Cumulus-oocyte Complexes Research Articles

Porcine sperm bind to an oviduct glycan coupled to glass surfaces as a model of sperm interaction with the oviduct

During transport through the oviduct, sperm interact with epithelial cells by attaching to specific glycans, a mechanism believed to select sperm and prolong their viability. An in vitro model of sperm-oviduct interactions was developed, consisting of a glass surface (either a slide or a coverslip) to which an oviduct glycan (sulfated Lewis X trisaccharide; suLeX) is coupled. The ability of porcine sperm to attach to suLeX-surfaces and detach in response to progesterone and mature cumulus-oocyte complexes (COCs) was validated. The suLeX-coverslip was adapted for in vitro fertilization (IVF), termed glycan-IVF, by allowing porcine sperm to first bind suLeX before transferring mature COCs. The glycan-IVF method produced a percentage of fertilized oocytes comparable to that of conventional IVF (75.1 vs. 72.0%). Finally, the ability of the suLeX-coverslip to maintain sperm fertilizing ability over time was assessed. After 24 h of incubation, fertilization by sperm bound to the suLeX-coverslip was sustained, compared to sperm with unmodified coverslips (12.0 vs. 1.0%, p < 0.05). Furthermore, the percentage of polyspermic zygotes was reduced in the suLeX-coverslip method (17.7 vs. 41.3%, p < 0.05). This study validated an in vitro model for studying sperm-oviduct interactions, with potential applications in assisted reproductive technologies.

Characteristics of porcine oocyte-cumulus complexes derived from various sizes of antral follicles and classified by brilliant cresyl blue staining, and developmental competence of the oocytes.

The present study sought to determine the characteristics of porcine oocyte-cumulus complexes (OCCs) derived from very small and small antral follicles (with less than 1mm and 1-3mm in diameter, respectively; VSF and SF) in comparison with controls from medium ones (with 3-6mm in diameter; MF). Additionally, the present study examined the utility of brilliant cresyl blue (BCB) staining for assessing these OCCs. The incidence of BCB- oocytes in VSF- and SF-derived OCCs was higher than that in MF-derived OCCs. Although the meiotic and developmental competences of BCB+oocytes from MF were superior to those from VSF and SF, blastocysts were successfully obtained from BCB+oocytes even derived from VSF. The mean numbers of both total and viable cumulus cells surrounding an oocyte were significantly affected not only by the origin of the OCCs, but also by the BCB status of the oocytes (largest in MF-derived OCCs containing BCB+oocytes). Although the outer and inner diameters of zona pellucida were affected by the origin of OCCs and the BCB status of oocytes (largest in MF-derived oocytes), the ooplasmic diameter of BCB+oocytes did not differ among those derived from VSF, SF, and MF. Regardless of the BCB status, the transcriptional levels of G6PD and TKT in cumulus cells decreased during follicular development from VSF to MF, whereas the RPIA mRNA level in cumulus cells of MF-derived BCB+OCCs was lower than in the others. These results underscore the utility of BCB staining for selecting MF-, SF-, and even VSF-derived OCCs containing oocytes with relatively higher meiotic and developmental competences, as well as the importance of having a sufficient number of healthy cumulus cells expressing genes related to the pentose phosphate pathway at lower levels.

Curcumin Suppresses ROS Production and Increases Mitochondrial Activity in Cumulus Cells and Oocytes of COCs Derived From Non-Vascularized Follicles in Pigs.

In vitro maturation (IVM) produces offspring from domestic animals; however, the blastocyst rate after IVM was low. We previously reported that the developmental competence of oocytes derived from follicles with blood vessels absent on the surface (non-vascularized follicles: NVF) is quite low compared to those derived from follicles with blood vessels present on the surface (vascularized follicles: VF). Thus, it is important to develop technique to improve the quality of NVF-derived oocyte by IVM. Since it has been reported that reactive oxygen species (ROS) reduces oocyte quality, in this study, we investigated whether curcumin that is known as antioxidant could improve oocyte quality derived from NVF. As results, cultivation of NVF Cumulus-oocyte complexes (COCs) with curcumin significantly improved cumulus expansion and oocyte meiotic maturation of NVF COCs compared to those of NVF COCs without curcumin. Cultivation with curcumin of NVF COCs significantly improved the proliferative activity of cumulus cells. Furthermore, the cultivation significantly reduced ROS activity and increased mitochondrial activity. Hence, it was revealed that the addition of curcumin to the maturation medium increased mitochondrial activity and reduced ROS levels in NVF-derived cumulus cells and oocytes, thereby improving the maturation of oocytes within COCs.

