Expansion Of Cumulus-oocyte Complexes Research Articles

Curcumin protects porcine granulosa cells and mouse ovary against reproductive toxicity of aflatoxin B1 via PI3K/AKT signaling pathway

Aflatoxin B1 (AFB1) is a widespread food contaminant with toxic effects on female reproductive system. This concerns the human public health and the development of the livestock industry. Effective and feasible measures against reproductive toxicity of AFB1 are also unknown. Curcumin has been shown to improve animal production performance and alleviate toxicity of AFB1 in other tissues. The study aimed to reveal the potential mechanism of curcumin in alleviating AFB1 exposure-triggered reproductive toxicity in porcine. Porcine GCs and matured oocytes in vitro and mouse ovary in vivo were cultured as model and then exposed to AFB1 and curcumin. The transcriptomics for porcine GCs was performed. The apoptosis, cell cycle, ROS content and mitochondrial membrane potential level analysis were determined using probe staining and flow cytometry. QRT-PCR and western blotting were analyzed for the genes expression. Oocytes maturation and parthenogenetic activation were performed to evaluate the quality of oocytes. The data showed that AFB1 exposure significantly inhibited porcine GCs viability and exhibited concentration-dependent relationship from 1 to 16 μM. Curcumin supplementation efficiently reversed the inhibition of cell viability. Further analysis indicated that curcumin application alleviated AFB1-exposed porcine GCs and mouse ovary mitochondria dysfunction, oxidative stress, cell cycle arrest, and apoptosis. We found that PI3K/Akt signaling pathway plays a vital role in AFB1-induced porcine GCs toxicity through bioinformatic analysis. The data not only confirmed the correlation of PI3K/Akt signaling pathway and oxidative stress, cell cycle arrest and apoptosis, but also consistent with the results that curcumin supplementation could markedly activate the PI3K/Akt signal inhibited by AFB1. Moreover, curcumin reversed AFB1-impaired cumulus-oocyte complex expansion, oocytes maturation rate, blastocyst rate and formation. Our study demonstrated that curcumin supplementation can protect porcine GCs and mouse ovary from toxicity of AFB1 by targeting the PI3K/AKT signaling pathway and provides a viable treatment approach for AFB1-induced female reproductive toxicity.

MiR-29b inhibits COC expansion and oocyte in vitro maturation via induction of ROS by targeting CYCS

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

PSXIII-14 Effect of melatonin supplementation during in vitro embryo culture on cleavage rates following in vitro maturation and fertilization of bovine oocytes

Abstract In vitro embryo production (IVEP) is an important advanced reproductive technology and has great potential in enhancing livestock production. However, in vitro culture (IVC) of embryos exposes them to stress resulting in compromising quality and efficiency of embryo production. Melatonin has been reported in several studies to reduce oxidative stress and improve the efficiency of IVEP. Our objective was to evaluate the effect of melatonin supplementation in IVC media on cleavage rates of bovine embryos. Bovine ovaries (n = 22) were obtained from local abattoir with ovarian weight of 7.29 ± 3.31 g, and size of 3.65 ± 0.69 cm. Cumulus-oocyte complexes (COCs) were recovered from 2 to 8 mm size follicles by aspiration using an 18-gauge needle attached with a 10 mL syringe. COCs were matured, fertilized, and cultured in vitro using IVF Bioscience bovine media and their protocols. Oocytes were in vitro matured for 23 h at 38.8°C, 5.5% CO2 in a humidified atmosphere and assessed for maturation by assessing COC expansion. Fully expanded and partially expanded COCs were fertilized for 20 h in a 4-well dish with frozen/thawed bull semen. Following IVF, the cumulus cells were removed by vertexing, and the presumptive zygotes were cultured in vitro with and without 1 µM melatonin in 4-well dishes in triplicates under the oil for embryo development at 38.8°C and 5.5% CO2. Cleavage rates were determined on d 3 of culture. The percentage of cleaved embryos was calculated as (number of zygotes cleaved/total number of fertilized oocytes) x100. Data were analyzed by student’s unpaired t- test with Welch’s correction using GraphPad Prism software. The cleavage rate was greater in melatonin supplemented group (54.17 ± 11.24%) than the control (49.83 ± 7.36%) but the difference was not significant (P &amp;gt; 0.05). In conclusion, under our experimental conditions, melatonin supplementation during embryo culture, following IVM/IVF of the bovine oocytes, did not significantly improve cleavage rates.

