Cumulus-oocyte Complexes Research Articles

Assessment of loliolide extracted from Biserula pelecinus, present during in vitro oocyte maturation, on fertilisation and embryo development in sheep

Context As a ‘duty of care’, it is important to test whether new forage plants for ruminants contain secondary compounds (PSCs) that affect reproductive performance. We have previously observed, a posteriori, that the presence of a methanolic extract of Biserrula pelecinus during maturation of sheep oocytes increased fertilisation rate and blastocyst development. This result needed to be verified a priori and, if the outcome was repeated, we needed to identify the plant secondary metabolite responsible. Aims To test whether PSCs from B. pelecinus, when added to the oocyte maturation medium, improve fertilisation rate and blastocyst development; to test whether loliolide is the active molecule produced by B. pelecinus. Methods Methanol–chloroform extracts of B. pelecinus were fractionated using rapid silica filtration and solvents of increasing polarity. Fractions at final concentrations of 0, 100 or 200 μg mL−1 were added to the medium used to mature sheep cumulus–oocyte complexes (COCs) and effects were determined for maturation, subsequent cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and total blastocyst cell number (TCN). Results Fraction BP-6 at 100 μg mL−1 reduced blastocyst rate (P < 0.05), but had no effect when the dose was doubled to 200 μg mL−1. Further fractionation using semi-preparative high-performance liquid chromatography showed loliolide as the most abundant compound in BP-6. Supplementation of the in vitro maturation medium with loliolide (0, 2.5, 5, 10 and 25 μg mL−1) did not affect any measure of embryo development. All COCs treated with B. pelecinus fractions reached the final stage of embryo development, blastocyst hatching. Total blastocyst cell number was not affected. Conclusion The presence of fractions of B. pelecinus extract during in vitro oocyte maturation can reduce embryo development. Implications In vitro techniques can detect potential effects of forages on reproduction. Some fractions from an extract of B. pelecinus when present during oocyte maturation can reduce embryo development. The abundant PSC, loliolide, was not responsible. There was no indication that a PSC in B. pelecinus improves outcomes.

Chronic psychosocial stress affects insulin-like growth factor 1 and its receptors in mouse ovaries.

Context Chronic psychosocial stress negatively affects folliculogenesis and oogenesis. Intraovarian mechanisms mediating these effects are poorly understood. Aims This work aimed to find out how chronic psychosocial stress affects ovarian IGF1 and its receptor (IGF1R), as well as Igf1 and Igf1r gene expression in cumulus-oocyte complexes (COCs). It also aimed to address possible protective effects of gonadotropin stimulation on IGF1 ovarian signalling. Methods Female CD1 mice experienced chronic psychosocial stress of 11-day isolation followed by overcrowding for 10days. To verify the model, blood corticosterone levels and the quality of oocytes were evaluated in stressed females. The levels of IGF1/IGF1R, blood IGF1 concentration, and expression of Igf1 /Igf1r in the ovaries were compared in stressed and unstressed females. Key results Psychosocial stress caused an elevation of corticosterone level, which was alleviated by gonadotropin treatment. The stressed mice showed a decreased IGF1 level in the ovaries and a decreased expression of Igf1 and Igf1r in COCs. In the unstressed females, gonadotropin injection decreased the expression of Igf1 and Igf1r ; in the stressed females, the same treatment increased Igf1r expression. Neither stress nor ovarian stimulation with gonadotropins affected the serum IGF1 level. Conclusions Psychosocial stress suppresses IGF1 signalling in the ovaries. Gonadotropin treatment modulates these effects differently in stressed and unstressed animals. Implications The results may have translational value for human reproduction. Ovarian IGF1 can be considered a candidate for further improvement of IVF results in women under conditions of chronic stress.