Epitalon-activated telomerase enhance bovine oocyte maturation rate and post-thawed embryo development.

Telomerase is highly expressed in oocyte cumulus cells and plays a significant role in follicular development and oocyte maturation. In this study, we hypothesized that in vitro culture conditions may affect telomerase activity during in vitro embryo production (IVP) and that its activation may improve embryo quality. We first examined telomerase protein levels and localization in bovine cumulus-oocyte complexes via immunofluorescence assays. The results showed that healthy cumulus-oocyte complexes have the nuclear localization of the telomerase while the degraded cumulus-oocyte complex had reduced telomerase levels and that telomerase was localized in the cytoplasm. We activated telomerase via Epitalon, a tetrapeptide with the amino acid sequence Ala-Glut-Asp-Gly. We observed a significant improvement in the oocyte maturation rate compared with the control group (p < 0.05). Furthermore, telomerase activity was significantly compromised in post-thawed embryos, and Epitalon treatment significantly improved blastocyst hatching rate and implantation potential (p < 0.05). Moreover, we performed qPCR, reactive oxygen species, and JC-1 (ΔΨm) assays to evaluate the effect of Epitalon on the health of in vitro mature oocytes, cumulus cells, and post-thawed blastocysts, and the result showed that Epitalon highly enhances the quality and health of the oocyte, cumulus cell, and post-thawed blastocyst. Our results suggest that telomerase activation via Epitalon improves bovine in vitro embryo production.

Effect of Time-Lapse Incubation System on In Vitro Development of Alpaca Embryos.

Currently, incubators with a time-lapse system are widely used for invitro embryo production in several species, however, their effect on alpaca embryo development compared to conventional incubators remains unknown. The aim of this study was to compare early invitro embryo development in alpacas using a time-lapse incubator system versus a conventional incubator. Ovaries were obtained from a slaughterhouse and 1048 cumulus-oocyte complexes (COCs) were collected and invitro matured for 26 h in either a time-lapse system (n = 542) or a conventional incubator (n = 542). All the matured COCs were fertilised for 24 h with fresh spermatozoa recovered from epididymis obtained from the slaughterhouse. The presumptive zygotes in both systems were cultured in a commercial one-step culture medium, and the embryo development was evaluated at 48, 120 and 168 h after fertilisation. Our results showed no differences in maturation rates between the time-lapse system and the conventional incubator (79.9% vs. 55.0%, p = 0.080). Cleavage rates at 48 h were also similar between the time-lapse and conventional systems: 20.5% vs. 20.8% for embryos at the two- to four-cell stage, and 6.0% vs. 7.0% for embryos above the four-cell stage (p > 0.05). At 120 h after fertilisation, the percentage of embryos were more evenly distributed across all stages (below eight-cell, eight-cell and above eight-cell stages) in both systems (p > 0.05). At 168 h, no differences in the blastocyst rates and blastocyst quality were observed between the time-lapse and conventional systems. In conclusion, invitro embryo production in alpacas can be successfully carried out using the time-lapse incubation system or a conventional incubator with comparable results. Further study would focus on the correlation between the developmental patterns and the quality of alpaca embryos, as well as the comparison of invivo embryo development between the two incubation systems.

The effect of single versus group culture on cumulus-oocyte complexes from early antral follicles.

