Expansion Of Cumulus-oocyte Complexes Research Articles

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan.

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus-oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.

Silibinin supplementation ameliorates the toxic effects of butyl benzyl phthalate on porcine oocytes by eliminating oxidative stress and autophagy

Butyl benzyl phthalate (BBP) is a common environmental pollutant, it is high in paints, adhesives and other decorative materials, food packaging bags, cleaning agents, is a plasticizer is very widely used in daily life. However, it remains unknown whether BBP causes damage to oocytes cultured in vitro and whether there is an effective rescue strategy. Here, we evaluated the effects of exposure to different concentrations of BBP (10, 50, and 100 μM) on the meiosis of porcine oocytes. The results showed that exposure to BBP (100 μM) severely impaired expansion of cumulus-oocyte complex (COCs) and PBE (control:71.6% vs 100 μM: 48.8%). Spindle conformation and chromosome alignment were also significantly abnormal (34.8% and 46.0%, respectively) compared to the control (11.1% and 17.5%, respectively), and BBP caused damage to microfilaments and cortical granules (CGs). In addition, oocyte exposure to BBP induced impaired mitochondrial function and disrupted mitochondrial integrity. Silibinin is a natural active substance isolated from the seeds of Silybum marianum (L.) Gaertneri with strong antioxidant and anti-inflammatory effects. Noteworthy, we added different concentrations of silibinin (10, 20, and 50 μM) to BBP-exposed oocytes for rescue experiments, where 50 μM effectively rescued BBP-induced meiotic failure (70.6%). It also prevented the generation of excessive autophagy and apoptosis in oocytes by inhibiting the production of ROS. In a word, our results suggest that supplementation of silibinin attenuates the impaired oocyte development caused by BBP exposure，which provides a potential strategy to protect oocytes from environmental pollutants.

Bioactivity and Pharmacodynamics of X002, a Follicle-stimulating Hormone-IgG4 Fc Fusion Protein.

Current follicle-stimulating hormone (FSH) drugs meet safety criteria but have suboptimal efficacy, poor patient compliance, and high cost. Alternative FSH-like drugs would help to meet the high market demand. Here, we evaluated X002, an FSH-Fc fusion protein, for bioactivity and half-life in vitro and in vivo. In all cases, the effects of X002 were compared with those of a commercially available short-acting FSH recombinant hormone. First, female Kunming mice (age, 21 to 24 d) were stimulated with pregnant mare serum gonadotropin (PMSG) for 46 h, after which naked oocytes were harvested, treated with X002 or the comparison agent at 37 °C for 4 h, and then evaluated for germinal vesicle breakdown. Second, cumulus-oocyte complexes (COC) were collected from PMSG-stimulated mice and cocultured with X002 or the comparison agent for 14 h; the COC diameters were then measured, and the expression of genes involved in COC expansion were evaluated using quantitative RT-PCR analysis. Third, to assess the pharmacokinetics of X002, female Sprague-Dawley rats (age, 6 to 8 wk) were injected subcutaneously with X002 or the comparison agent; serum samples then were collected at various times and assessed via ELISA. Fourth, to evaluate X002 pharmacodynamics, 26-d-old female Sprague-Dawley rats were treated with X002 or the comparison agent; 84 h later, the rats were stimulated with human chorionic gonadotropin (hCG). At 12 h after hCG injection, euthanasia was performed. Ovaries were removed and weighed, and serum levels of estradiol and progesterone were measured. Finally, to assess superovulation, the oocytes in the fallopian tubes were counted at 108 h after in vivo treatment of rats with X002 or the comparison agent. The data show that X002, a long-acting agent, promoted germinal vesicle breakdown and COC expansion in vitro and in vivo ovarian weight gain and superovulation to a degree similar to the short-acting comparison agent.

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 sequencingand 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.

Decreased Expression of EZH2 in Granulosa Cells Contributes to Endometriosis-Associated Infertility by Targeting IL-1R2.