Effects of Ovarian Stimulation with Gonadotropins in the Conditions of Chronic Psychosocial Stress on the Quality of Murine Oocyte

Chronic psychosocial stress may negatively affect the female reproductive system. Meanwhile, the effect of ovarian stimulation with gonadotropins during stress on the quality of oocytes remains poorly studied. The purpose of this work was to investigate the effects of chronic psychosocial stress on the quality of murine cumulus-oocyte complexes during natural estrus cycle, as well as during ovarian stimulation with exogenous gonadotropins; the latter is an important part of modern assisted reproductive technologies. The results of the study demonstrate that psychosocial stress does not affect the number of ovulating oocytes, but worsens their quality, i. e. reduces the percentage of mature oocytes. In addition, stressed mice exhibited the increased accumulation of reactive oxygen species in oocytes, which is accompanied by the enhanced rate of apoptosis in cumulus cells. Hormonal stimulation of the ovaries with gonadotropins alleviates the negative changes associated with the psychosocial stress, normalizing the level of reactive oxygen species in oocytes and reducing the rate of apoptosis in cumulus cells.

Efficiency of oocytes retrieval during a repeated series of transvaginal ultrasound-guided puncture of follicles in Simmental heifers

The article presents data on the efficiency of a repeated series of transvaginal ultrasound-guided recovery of oocytes (OPU) in donor heifers of the Simmental breed. Two series were performed — five OPU sessions with an intensity of once a week and a two-week rest period between series. During the repeated series of OPUS, 1.7 fewer follicles were visualized in donor heifers on average per session compared to the value of this indicator established in the first series (7.0 versus 5.3 follicles, p < 0.05). A decrease in the number of ultrasound-visible follicles during repeated series at the trend level was noted in all five OPU sessions. During the repeat series, 0.4 fewer oocyte-cumulus complexes (OCCs) were obtained from one donor on average per session (3.1 versus 2.7 OCCs). There were no differences in the quality of OCCs between the two OPU series: the proportion of OCCs suitable for further use was 67.0% and 63.5%, respectively, while in individual OPU sessions the differences between the series were multidirectional. Fertilization of oocytes and further in vitro cultivation showed a significantly higher degree of the first division — crushing during the 1st series of OPU (p < 0.05), however, these differences were leveled with further cultivation: the yield of blastocysts was, respectively, 23.6% and 21.7%. Considering the absence of significant differences in the quantitative characteristics of the obtained oocytes, as well as their developmental competencies in vitro, conducting a repeat series of OPU after a two-week rest period may be recommended for obtaining oocytes in donor heifers of the Simmental breed.

Gene expression levels in cumulus cells are correlated with developmental competence of bovine oocytes

The generation of mammalian embryos by in vitro culture is hampered by the failure of many of the embryos to develop to the blastocyst stage. This problem occurs even when cumulus–oocyte complexes (COCs) with good morphology are visually selected and used for culture. Because cumulus cells are important for oocyte maturation and subsequent embryo development, here we compared gene expression patterns in cumulus cells of COCs that developed in vitro to the blastocyst stage with those of COCs that failed to develop. Cumulus cells were aspirated from bovine COCs selected for in vitro culture. Oocyte developmental competence was evaluated by screening for cleavage and development to the blastocyst stage. The collected cumulus cells were used to quantify mRNA levels of FSH receptor (FSHR), insulin-like growth factor-1 receptor (IGF-1R), anti-Müllerian hormone (AMH), AMH receptor II (AMHRII), epidermal growth factor receptor (EGFR), estrogen receptor β (ERβ), B cell lymphoma/leukemia-2 associated X (Bax), and cysteine-aspartic acid protease-3 (Caspase-3). We found that the expression levels of FSHR, IGF-1R, AMH, and EGFR were higher in cumulus cells from COCs that developed to blastocysts as compared with those that failed to develop, whereas expression levels of Bax and Caspase-3 were lower in cumulus cells of COCs that matured to the blastocyst stage. Positive correlations were found between FSHR and IGF-1R expression (r = 0.59) and between ERβ and EGFR expression (r = 0.43) in cumulus cells from COCs that developed to the blastocyst stage. Our findings indicate that gene expression levels in cumulus cells are correlated with the developmental competence of bovine oocytes. Measurement of gene expression in cumulus cells therefore offers a non-invasive means of predicting oocyte developmental competence.