This study aimed to evaluate the effectiveness of single versus group culture strategies for cumulus-oocyte complexes (COCs) derived from early antral follicles (EAFs), with the goal of optimizing culture conditions to increase oocyte availability for assisted reproductive technologies. COCs isolated from EAFs (350-450µm) from sheep ovarieswere cultured in TCM199 medium supplemented with 0.15µg/mL Zn++ as zinc sulfate, 10-4IU/mL FSH, 10ng/mL estradiol, 50ng/mL testosterone, 50ng/mL progesterone, and 5µM Cilostamide. After 5days of long in vitro culture (LIVC), COCs underwent in vitro maturation. This study investigated the effects of single and group culture conditions on COCs, focusing on morphology (integrity of oocyte-granulosa cell complex), viability, oocyte diameter, chromatin configuration, and ultrastructure. Additional factors influencing developmental competence were assessed, including global transcriptional activity, gap junction communication, and meiotic competence. Intracellular reactive oxygen species (ROS) levels and mitochondrial activity were also measured. No significant differences were observed between groups in terms of morphology, viability, oocyte diameter, chromatin configuration, ROS levels, or mitochondrial activity. However, group culture resulted in ultrastructural changes, with a notable reduction in global transcriptional activity, an increase in active gap junctions, and a higher rate of meiosis resumption (p < 0.01). Overall, group culture of COCs derived from sheep EAFs promoted meiosis resumption, suggesting that this approach could improve in vitro culture techniques, increase the availability of mature gametes, and support fertility preservation programs.

Pro-cumulin addition in a biphasic in vitro oocyte maturation system modulates human oocyte and cumulus cell transcriptomes.

Biphasic in vitro oocyte maturation (IVM) can be offered as a patient-friendly alternative to conventional ovarian stimulation in in vitro fertilization (IVF) patients predicted to be hyper-responsive to ovarian stimulation. However, cumulative live birth rates after IVM per cycle are lower than after conventional ovarian stimulation for IVF. In different animal species, supplementation of IVM media with oocyte-secreted factors (OSFs) improves oocyte developmental competence through the expression of pro-ovulatory genes in cumulus cells. Whether the addition of OSFs in human biphasic IVM culture impacts the transcriptome of oocytes and cumulus cells retrieved from small antral follicles in minimally stimulated non-hCG-triggered IVM cycles remains to be elucidated. To answer this, human cumulus oocyte complexes (COCs) that were fully surrounded by cumulus cells or partially denuded at the time of retrieval were cultured in a biphasic IVM system either without or with the addition of pro-cumulin, a GDF9: BMP15 heterodimer. Oocytes and their accompanying cumulus cells were collected separately, and single-cell RNA-seq libraries were generated. The transcriptomic profile of cumulus cells revealed that pro-cumulin upregulated the expression of genes involved in cumulus cell expansion and proliferation while downregulating steroidogenesis, luteinization and apoptosis pathways. Moreover, pro-cumulin modulated the immature oocyte transcriptome during the prematuration step, including regulating translation, apoptosis and mitochondria remodeling pathways in the growing germinal vesicle (GV) oocytes. The addition of pro-cumulin also restored the transcriptomic profile of matured metaphase II (MII) oocytes that were partially denuded at collection. These results suggest that cumulus cell and oocyte transcriptome regulation by pro-cumulin may increase the number of developmentally competent oocytes after biphasic IVM treatment. Future studies should assess the effects of pro-cumulin addition in human biphasic IVM at the proteomic level and the embryological outcomes, particularly its potential to enhance outcomes of oocytes that are partially denuded at COC collection.

Cumulus cells and the TNF-alpha signaling facilitate aging of ovine oocytes.

Post-maturation oocyte aging (PMOA) is known to significantly impair the developmental potential of oocytes; however, comprehensive studies on ovine PMOA remain limited. In mice, cumulus cells (CCs) accelerate oocyte aging by releasing cytokines, but the roles of CCs and cytokines in PMOA of domestic animals are poorly understood. This study aimed to elucidate the involvement of CCs and tumor necrosis factor (TNF)-α in the PMOA of ovine oocytes. Our findings reveal that PMOA significantly reduced blastocyst rates and the expression of development-promoting genes, while increasing oocyte degeneration and activation rates, along with expression of development-inhibiting genes, compared to newly matured oocytes. These detrimental effects were more pronounced in oocytes aged as cumulus-oocyte complexes than as cumulus-denuded oocytes. Additionally, PMOA led to increased apoptotic rates, TNF-α production, and TNF receptor 1 (TNFR1) expression in CCs, coupled with a significant reduction in the expression of anti-apoptotic genes. Mature oocytes expressed TNFR1, with levels decreasing significantly during PMOA. Importantly, the addition of the TNF-α antagonist Etanercept to the aging medium markedly improved parthenogenetic embryo development and the expression of competence-related genes, while mitigating CC apoptosis during PMOA of COCs. In conclusion, PMOA compromises developmental potential while heightening oocyte degeneration and activation sensitivity in ovine oocytes. Cumulus cells exacerbate PMOA through increased TNF-α signaling activity, highlighting the potential of TNF-α antagonists as therapeutic agents to counteract the deleterious effects of PMOA.