Use of excised ovaries for oocyte recovery by ultrasound guided follicular aspiration – Validation of an experimental model for research purposes in live mares ovum pick up

Ovum pick-up (OPU) by transvaginal ultrasound guided follicle aspiration in mares is a common assisted reproductive technique used for oocyte recovery and in vitro production of horse embryos. There has been relatively little research into the factors influencing oocyte recovery in OPU from live mares. The objective of this study was to compare oocyte recovery and morphology of ultrasound-guided follicle puncture and aspiration in live mares and in postmortem excised ovaries, in order to validate an experimental model for research purposes of the efficiency of OPU in mares. Data from OPU performed in 12 mares from a commercial program (follicle numbers, oocyte recovery and oocyte morphology) were compared to that obtained from ultrasound-guided follicle puncture of 13 postmortem excised ovaries from slaughtered mares processed within 2 h of slaughter. In both groups, the OPU was performed by the same operator using the same equipment and OPU technique. The recovered oocytes per aspirated follicle was higher (P < 0.05) in the postmortem group (105/166, 63.2 %) than in live mares (138/261, 52.9 %). There was more (P < 0.05) expanded cumulus oocyte complexes in the postmortem than in the live mares (18 % vs. 2.9 %). Several oocytes (5 oocytes from 81 aspirated follicles) were found in the leaked fluid which overflowed during follicle flushing of postmortem ovaries. In conclusion, the higher recovery rate obtained in the excised ovaries and the finding of oocytes in the leaked fluid during OPU, suggests that there is still room for improvement in the in vivo OPU technique. Utilizing postmortem excised ovaries could offer an alternative for further research into factors affecting oocyte recovery and oocyte leakage during OPU procedures.

Effect of supplementing epinephrine in maturation media on in-vitro developmental competence of cattle and buffalo oocytes

During in-vitro maturation, the oocyte experiences stressful conditions that likely compromise its development. Epinephrine is a catecholamine that plays a vital role during cellular stress by scavenging free radicals. The hypothesis is that epinephrine addition in maturation media improves the developmental competence of oocytes in cattle and buffalo. The objectives of the experiments were to investigate the effect of epinephrine addition in maturation media on nuclear maturation, developmental competence, and oocyte mRNA abundance of genes related to antioxidants and growth pathways in cattle and buffalo. In experiment 1, cattle oocytes were matured for 24 h in maturation media supplemented with increasing concentrations of epinephrine 0, 0.01, 1.0, and 100 μM. Oocytes were cultured to assess cleavage at 48 h and blastocyst on day 7 of the culture. The cumulus-oocyte complexes (COCs) expansion, nuclear maturation, and oocyte mRNA abundance of genes (SOD1, GPX4, GDF9, CASP9) were evaluated. In experiment 2, buffalo oocytes were matured and assessed for development and mRNA abundance as described for cattle. In addition, the blastomere number was counted in the hatched blastocyst. The data were analyzed using GLIMMIX and MIXED procedures of SAS. Results revealed that the supplementation of epinephrine increased (P ≤ 0.03) the COCs expansion, nuclear maturation, and developmental competence of oocytes in cattle. Interestingly, all the responses were maximized (quadratic effect; P ≤ 0.08) at 1 μM concentrations. The mRNA abundance of genes in cattle oocytes was not affected by the treatment. The experiment in buffalo revealed that epinephrine increased blastocyst formation without affecting COCs expansion, and nuclear maturation. The higher blastocyst was achieved at 0.01 μM concentrations of epinephrine. Interestingly, the addition of epinephrine increased the mRNA abundance of genes related to antioxidant pathways (SOD1, GPX4). Moreover, supplementation of epinephrine increased the blastomere count of the hatched blastocyst in buffalo. In conclusion, epinephrine addition in maturation media benefited oocyte development in cattle and blastocyst yield in buffalo at 1 and 0.01 μM concentrations, respectively. It appears that the addition of epinephrine affected different cellular pathways, COCs expansion, and nuclear maturation in cattle and increased antioxidant genes for buffalo.

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.

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.

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.

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.