The mechanism by which endometriosis, a common gynecological disease characterized by chronic pelvic pain and infertility, causes infertility remains elusive. Luteinized unruptured follicle syndrome, the most common type of ovulatory dysfunction, is a cause of endometriosis-associated infertility involving reduced numbers of retrieved and mature oocytes. Ovulation is controlled by luteinizing hormone and paracrine signals produced within the follicle microenvironment. Generally, interleukin (IL)-1β is elevated in endometriosis follicular fluid, whereby it amplifies ovulation signals by activating extracellular-regulated kinase 1/2 and CCAAT/enhancer binding protein β pathways. However, this amplification of ovulation by IL-1β does not occur in patients with endometriosis. To illuminate the mechanism of ovulatory dysfunction in endometriosis, we analyzed the effect of oxidative stress and IL-1β expression on endometriosis follicles. We found that oxidative stress decreased EZH2 expression and reduced H3K27Me3 levels in endometriosis ovarian granulosa cells (GCs). Selective Ezh2 depletion in mice ovarian GCs reduced fertility by disturbing cumulus-oocyte complex expansion and reducing epidermal growth factor-like factor expression. Gene expression and H3K27Me3 ChIP-sequencing (ChIP-Seq) of GCs revealed IL-1 receptor 2 (IL-1R2), a high-affinity IL-1β-receptor that suppresses IL-1β-mediated inflammatory cascades during ovulation, as a crucial target gene of the EZH2-H3K27Me3 axis. Moreover, IL-1β addition did not restore ovulation upon Ezh2 knockdown, indicating a vital function of IL-1R2 in endometriosis. Thus, our findings show that reducing EZH2 and H3K27Me3 in GCs suppressed ovulatory signals by increasing IL-1R2 expression, which may ultimately contribute to endometriosis-associated infertility.

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.

P-610 Endoplasmic reticulum stress-induced Notch signaling stimulates cumulus-oocyte complex expansion in PCOS

Abstract Study question Does endoplasmic reticulum (ER) stress and Notch signaling affect cumulus-oocyte complex (COC) expansion in pathophysiology of polycystic ovary syndrome (PCOS)? Summary answer Notch signaling is induced via activation of ER stress in granulosa cells (GCs) of PCOS and stimulates COC expansion that is abrogated by Notch inhibition. What is known already PCOS presents a variety of symptoms including ovarian dysfunction which is caused by various local factors in follicular microenvironment; among them, ER stress and following activation of unfolded protein response are critical, causing ovarian fibrosis, growth arrest of antral follicles and other ovarian dysfunctions. While Notch signaling pathway plays an important role of various ovarian functions such as ovarian development, follicle growth, luteinization and steroid hormone synthesis, the potential interaction between Notch signaling and ER stress in ovarian function is not determined. Study design, size, duration To examine expression levels of Notch signaling, ovaries and granulosa-lutein cells (GLCs) were collected from PCOS patients undergoing surgery or IVF. Human GLCs were collected from follicular fluid of IVF patients and cultured under ER-stressed condition. COCs obtained from PMSG-primed mice were subjected to examine the in vitro effects of ER stress activation and Notch inhibition on COC expansion. To examine the in vivo effects of Notch inhibition, dehydroepiandrosterone-induced PCOS mouse model was used. Participants/materials, setting, methods The expression levels of Notch signaling in ovaries and GLCs were investigated by immunohistochemistry and real time qPCR. To examine whether Notch signaling is activated by ER stress, human GLCs were incubated with ER stress inducer or inhibitor and ATF4 was knocked down by RNA interference. To investigate COC expansion level, murine COCs were cultured under ER stress condition with/without Notch signaling inhibitor. The COCs were collected from PCOS mice treated with/without Notch inhibitor. Main results and the role of chance We found that the expression levels of Notch2 and Hey2, a transcription factor activated by Notch signaling, were upregulated in GCs of antral follicles from PCOS patients and PCOS mice by using immunohistochemical analysis. Similarly, mRNA levels of these genes were higher in GLCs from PCOS patients than those from control patients. Notch signaling was induced in cultured human GLCs incubated with an ER stress inducer, tunicamycin; the effect was abrogated by incubation with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), or knockdown of activating transcription factor 4 (ATF4, a transcription factor induced by ER stress). These findings suggest that Notch signaling is induced by ER stress via ATF4 pathway in human GCs. Measuring under a microscope, the area of expanded COCs was increased in cultured murine COCs incubated with tunicamycin, while this stimulatory effect of tunicamycin was abrogated by adding a Notch signaling inhibitor, DAPT. The area of expanded COCs obtained from PCOS model mice was increased compared to control mice, while administration of DAPT to these mice reduced the area. These results suggest that ER stress-induced Notch signaling stimulate COC expansion contributing PCOS pathophysiology. Limitations, reasons for caution COC expansion area was measured only in PCOS model mouse; it is unknown whether COC expansion is induced in PCOS patients. This point requires further investigation in PCOS patients. Wider implications of the findings Our findings suggest that ER stress-induced Notch signaling affects COC expansion, associated with ovulatory dysfunction in PCOS. The detailed understandings of PCOS pathophysiology may be beneficial for substantial clinical implications and inhibition of ER stress or Notch signaling may serve as a novel therapeutic approach for PCOS. Trial registration number This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (19k09749, 19k24045, 19k24021, 21k16808, 21j12871,), a grant from the Takeda Science Foundation, a grant from The Tokyo Society of Medical Science, a grant from The Japan Society of Fertility Preservation, and a grant from The Japan Society for Menopause and Women’s Health (JMWH) (a JMWH Bayer Grant).