Effect of Liquid Marble 3D Culture System on In Vitro Maturation and Embryo Development of Prepubertal Goat Oocytes.

Suboptimal culture conditions during in vitro maturation (IVM) affect oocyte developmental competence and the viability of the resulting embryo. Three-dimensional (3D) culture systems provide a more biologically appropriate environment compared to traditional two-dimensional (2D) cultures. The aim of this study was to evaluate the effect of liquid marble (LM) microbioreactors as a 3D culture system on IVM and the subsequent embryo development of prepubertal goat oocytes. The cumulus-oocyte complexes (COCs) recovered from prepubertal goat ovaries underwent IVM in drops under oil (the 2D system and the control group) and in the 3D LM system (the LM group). After IVM, oocytes were parthenogenetically activated and cultured until the blastocyst stage. The control and LM groups showed similar rates of nuclear maturation (52.17% and 44.12%) and blastocyst formation (10.64% and 10.10%). Reactive oxygen species and glutathione levels and the density of transzonal projections (TZPs) in oocytes did not differ between groups. The LM system increased mitochondrial activity and modified the organization of these organelles in the oocyte cytoplasm compared to the control group. The LM microbioreactor demonstrated the ability to improve the mitochondrial status of the oocytes and was not harmful for oocyte IVM and subsequent embryo development. Therefore, LM could be used as a 3D cost-effective culture system for the IVM of prepubertal goat oocytes.

Region-specific roles of oviductal motile cilia in oocyte/embryo transport and fertility

Abstract The physiological and clinical importance of motile cilia in reproduction is well recognized, however, the specific role they play in transport through the oviduct and how ciliopathies lead to subfertility and infertility is still unclear. The contribution of cilia beating, fluid flow, and smooth muscle contraction to overall progressive transport within the oviduct remains under debate. Therefore, we investigated the role of cilia in the oviduct transport of preimplantation eggs and embryos using a combination of genetic and advanced imaging approaches. We show that the region of the oviduct where cumulus-oocyte complex circling occurs, around the time of fertilization, is correlated with asymmetrical mucosal fold arrangement and non-radially distributed ciliated epithelium. Our results suggest that motile cilia, as well as mucosal fold asymmetry, may contribute to the local flow fields that help steer luminal contents away from the epithelial walls. We also present in vivo, volumetric evidence of delayed egg transport in a genetic mouse model with disrupted motile cilia function in the female reproductive system. Females with Dnah5 deleted in the oviduct epithelium are subfertile and demonstrate disrupted motile cilia activity within the oviduct mucosa. 50% of Dnah5 mutant females have delayed egg transport where COCs did not progress to the ampulla at the expected time-point and remained within the ovarian bursa. The integration of advanced imaging with genetic dysfunction of motile cilia activity provides valuable insights into oviductal transport processes. Potentially, these data could be valuable for better understanding and management of tubal pathologies and human infertility.

Melatonin protects porcine oocytes from gossypol-induced meiosis defects via regulation of SIRT1-mediated mitophagy.