The dose-dependent dual effects of alpha-ketoglutarate (AKG) on cumulus oocyte complexes during in vitro maturation

In this study, we reported for the first time the dose-dependent dual effects of Alpha-Ketoglutarate (AKG) on cumulus oocyte complexes (COCs) during in vitro maturation (IVM). AKG at appropriate concentration (30 µM) has beneficial effects on IVM. This includes improved cumulus expansion, oocyte quality, and embryo development. These effects are mediated through multiple underlying mechanisms. AKG reduced the excessive accumulation of reactive oxygen species (ROS) in cumulus cells, reduced the consumption of GSH and NADPH. Cumulus GSH and NADPH were transported to oocytes via gap junctions, thereby reducing the oxidative stress, apoptosis and maintaining the redox balance in oocytes. In addition, AKG improved the mitochondrial function by regulating the mitochondrial complex 1 related gene expression in oocytes to maintain mitochondrial membrane potential and ATP production. On the other hand, oocyte generated GDF9 could also be transported to cumulus cells to promote cumulus expansion. Conversely, a high concentration of AKG (750 µM) exerted adverse effects on IVM and suppressed the cumulus expansion as well as reduced the oocyte quality. The suppression of the cumulus expansion caused by high concentration of AKG could be rescued with GDF9 supplementation in COCs, indicating the critical role of GDF9 in IVM. The results provide valuable information on the variable effects of AKG at different concentrations on reproductive physiology.Graphical abstract

INCREASED IN VITRO PRODUCTION OF BOVINE EMBRYOS RESULTING FROM OOCYTE MATURATION IN THE PRESENCE OF TRICIRIBINE, A SPECIFIC INHIBITOR OF AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX).COCs were fertilizedin vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1μM of triciribine were stained at 3, 6, and 9 hours of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5% significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P<0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P<0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P<0.05). On the other hand, when COCs were matured in the presence of 1μM, there was an increase in the blastocyst rate (P<0.05), but without altering the timing of meiosis resumption (P<0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1μM of triciribine increases the in vitro production of bovine embryos.

NPR3 is regulated by gonadotropins and modulates bovine cumulus cell expansion.

The significant role of C-type natriuretic peptide (CNP) and its receptor 2 (NPR2) in regulating oocyte meiotic maturation and facilitating communication between oocytes and surrounding cumulus cells has been well-documented in various mammalian species, including mice, cattle and swine. However, further investigation is needed to ascertain whether natriuretic peptide receptors (NPRs) are involved in regulating other essential ovarian functions. Hence, this study aimed to explore the potential involvement of NPRs in the regulation of cumulus expansion and oocyte meiotic maturation in bovine cumulus-oocyte complexes (COCs). The findings revealed that NPR3 mRNA abundance was downregulated by FSH and LH in cumulus cells of bovine COCs during in vitro maturation (IVM), while NPR2 mRNA levels were not affected by gonadotropins. Inhibition of the epidermal growth factor receptor (EGFR) during IVM of COCs prevented the NPR3 mRNA downregulation induced by gonadotropins in cumulus cells. Additionally, treatment of COCs during IVM with an NPR3 agonist (cANP4-23) inhibited cumulus expansion induced by gonadotropins. This inhibitory effect was further intensified when COCs were co-treated with cANP4-23 and CNP. These findings provide robust evidence indicating that normal cumulus expansion in bovine COCs involves an inhibitory effect of gonadotropins on NPR3 mRNA expression, which is mediated via EGFR signaling. The study also provides evidence that CNP and NPR3 interact synergistically to regulate cumulus expansion in response to gonadotropins.