Anti-Müllerian Hormone Inhibits FSH-Induced Cumulus Oocyte Complex In Vitro Maturation and Cumulus Expansion in Mice.

Simple SummaryAnti-Müllerian hormone (AMH) is a homodimeric glycoprotein composed of two identical subunits, which inhibits the recruitment of primordial follicles and the development of antral follicles in females. Anti-Müllerian hormone can be used as a diagnostic and prognostic marker for ovarian reserve, superovulation, embryo quality, and conception rate. However, few studies have focused on the effect of AMH on oocyte maturation. In the present study, we found Anti-Müllerian hormone has no effect on the nuclear maturation and cumulus expansion of cumulus oocyte complexes (COCs), whereas it has an inhibitory effect on follicle-stimulating hormone (FSH)-stimulated COCs nuclear maturation and cumulus expansion. These findings expand our knowledge of the functional role of AMH in modulating folliculogenesis.Anti-Müllerian hormone (AMH) is secreted by the ovaries of female animals and exerts its biological effects through the type II receptor (AMHR2). AMH regulates follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH. Despite the considerable research on the actions of AMH in granulosa cells, the effect of AMH on the in vitro maturation of oocytes remains largely unknown. In the current study, we showed that AMH is only expressed in cumulus cells, while AMHR2 is produced in both cumulus cells and oocytes. AMH had no significant effect on COCs nuclear maturation, whereas it inhibited the stimulatory effects of FSH on COCs maturation and cumulus expansion. Moreover, AMH treatment effectively inhibited the positive effect of FSH on the mRNA expressions of Hyaluronan synthase 2 (Has2), Pentraxin 3 (Ptx3), and TNF-alpha-induced protein 6 (Tnfaip 6) genes in COCs. In addition, AMH significantly decreased the FSH-stimulated progesterone production, but did not change estradiol levels. Taken together, our results suggest that AMH may inhibit the effects of FSH-induced COCs in vitro maturation and cumulus expansion. These findings increase our knowledge of the functional role of AMH in regulating folliculogenesis.

L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1mM L-cysteine in 5% CO2 under humidified air for 24hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

L–carnitine improves developmental competence of buffalo oocytes in vitro

Objective: To investigate the effect of L-carnitine on in vitro maturation and subsequent in vitro embryo production of buffalo oocytes.Methods: Cumulus oocyte complexes (COCs) were aspirated from ovaries of slaughtered buffaloes. COCs were classified into good and fair qualities based on morphological observation of numbers and integrity of cumulus cells surrounding the oocyte. Both categories of COCs were placed in in vitro maturation medium with supplementation of different concentrations (0, 0.250, 0.375 or 0.500 mg/mL) of L-carnitine. Oocytes from both qualities were in vitro fertilized and in vitro cultured for 7 days, to examine the developmental competence.Results: Supplementation of L-carnitine to in vitro maturation medium increased the cumulus cell expansion rate of COCs to grade A, and reduced the cumulus cell expansion of COCs to grade B and grade C in both good and fair quality oocytes. Similarly, L-carnitine induced the in vitro meiotic progression of buffalo oocytes to metaphase Π in both good and fair quality oocytes. Additionally, L-carnitine reduced the rate of oocyte degeneration in both good and fair quality oocytes. L-carnitine increased the rate of cleaved formation at day 2 and blastocyst formation at day 7 during in vitro culture in both qualities of oocytes. Moreover, a higher rate of blastocyst production was observed in L-carnitine-treated fair quality oocytes, which was higher than the results in the untreated good quality oocytes.Conclusions: L-carnitine enhances meiotic maturation and subsequent embryo development from both good and fair quality buffalo oocytes.