Cottonseed meal (CSM) is an ideal source of protein feed ingredients. However, the gossypol contained in it has toxic effects on animals, limiting its use in livestock production. The underlying mechanisms remain largely unknown. This study aimed to investigate the adverse effects of gossypol exposure and assess whether melatonin, a natural antioxidant, could alleviate oocyte damage induced by gossypol. Porcine cumulus oocyte complexes (COCs) were treated with gossypol alone or co-treated with melatonin for 44h during in vitro maturation. The results demonstrated that gossypol exposure induced oxidative stress and mitochondrial dysfunction, leading to oocyte maturation failure. Conversely, melatonin co-treatment mitigated these detrimental effects, by promoting oocyte mitophagy, as evidenced by the upregulation of PINK1, Parkin, and LC3 expressions, along with the downregulation of P62. Further investigation revealed that gossypol treatment significantly decreased SIRT1 protein expression, while melatonin co-treatment markedly increased it. Using the SIRT1 inhibitor Ex527 confirmed that melatonin enhances mitophagy through SIRT1, improving mitochondrial function and rescuing oocyte maturation. This study revealed the potential harm of gossypol on mammalian reproductive health, provided experimental reference for the protective effect of melatonin, and provided theoretical basis for the effective prevention and treatment of reproductive damage caused by gossypol.

Recombinant FSH induced progesterone via partially regulating let-7 expression in human and mouse granulosa cells.

Serum progesterone may increase prior to ovulation trigger in in vitro fertilization patients, jeopardizing endometrial receptivity and therefore live birth rate. Recombinant FSH (rFSH) promotes progesterone production from human granulosa cells. Yet, the role of FSH on progesterone production need deeper exploration. Studies were conducted in human primary cumulus cells from IVF cycles, human granulosa cell line and mice primary granulosa cells. The relative expression of let-7 was evaluated using real time PCR. Human primary cumulus cells were collected from individual cumulus-oocyte complex of high-progesterone patients (serum progesterone level higher than 5 nM, n=18) and control group (serum progesterone level less than 5 nM, n=25). The expression of let-7a in human primary cumulus cells was markedly reduced in the high-progesterone group compared to the control. The serum progesterone level was augmented after rFSH treatment at dose of 0.5, 1 and 2.5 IU along with reduce expression of let-7a. Progesterone level in cultured medium from isolated mouse primary granulosa cells and human granulosa cell line were significantly elevated with rFSH at 12.5, 25, 50 IU/L concentrations with decreased expression of let-7a. Besides, there was a robustly increase of let-7a expression in let-7a mimics-transfected group and decrease in let-7a inhibitor-group with or without rFSH treatment and the opposite trend of progesterone. Collectively, our findings revealed the key role of let-7 in rFSH induced progesterone level both in human and mouse granulosa cells, providing potential mechanism for premature progesterone rise.

Cumulus cell expansion, nuclear maturation and embryonic development of bovine cumulus-oocyte complexes matured in varying concentrations of follicular fluid.

In this study, we tested the overall hypothesis that CC expansion and early embryo development would be improved by including follicular fluid (FF) from small or large follicles in the oocyte maturation medium. In the first experiment, FF aspirated from bovine abattoir ovaries was added to the maturation medium at 0, 25, 50, 75 or 100%. Images of individual COCs were captured at 0, 6, 12 and 19 hours (h) of the maturation period and analyzed to calculate change in the total area over time. Cumulus cell expansion was greatest in COCs matured in 75% and 50% FF, and these differences were detectable at 12 (75% FF only) and 19 h (50% and 75% FF) of maturation. The improvement in CC expansion was greatest when FF from small follicles was used. Treatments for the subsequent experiments were selected based upon the results of the first experiment. Oocyte nuclear maturation rates were observed after supplementing the maturation medium with 0 or 75% FF and maturing for 19 h. The rate of nuclear maturation as determined by the presence or absence of the first polar body was similar between control (0% FF) and treated (75% FF) groups. In the final experiment, COCs were matured in 0%, 50% or 75% FF in preparation for IVF. Duration of the maturation period (12, 19 or 22 h) and size of the follicles from which FF was collected (small or large) also varied. In general, FF supplementation at 50% did not affect the zygotes' developmental potential (neither increased nor decreased). Supplementation of maturation medium with 75% FF from small follicles consistently reduced measures of embryo development while 75% FF from large follicles yielded mixed results. It is concluded that FF supplementation improves CC expansion, but the greater CC expansion does not benefit subsequent embryo development. Notably, however, the 50% FF treatment did not reduce blastocyst rates, indicating that FF can be included in maturation media at concentrations of 50% or less with no detriment to IVF outcomes.

