothesized that ER stress activated in granulosa cells of antral follicles in PCOS induces Notch signaling in these cells, and that activated Notch signaling induces aberrant cumulus-oocyte complex (COC) expansion. Expression of Notch2 and Notch-target transcription factors was increased in granulosa cells of PCOS patients and model mice. ER stress increased expression of Notch2 and Notch-target transcription factors in cultured human granulosa-lutein cells (GLCs). Inhibition of Notch signaling abrogated ER stress-induced expression of genes associated with COC expansion in cultured human GLCs, as well as ER stress-enhanced expansion of cumulus cells in cultured murine COCs. Furthermore, inhibition of Notch signaling reduced the areas of COCs in PCOS model mice with activated ER stress in the ovary, indicating that Notch signaling regulates COC expansion in vivo. Our findings suggest that Notch2 signaling is activated in granulosa cells in PCOS and regulates COC expansion. It remains to be elucidated whether aberrant COC expansion induced by the ER stress-Notch pathway is associated with ovulatory dysfunction in PCOS patients.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography-high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Incorporation of a fixed-time artificial insemination protocol results in improved reproductive management and genetics of the beef herd. However, a subset of animals exposed to such protocols will not display estrus prior to insemination. Behavioral estrus is indicative of the preovulatory follicle's physiological maturity and is essential for both the production of an oocyte with optimal developmental competence and preparation of the maternal environment for pregnancy establishment. Animals that do not display estrus prior to insemination and are induced to ovulate a physiologically less advanced follicle have reduced oocyte developmental competence that leads to reduced embryo cleavage rates, embryo quality, and pregnancy rates. This study investigated the impacts of reduced follicle maturity at the initiation of ovulation on the energy production capacity of the oocyte as well as follicular fluid metabolic composition. Results from this study demonstrated that follicle maturity, indicated by increased serum estradiol concentration at the initiation of ovulation, resulted in increased ATP within the oocyte as well as an increased level of metabolites involved in glucose metabolism in the follicular fluid. Increased energy production ability in the oocytes from more mature follicles could contribute to the increased cleavage rates and embryo quality seen in previous studies.

Mammalian cumulus-oocyte complex communication: a dialog through long and short distance messaging.

Communications are crucial to ovarian follicle development and to ovulation, and while both folliculogenesis and oogenesis are distinct processes, they share highly interdependent signaling pathways. Signals from distant organs such as the brain must be processed and compartments within the follicle have to be synchronized. The hypothalamic-pituitary-gonadal (HPG) axis relies on long-distance signalling analogous to wireless communication by which data is disseminated in the environment and cells equipped with the appropriate receptors receive and interpret the messages. In contrast, direct cell-to-cell transfer of molecules is a very targeted, short distance messaging system. Numerous signalling pathways have been identified and proven to be essential for the production of a developmentally competent egg. The development of the cumulus-oocyte complex relies largely on short distance communications or direct transfer type via extensions of corona radiata cells through the zona pellucida. The type of information transmitted through these transzonal projections is still largely uncharacterized. This review provides an overview of current understanding of the mechanisms by which the gamete receives and transmits information within the follicle. Moreover, it highlights the fact that in addition to the well-known systemic long-distance based communications from the HPG axis, these mechanisms acting more locally should also be considered as important targets for controlling/optimizing oocyte quality.

Calcium (Ca2+) expression and intensity in cumulus-oocyte complex (COCs)in Kacang goat after vitrification.

The process of vitrification of the cumulus-oocyte complex (COCs) often results in cold shock. When warming, heat shock occurs which can disrupt the balance of intracellular calcium (Ca2+) intensity. Drastic changes in temperature cause Reactive Oxygen Species (ROS), affecting changes on Ca2+ in COCs. The role of calcium is needed for oocyte activation in the fertilization process. The purpose of this study was to measure the expression of Ca2+ and the intensity of Ca2+ in COCs after vitrification. The study was divided into 2 groups, the control group (C) of fresh COCs, and the treatment group (T) of COCs after vitrification. After vitrification for 24 hours, then thawing, the expression of Ca2+ was examined using the Immunocytochemistry (ICC) method and the intensity of calcium (Ca2+) with a Confocal Laser Scanning Microscope (CLSM). The research data obtained were analyzed statistically by T-Test. The results showed that the expression of Ca2+ in the control group (12.00±0.00) was different from the treatment group (0.35±0.79). The intensity of Ca2+ in the control group (1059.43±489.59) was different from the treatment group (568.21±84.31). The conclusion of this study is that cryopreservation affects calcium in COCs; there were differences in the expression and the intensity of Ca2+ between fresh COCs and COCs after vitrification. Ca2+ intensity of COCs after vitrification was concentrated in the nucleus, while in fresh COCs it was concentrated in the cytoplasm.