Compromised Cumulus-Oocyte Complex Matrix Organization and Expansion in Women with PCOS

The cumulus-oocyte complex (COC) matrix plays a critical role in the ovulation and fertilization process and a major predictor of oocyte quality. Proteomics studies of follicular fluid showed differential expression of COC matrix proteins in women with polycystic ovary syndrome (PCOS), indicating altered COC matrix in these women. In the present study, we aimed to understand COC matrix gene induction in humans and its probable dysfunction in women with PCOS. Animal studies have shown that amphiregulin (AREG) and growth differentiation factor-9 (GDF-9) are important in the induction of COC matrix genes which are involved in cumulus expansion. The effects of AREG and GDF-9 on expression of tumor necrosis factor alpha induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) on human cumulus granulosa cells (CGCs) and murine COC expansion were evaluated. Further time-dependent effects of growth factor supplementation on these gene expressions in CGCs from PCOS and control women were compared. Follicular fluid from PCOS showed reduced COC matrix expansion capacity, using murine COCs. Expression of COC matrix genes TNFAIP6 and HAS2 were significantly reduced in CGCs of PCOS. Treatment of CGCs with AREG and GDF-9 together induced expression of both these genes in controls and could only restore HAS2 but not TNFAIP6 expression in PCOS. Our results suggest that the reduced potential of follicular fluid to support COC expansion, altered expression of structural constituents, and intrinsic defects in granulosa cells of women with PCOS may contribute to the aberrant COC organization and expansion in PCOS, thus affecting fertilization.

Integrated Analysis of Transcriptome and Histone Modifications in Granulosa Cells During Ovulation in Female Mice.

The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation-sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.

돼지 난자난구세포 복합체의 난구세포 분포에 따른 단백질체 차이 분석

Porcine cumulus cell layers are thought to influence various fundamental functions during oocyte maturation. The expansion of cumulus-oocyte complexes (COCs) is known to highly correlate with oocyte maturation, fertilization, and embryonic development. In this study, we performed two dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis using porcine COCs proteins. Porcine COCs were collected from ovary follicles and classified as the good-quality group and the poor-quality group based on cumulus cell layers around oocyte. The COCs protein samples were separated in a pH 3.0–10.0 IPG gels for the first dimension, and then 8%–16% SDS-PAGE gel for the second dimension. Gel image analysis was performed in protein spots between good- and poor-quality groups, and then differential protein spots were identified using MALDI-TOF analysis. There were more than 300 protein spots in the 2-DE gel image obtained from 1 mg protein samples of COCs. Five differential protein spots were identified by gel image analysis with more than 1.5-fold differences. All 5 protein spots were up-regulated in good quality group and revealed as transferrin, tight junction protein ZO-2, tubulin beta chain, meiosis-specific nuclear structural protein 1 and dynamin 1-like isoform 4 in good. Our results indicated the identification of 5 differentially expressed proteins in the porcine good-quality COCs and these protein profiles will make available important guides for porcine good oocyte selection for the production of in vitro embryos.

The Calcium-Sensing Receptor Is Involved in Follicle-Stimulating Hormone-Induced Cumulus Expansion in in vitro Cultured Porcine Cumulus-Oocyte Complexes.