Comparative analysis of short versus long co-incubation of gametes on post-insemination outcomes and embryo morphokinetics: a sibling oocyte randomized study

Research questionDoes a short co-incubation of gametes in conventional IVF impact post-insemination outcomes and embryo morphokinetics? DesignSibling oocyte randomized pilot study performed between December 2020 and March 2023. Eligible couples (n=55) were female aged 18-43 years with body mass index (BMI) ≤35 and male normal semen parameters. Cumulus oocyte complexes (COCs) (min 6 - max 12) were randomized in a 1:1:2 proportion in long (16-18 hrs), or short (2 hrs) co-incubation IVF exposure, and ICSI, respectively. All oocytes inseminated via IVF were monitored by time-lapse (TL). Blastocysts of ≥BL3CC were subjected to trophectoderm (TE) biopsy on day (D)5, D6 or D7 by next generation sequencing (NGS). ResultsA total of 954 COCs were distributed to long co-incubation (n=235, 24.6%), short co-incubation (n=235, 24.6%), and ICSI (n=484, 50.3%). In total, 202 were MII after long co-incubation, 209 after short co-incubation and 394 at ICSI. Regression analyses showed both short co-incubation groups (OR: 0.72, 95% CI: 0.46–1.11, P=0.139) and ICSI (OR: 1.43, 05% CI: 0.95–2.15, P=0.090) did not significantly impact normal fertilization rates (2PN) when compared to long co-incubation. Usable blastocysts per MII, per 2PN, and euploid blastocysts per MII, per 2PN and per tested blastocysts were similar among the groups. The rate of embryo arrest (HR: HR: 0.79, 95% CI: 0.57-1.11, P=0.183) and timings to reach morphokinetic milestones among embryos reaching blastulation were similar between IVF groups (P>0.05, for all). ConclusionA short 2-hour co-incubation of gametes in IVF yields comparable fertilization, blastocyst, and euploidy rates without adverse embryo morphokinetic events, compared to standard overnight co-incubation.

New Insights on In Vitro Maturation of Oocytes for Fertility Preservation

In the last decade, the evolution of oncofertility has sparked a resurgence of interest in in vitro maturation (IVM) due to its suitability in certain oncological scenarios where controlled ovarian hyperstimulation may not be feasible. The retrieval of immature cumulus–oocyte complexes from small antral follicles, regardless of the menstrual cycle phase, presents a swift opportunity to vitrify mature oocytes or embryos post-IVM in urgent situations or when stimulation is not advisable. Harvesting immature cumulus–oocyte complexes and immature oocytes can be achieved transvaginally or directly in the laboratory from extracorporeal ovarian tissue. Although IVM has transitioned from an experimental status due to safety validations, it relies on the intricate process of oocyte maturation. Despite successful live births resulting from IVM in fertility preservation contexts, the comparatively lower developmental competence of in vitro matured oocytes highlights the necessity to enhance IVM culture systems. Recent advancements in IVM systems hold promise in bolstering oocyte competence post-IVM, thereby narrowing the gap between IVM and outcomes from ovarian stimulation. Additionally, for optimizing the chances of conception in cancer survivors, the combination of IVM and ovarian tissue cryopreservation stands as the favored choice when ovarian stimulation is unfeasible.

Lack of effect of cAMP modulation during pre-maturation in vitro on development to the blastocyst stage and expression of specific genes associated with lineage commitment