Luteinizing hormone receptor deficiency in immature cumulus-oocyte complexes retrieved for assisted reproduction.

This study investigated whether luteinizing hormone receptor (LHR) expression varies in the granulosa cells of individual follicles according to the maturation stage of the oocytes harvested for assisted reproductive technology treatment. We observed minimal to no LHR messenger ribonucleic acid and protein expression in cumulus cells surrounding oocytes arrested in the germinal vesicle stage. Interestingly, their ability to mature was confirmed by rescue invitro maturation, suggesting somatic cell LHR deficiency as a key factor for the retrieval of germinal vesicle oocytes in assisted reproductive technology procedures.

In Vitro Toxicity of a DEHP and Cadmium Mixture on Sheep Cumulus-Oocyte Complexes.

Di-(2-ethylhexyl) phthalate (DEHP) and Cadmium (Cd) affect female reproduction. To date, toxicological research has focused on the effects of individual contaminants, whereas living beings are exposed to mixtures. This study analyzed the effects of a DEHP/Cd mixture on nuclear and cytoplasmic maturation of sheep cumulus-oocyte complexes (COCs) compared with single compounds. COCs recovered from slaughterhouses-derived sheep ovaries were in vitro exposed to 0.5 μM DEHP, 0.1 μM Cd, or DEHP/Cd mixture at the same concentrations during 24 h of in vitro maturation (IVM). After IVM, oocyte nuclear chromatin configuration was evaluated, and bioenergetic/oxidative parameters were assessed on expanded cumulus cells (CCs) and matured oocytes (chi-square test and one-way ANOVA; p < 0.05). Under examined conditions, oocyte nuclear maturation was never impaired. However, COC bioenergetics was affected with stronger effects for the mixture than single compounds. Indeed, the percentages of matured oocytes with healthy mitochondrial distribution patterns were reduced (p < 0.001 and p < 0.05 for mixture and single compounds, respectively). Oocyte mitochondrial membrane potential, intracellular ROS levels, and mitochondria/ROS co-localization were reduced, with the same significance level, in all contaminated conditions. CCs displayed increased ROS levels only upon mixture exposure (p < 0.001). In conclusion, in vitro exposure to the DEHP/Cd mixture affected COC quality in the sheep to a greater extent than separate compounds.

Vitamins, Coenzyme Q10, and Antioxidant Strategies to Improve Oocyte Quality in Women with Gynecological Cancers: A Comprehensive Review.

Gynecological cancers, including ovarian, cervical, and endometrial cancers, significantly affect both survival and reproductive health in women. Cancer treatments such as chemotherapy and radiotherapy can impair ovarian function, reducing oocyte quality and fertility potential. This review aims to evaluate how vitamins and antioxidants can enhance fertility and fertility preservation outcomes for women diagnosed with gynecological cancers, particularly in the context of assisted reproductive technologies (ART). Standard treatments for these cancers, including hysterectomy, bilateral salpingo-oophorectomy, radiation, and chemotherapy, often compromise ovarian function and oocyte quality. This review focuses on the potential role of these interventions in improving oocyte quality, thereby supporting successful fertility preservation and ART outcomes. A comprehensive narrative review of the current literature was conducted, examining the effects of vitamins A, C, D3, E, and Coenzyme Q10 on oocyte quality, particularly in the context of oxidative stress and inflammation induced by cancer and its treatments. The evidence suggests that certain vitamins and antioxidants may mitigate oxidative damage and enhance oocyte quality. Vitamin A supports cumulus-oocyte complex integrity, while vitamins C and E act as potent antioxidants, reducing oxidative stress in ovarian tissues. Vitamin D3 enhances ovarian reserve markers and modulates inflammatory cytokines. Coenzyme Q10 improves mitochondrial function and reduces DNA damage, increasing oocyte viability and fertilization potential. The incorporation of specific vitamins and antioxidants into fertility preservation strategies may enhance oocyte quality in women with gynecological cancers. Although the preliminary findings are promising, further research is needed to determine optimal dosages and establish standardized protocols for clinical use.