The Calcium-Sensing Receptor (CASR) is a G protein-coupled receptor of the C family that reportedly promotes maturation of porcine oocytes. However, its role in cumulus expansion of cumulus-oocyte complexes (COCs) is not well known. This study was conducted to determine the role of CASR and potential mechanisms involved during in vitro maturation (IVM) of porcine COCs. After culture of COCs in follicle-stimulating hormone (FSH)-supplement maturation medium for 24 h, the time of breakdown of the germinal vesicle (GVBD), indicative of initiation of meiotic maturation, resulted in an increased (p < 0.05) CASR mRNA expression level in cumulus cells. Moreover, IVM of COCs in 10 μM of the CASR agonist NPS R-568 promoted (p < 0.05) cumulus expansion but only in FSH-containing medium. Conversely, 20 μM of the CASR inhibitor NPS2390 precluded cumulus expansion. We next tested the effect of the CASR agonist/inhibitor on the expression of cumulus expansion-related genes. The CASR agonist significantly upregulated the expression of hyaluronan acid synthase 2 (HAS2), whereas the CASR inhibitor downregulated the expression of all HAS2, prostaglandin-endoperoxide synthase 2 (PTGS2), and tumor necrosis factor a-induced protein 6 (TNFAIP6). Altogether, these results suggest that CASR activity is involved in FSH-stimulated porcine cumulus expansion.

152 The Effect of Nerve Growth Factor Beta on Embryonic Cleavage Rates

Abstract Nerve growth factor, (NGF) beta is a seminal plasma protein that has been associated with sire conception rates in cattle. Previous research showed that the administration of NGFβ, via culture media, to cows resulted in improved conceptus development. Though this finding was thought to be an indirect effect of improved corpus luteum (CL) function, questions raised if NGFβ could act directly on the embryo to promote development. This work seeks to determine the effect of NGFβ supplementation during in-vitro fertilization (IVF) on cleavage and blastocyst rates. How does the administration of NGFβ in culture media affect cleavage and blastocyst rates during in-vitro fertilization? Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COC). Selected COC were placed in the maturation medium. Expanded COC were inseminated with frozen-thawed spermatozoa, and IVF media was supplemented with either 0 ng/mL or 100 ng/mL NGF. Presumptive zygotes were transferred to development medium in a tri-gas chamber with 5% CO2, 5% O2, and 90% N2 in a humidified atmosphere at 39°C, mimicking the bovine uterine climate, until 8 days. Treatment with NGFβ increased the percentage of cleaved embryos at 48 hours and the percentage of hatched embryos at 8 days per oocyte. Treatment of NGFβ did not alter the percentage of blastocysts per cleaved embryo or the percentage of hatched blastocysts. These results show that the NGFβ can act directly on the embryo during fertilization to alter embryonic development, specifically embryonic cleavage rates. Future in vivo studies should assess the downstream effects of NGF treatment on conception rates in cattle.

153 The Effect of Nerve Growth Factor Beta on Embryonic Cleavage Rates

Abstract Nerve growth factor (NGF) beta is a seminal plasma protein that has been associated with sire conception rates in cattle. Previous research showed that the administration of NGFβ, via culture media, to cows resulted in improved conceptus development. Though this finding was thought to be an indirect effect of improved corpus luteum (CL) function, questions raised if NGFβ could act directly on the embryo to promote development. This work seeks to determine the effect of NGFβ supplementation during in-vitro fertilization (IVF) on cleavage and blastocyst rates. How does the administration of NGFβ in culture media affect cleavage and blastocyst rates during in-vitro fertilization? Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COC). Selected COC were placed in the maturation medium. Expanded COC were inseminated with frozen-thawed spermatozoa, and IVF media was supplemented with either 0 ng/mL or 100 ng/mL NGF. Presumptive zygotes were transferred to development medium in a tri-gas chamber with 5% CO2, 5% O2, and 90% N2 in a humidified atmosphere at 39°C, mimicking the bovine uterine climate, until 8 days. Treatment with NGFβ increased the percentage of cleaved embryos at 48 hours and the percentage of hatched embryos at 8 days per oocyte. Treatment of NGFβ did not alter the percentage of blastocysts per cleaved embryo or the percentage of hatched blastocysts. These results show that the NGFβ can act directly on the embryo during fertilization to alter embryonic development, specifically embryonic cleavage rates. Future in vivo studies should assess the downstream effects of NGF treatment on conception rates in cattle.

Synergistic effect between LH and estrogen in the acceleration of cumulus expansion via GPR30 and EGFR pathways.