Pharmacological elevation of cAMP of cultured cumulus-oocyte complexes (COC) before or coincident with initiation of maturation has been reported to improve outcomes for various systems for in vitro production of embryos. Here it was hypothesized that artificial elevation of cAMP in the oocyte for a 2-h period of pre-maturation would improve developmental competence of matured oocytes and result in increased blastocyst yield and alter expression of genes important for embryonic differentiation. Treated COC were cultured for 2 h with dibutyryl cAMP (dbcAMP), a membrane-permeable form of cAMP, and 3-isobutyl-1-methylxanthine (IBMX), which inhibits phosphodiesterases that convert cAMP to ATP. Subsequently, oocytes were matured and fertilized and the resultant embryos cultured for 7.5 d. There was no effect of treatment on the percent of oocytes or cleaved embryos becoming blastocysts or on transcript abundance of EOMES, GATA4, NANOG, SOX2, or SOX17 in the blastocyst. Results do not support the use of pharmacological enhancers of cAMP concentrations in the oocyte for improving the blastocyst yield following in vitro oocyte maturation, fertilization, and embryo culture.

Quality of IVM ovarian tissue oocytes: impact of clinical, demographic, and laboratory factors.

To determine how clinical, demographic, and laboratory characteristics influence ovarian tissue oocyte quality. Immature cumulus-oocyte complexes were isolated from removed ovaries and cultured for 48-52h in either monophasic standard or biphasic CAPA media for fertility preservation. A total of 355 MII oocytes from 53 patients were described for intracytoplasmic and extracytoplasmic anomalies. Multiple clinical, laboratory, and demographic characteristics were analyzed. Statistically significant differences between independent groups in qualitative variables were identified using Pearson's χ2 and Fisher's exact tests. The diagnostic value of quantitative variables was assessed using the ROC curve analysis. Factors associated with the development of dysmorphism, taking patient age into account, were identified using the binary logistic regression analysis. Dysmorphisms were observed in 245 oocytes (69.0%), with a median number of dysmorphisms of 2. Oocyte dysmorphisms were found to be 2.211 times more likely to be detected in patients with ovarian cancer, while the presence of dark-colored cytoplasm was associated with gynecologic surgery in the anamnesis (p = 0.002; OR 16.652; 95% CI, 1.977-140.237; Cramer's V 0.187). Small polar bodies developed 2.717 times more often (95% CI, 1.195-6.18) in patients older than 35. In the case of ovarian transportation on ice at 4 ℃, the chances of development of cytoplasmic granularity increased 2.569 times (95% CI, 1.301-5.179). The use of biphasic CAPA IVM media contributed to a decrease in the probability of large polar body formation (p = 0.034) compared to the standard monophasic IVM media. Both patients' characteristics and laboratory parameters have an impact on the quality of IVM ovarian tissue oocytes.

The role of microenvironmental factors in the pre-ovulatory oocyte in the pathogenesis of “poor” ovarian response to in vitro fertilization (IVF) programs

Background. The “poor” response to ovarian stimulation during IVF (in vitro fertilization) cycles depends on various factors, including the biochemical composition of the follicle microenvironment. Recent studies have focused on the relationship between vascular tone regulators, angiogenic factors, oxidative stress, and the quality of oocytes. Despite this, there is still no consensus on the exact effect of these factors on folliculogenesis or their role in “poor” responses.Objective. To evaluate the content of biochemical factors and regulators of vascular tone and angiogenesis in follicular fluid in patients with a “poor” ovarian response to stimulation in IVF programs, compared to similar indicators in women with a normal ovarian response.Materials and methods. An open prospective cohort study was conducted, including 56 patients who were part of the IVF program. The criterion for separating the cohort of women was their response to controlled ovarian stimulation during the IVF cycle. Women with a “poor” response (n=32) were placed in the main group, while those with a normal response (n=24) were in the comparison group. Follicular fluid samples from women with a “poor” response were divided into two subgroups based on the presence or absence of an oocyte-cumulus complex in the follicle. The biochemical and soluble regulatory factors in follicular fluid were analyzed in both groups after controlled ovarian stimulation was completed. Statistical analysis of the data was done using methods from variation statistics. A critical level of significance for the differences was set at p ≤0.05.Result. The levels of urea, glucose, high-density lipoprotein, and total antioxidant status (TAOS) in follicular fluid (FF) from patients with a “poor” ovarian response were statistically significantly higher compared to those in the control group. The levels of bilirubin, low-density lipoprotein, and uric acid were also significantly lower in VF from patients in the main group compared to the control group, p&lt;0.05. The indices of vascular endothelial growth factor (VEGF-A) and vascular endothelial growth receptor-2 (VEGFR-2) in FF from women with a “poor” response were significantly lower than those from patients with a normal response, while the levels of endogenous nitric oxide were significantly higher in the former group compared to the latter, p&lt;0.05.Conclusion. The absence of an oocyte-cumulus complex in the pre-ovulatory follicle, which is associated with a “poor” response of the ovary to stimulation, is likely due to a disruption in vascularization processes. This is supported by a decrease in the levels of pro- and anti-angiogenic factors, such as VEGF-A and VEGFR-2.