Media Supplementation With Gamma-Oryzanol Improves the Outcome of Ovine Oocyte Maturation In Vitro.

The process of maturing ovine oocyte in vitro has not yet been raised with acceptable results. This study was designed to evaluate the γ-oryzanol effect as a supplement of maturation media on the development of ovine oocytes to blastocyst. Aspirated from ovine ovaries, morphologically normal cumulus-oocyte complexes (COCs) were matured in media supplemented with or without 5µM γ-oryzanol. Matured oocytes were divided into two parts: one evaluated for their nuclear maturation, the level of GSH and ROS, mitochondrial membrane potential (MMP) and the pattern of transcription in oocytes and respective cumulus cells (CCs), and another subjected to fertilisation and culture to assess the development of oocytes to the blastocyst. γ-Oryzanol improved the proportion of cleaved embryos and total blastocysts in the treated group, which was linked to improved MMP, higher levels of intracellular GSH and lower levels of ROS. A lower proportion of MI and GVBD was recorded for treated oocytes in comparison with control, although the proportion of MII oocytes was not different between groups. The treated oocytes and CCs showed downregulation of genes related to apoptosis (BAX and CASP-9) and upregulation of genes related to antioxidative status (NRF2, CAT and SOD). In conclusion, our results demonstrated the improved developmental outcome of supplemented oocytes so that the antioxidant response and higher enzymatic activity were maintained, and the generation of ROS was turned off; therefore, a novel alternative for counteracting oxidative stress in ovine oocytes undergoing maturation was offered by γ-oryzanol through an antioxidative pathway.

Interaction of cumulus-oocyte complex (coc) number, oocyte quality, and blastocyst numbers by repeated ovum pick-up (opu) in in vitro embryo production

Interaction of cumulus-oocyte complex (coc) number, oocyte quality, and blastocyst numbers by repeated ovum pick-up (opu) in in vitro embryo production

Influence of thermal stress during in vitro maturation on the developmental competence of oocytes and embryos and the expression of Sirtuins in cumulus oocyte complexes in cattle

Sirtuins are of central importance in many cellular functions and promote cell survival under stress. However, little information is available regarding the relationship between sirtuins and female reproductive biology, especially in response to thermal stress. This study investigated the influence of moderately high (40°C) and low (37°C) thermal stress during in vitro maturation on the development competence of bovine oocytes and embryos. The expression and abundance of sirtuins and other proteins involved in stress response were also studied. The cumulus-oocyte complexes (COCs) of Simmental (Bos taurus) cows underwent in vitro maturation (IVM) at different temperatures (37°C, 38.5°C and 40°C). Before maturation, the oocytes were stained with Brilliant Cresyl Blue (BCB) and categorized as labeled (BCB+) or unlabeled (BCB-). Embryo production was analyzed at the different IVM temperatures. Polar body extrusion was evaluated following IVM, and the mRNA and protein abundance of sirtuins and P53 in oocytes and cumulus cells were analyzed. The differing temperatures during IVM did not significantly alter polar body extrusion and cleavage rates; however, significant differences in blastocyst production were observed. COCs matured at 38.5°C (control, 37.3%) had the highest blastocyst rate, in contrast to those matured at 37°C (33.2%) and 40°C (21.5%). In all groups, the blastocyst rates were higher for BCB+ oocytes than for BCB- oocytes. In BCB+ oocytes, the expression of SIRT1, SIRT2, SIRT3, and SIRT5 genes was higher after maturation than that before maturation and in most of the cases, the expression was higher when IVM was performed at 38.5°C. In the cumulus cells of BCB+ COCs, only SIRT2 remained unaffected by the maturation temperature. In summary, the temperature change of ±1.5°C for 24 h during bovine oocyte maturation impaired in vitro embryo development. This lead to several cellular biochemical alterations in oocytes and granulosa cells from COCs with higher developmental competence (BCB+). Thus, SIRT1 is important for in vitro embryonic development and may protect against cold and heat stress.

11 Does pregnancy status affect cumulus–oocyte complex recovery and embryo production in cycling Girolando heifers?

11 Does pregnancy status affect cumulus–oocyte complex recovery and embryo production in cycling Girolando heifers?