The estrogen membrane receptor GPR30 (also known as G-protein coupled receptor 30) has recently been shown to be involved in the regulation of oocyte maturation and cumulus expansion. However, whether GPR30 expression is regulated by gonadotropin stimulation and how it participates in the regulation of the maturation process is still not clear. In this study, we explored the mechanism underlying the synergy between luteinizing hormone and 17β-estradiol (17β-E2) to improve the epidermal growth factor (EGF) response in cumulus oocyte complexes (COCs) during oocyte maturation in mice. The expression and distribution of GPR30, EGFR, and EGF-like growth factors were examined by real-time quantitative PCR, western blot, and immunofluorescence staining. Lyso-Tracker Red labeling was performed to detect the lysosomal activity in follicle granular cells (FGCs). Cumulus expansion of COCs was evaluated after in vitro maturation for 16 h. We found that EGF-like growth factors transmit LH signals to increase GRP30 levels by inhibiting protein degradation in lysosomes. Meanwhile, 17β-E2 stimulates the GPR30 signaling pathway to increase EGF receptor levels, enhancing the response ability of EGF signaling in COCs and thus promoting cumulus expansion. In conclusion, our study reveals the synergistic mechanism between LH and estrogen in the regulation of cumulus expansion during oocyte maturation process.

Estrogen improves the development of yak (Bos grunniens) oocytes by targeting cumulus expansion and levels of oocyte-secreted factors during in vitro maturation.

The estrogen-signalling pathway is critical for normal follicular development; however, little is known about its importance during in vitro maturation (IVM) in large animals, particularly yaks (Bos grunniens). Through the present study, we aimed to determine the mechanisms underlying estrogen involvement in cumulus expansion and the subsequent development of cumulus-oocyte complexes (COCs). COCs were cultured in the maturation medium supplemented with different concentrations (10−6–10−3 mM) of 17β-estradiol (E2) or its receptor antagonist, fulvestrant, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine the expression of cumulus-expansion related factors and oocyte-secreted factors (OSFs). The cumulus expansion of COCs was observed using an inverted microscope, and COCs developmental ability were judged by the evaluation of cleavage and blastulation rates per inseminated oocytes by IVF, and the number of cells in the blastocyst. Cumulus expansion increased with 10−6–10−3 mM E2, but decreased with fulvestrant. HAS2, PTGS2, PTX3 and OSFs expression increased in the 10−6–10−3 mM E2 groups. Significantly higher cleavage and blastocyst rates were observed in the 10−4 mM E2 group than in the fulvestrant and 0 mM E2 groups. Moreover, in the 10−4 mM group, blastocysts at 7 days had higher cell counts than the other groups. In conclusion, the increase in cumulus expansion and subsequent oocyte development after the addition of E2 to IVM medium may have resulted from increased cumulus-expansion-related factor expression and OSF levels.

Erratum. Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

Study question Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? Summary answer Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. What is known already Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. Study design, size, duration Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. Participants/materials, setting, methods The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. Main results and the role of chance The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. Limitations, reasons for caution The number of PCOS patients in this study was small. Wider implications of the findings This study provides further evidence for metformin as a PCOS treatment. Study funding/competing interest(s) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. Trial registration number N/A.

Ovulation and ovarian wound healing are impaired with advanced reproductive age.

Aging is associated with reduced tissue remodeling efficiency and increased fibrosis, characterized by excess collagen accumulation and altered matrix degradation. Ovulation, the process by which an egg is released from the ovary, is one of the most dynamic cycles of tissue wounding and repair. Because the ovary is one of the first organs to age, ovulation and ovarian wound healing is impaired with advanced reproductive age. To test this hypothesis, we induced superovulation in reproductively young and old mice and determined the numbers of eggs ovulated and corpora lutea (CLs), the progesterone producing glands formed post-ovulation. Reproductively old mice ovulated fewer eggs and had fewer CLs relative to young controls. Moreover, reproductively old mice exhibited a greater number of oocytes trapped within CLs and expanded cumulus oocyte complexes within unruptured antral follicles, indicative of failed ovulation. In addition, post-ovulatory tissue remodeling was compromised with age as evidenced by reduced CL vasculature, increased collagen, decreased hyaluronan, decreased cell proliferation and apoptosis, impaired wound healing capacity, and aberrant morphology of the ovarian surface epithelium (OSE). These findings demonstrate that ovulatory dysfunction is an additional mechanism underlying the age-related loss of fertility beyond the reduction of egg quantity and quality.