Robotic Manipulation of Cumulus–Oocyte Complexes for Cumulus Cell Removal

The removal of cumulus cells from cumulus–oocyte complexes is a critical step in clinical in vitro fertilization. Since the oocyte is partially occluded by the surrounding cumulus cells and individual cumulus cells are small in size, it is difficult for embryologists to assess the oocyte's maturity before cumulus cell removal and to completely remove all the cumulus cells manually . Furthermore, it is easy for the oocyte to become lost inside the micropipette during aspiration due to the inaccuracy of manual control. To deal with these difficulties, a robotic system was developed to completely remove cumulus cells from mature oocytes. In this study, an EPSANet50 network was developed to accurately assess the maturity of oocytes, avoiding the removal of cumulus cells around the immature oocyte. An adaptive controller was designed to accurately position oocytes at the target position, reducing the loss of oocytes inside the micropipette. An improved Yolov5s network was proposed to quantify the number and size of cumulus cells and assess the completeness of cumulus cell removal. The experimental results on mouse cumulus–oocyte complexes showed that the robotic system had a higher success rate (98.0 ± 1.8% vs. 85.3 ± 2.4%) and lower discard rate (4.1 ± 2.7% vs. 19.6 ± 3.5%) than the manual operation. Moreover, a higher amplification rate and lower non-specific rate were also achieved by the robotic system in the subsequent genetic testing procedure, indicating reduced genetic contamination from the cumulus cells.

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.

Maturational competence of equine oocytes is associated with alterations in their "cumulome".

Assisted reproductive technologies are an emerging field in equine reproduction, with species dependent peculiarities, such as the low success rate of conventional in vitro fertilisation. Here, the "cumulome" was related to the developmental capacity of its corresponding oocyte. Cumulus oocyte complexes (COCs) collected from slaughterhouse ovaries were individually matured, fertilised by intracytoplasmic sperm injection (ICSI), and cultured. After maturation, the cumulus was collected for proteomics analysis using label-free mass spectrometry (MS) based protein profiling by nano-HPLC MS/MS and metabolomics analysis by UPLC-nanoESI MS. Overall, a total of 1671 proteins and 612 metabolites were included in the quantifiable "cumulome". According to the development of the corresponding oocytes, three groups were compared with each other: not matured (NM; n = 18), cleaved (CV; n = 15) and blastocyst (BL; n = 19) groups. CV and BL were also analysed together as the matured group (M; n = 34). The dataset revealed a closer connection within the two M groups and a more distinct separation from the NM group. Over-representation analysis detected enrichments related to energy metabolism as well as vesicular transport in the M group. Functional enrichment analysis found only the KEGG pathway of oxidative phosphorylation as significantly enriched in NM group. A compound attributed to ATP was observed with significantly higher concentrations in the BL group compared with the NM group. Finally, in the NM group, proteins related to degradation of glycosaminoglycans were lower and components of cumulus extracellular matrix were higher compared to the other groups. In summary, the study revealed novel pathways associated with the maturational and developmental competence of oocytes.

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.

Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

Flavopiridol inhibits oocyte maturation, reduces oocyte quality and blocks cumulus cell function

Flavopiridol (FP) is a plant-derived flavonoidis and used to treat cancers, fungal infections and inflammation-related diseases. However, it is not clear whether it has side effects on the female reproductive system. In this study, we aimed to investigate the toxic effects and potential underlying mechanisms of FP on oocyte maturation and cumulus cell expansion in mice. Cumulus-oocyte complexes (COCs) were cultured in vitro with FP of gradient concentration (50–1000 nM), according to the plasma concentration of FP in the clinical trial. The maturation rate and cumulus expansion index of oocytes were counted and studied by immunofluorescence staining, qRT-PCR, oocyte chromosome preparation and so on. The results showed that the FP-exposed COCs inhibited the oocyte maturation and cumulus cell expansion, leading to cell apoptosis in a dose dependent way. Oocytes exposed to 500 nM FP showed abnormalities in the spindle structure and chromosome arrangement, ultimately leading to the oocyte maturation arrest and aneuploidy. This may be due to the excessive oxidative stress caused by mitochondrial membrane potential damage and mislocalization. Therefore, when FP is used for cancer treatment, its side effects on the female reproductive system should be seriously considered.

7 Role of the preovulatory follicle in oocyte metabolic and developmental competence for pregnancy

Abstract A majority of bovine pregnancy loss occurs in the first 7 d of gestation. The oocyte is a key driver of embryo viability during this time, and its competence for embryo development and pregnancy establishment is greatly influenced by the surrounding microenvironment during oocyte maturation. Assisted reproductive technologies allow for more efficient propagation of superior genetics and improved reproductive management of the cattle herd; however, many assisted reproductive technologies alter the normal microenvironment of the maturing oocyte and negatively impact oocyte competence for early embryo development. For example, oocytes induced to ovulate from preovulatory follicles of lesser physiological maturity (decreased estradiol production and smaller diameter) have reduced blastocyst development, poorer embryo quality, and a hastened rate of embryo development compared with those ovulated from follicles of greater maturity. Our overarching hypothesis is that the intrafollicular milieu of physiologically mature preovulatory follicles provides the necessary microenvironment to metabolically program maturing oocytes for successful embryo development. Studies by our team have demonstrated that the follicular fluid metabolite milieu during in vivo oocyte maturation differs between preovulatory follicles of greater and lesser maturity and that such differences have direct effects on the maturing oocyte. Numerous follicular fluid metabolites involved in glucose and amino acid metabolism were more abundant in preovulatory follicles of greater versus lesser physiological maturity. Additionally, abundance of transcripts encoding glycolytic enzymes was higher in cumulus cells from follicles of greater maturity, and both ATP and levels of key transcripts in the mitochondrial respiratory chain were higher in oocytes collected from preovulatory follicles of greater versus lesser maturity. We supplemented in vitro maturing oocytes with follicular fluid from preovulatory follicles of greater or lesser maturity to determine causality of differing follicular fluid milieu on cumulus-oocyte metabolism and oocyte competence for early embryo development. Follicle maturity status had robust effects on cumulus-oocyte complex consumption or production of key metabolites in glucose, purine, and amino acid metabolism pathways during oocyte maturation. We also observed hastened development rate in embryos arising from in vitro matured oocytes supplemented with lesser maturity follicular fluid, mirroring past in vivo findings by others. Furthermore, RNA-sequencing of resulting blastocysts of identical stage and quality suggested more efficient metabolism and Wnt/β-catenin signaling in blastocysts derived from oocytes matured in preovulatory follicular fluid from greater maturity follicles. In contrast, transcriptome profiles of blastocysts from oocytes matured in preovulatory follicular fluid from lesser maturity follicles indicated oxidative stress and dysregulated cell division. These recent findings by our lab indicate that impacts of follicle maturity and the resulting follicular fluid microenvironment on the maturing oocyte withstand throughout early embryo development and hold promising roles to improve reproductive efficiency in cattle.