Early Developmental Competence Research Articles

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 μM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Ochratoxin A triggers endoplasmic reticulum stress through PERK/NRF2 signaling and DNA damage during early embryonic developmental competence in pigs

Ochratoxin A (OTA), a mycotoxin found in foods, has a deleterious effect on female reproduction owing to its endocrine-disrupting activity mediated through endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the mechanisms of OTA-induced ER stress in pig embryos during in vitro culture (IVC) are not yet fully understood. In the present study, porcine embryos were cultured for two days in an IVC medium supplemented with 0.5, 1.0, and 5.0 μM OTA, which led to an OTA-induced reduction in the developmental rate of blastocysts. The mRNA-seq transcriptome analysis revealed that the reduced blastocyst development ability of OTA-exposed porcine embryos was caused by ER stress, ultimately resulting in the accumulation of ROS and the occurrence of apoptosis. The expression levels of some UPR/PERK signaling-related genes (DDIT3, EIF2AK3, EIF2S1, NFE2L2, ATF4, EIF2A, and KEAP1) were found to differ in OTA-exposed pig embryos. OTA induces DNA damage by triggering an increase in RAD51/γ-H2AX levels and suppressing p-NRF2 activity. This effect is mediated through intracellular ROS and superoxide accumulation in the nuclei of porcine embryos. The cytotoxicity of OTA increased the activation of the PERK signal pathways (p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP) in porcine embryos, with abnormal distribution of the ER observed around the nucleus. Collectively, our findings indicate that ER stress is a major cause of decline in the development of porcine embryos exposed to OTA. Therefore, OTA exposure induces ER stress and DNA damage via oxidative stress by disrupting PERK/NRF2 signaling activity in the developmental competence of porcine embryos during IVC.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Inhibition of FSP1 impairs early embryo developmental competence in pigs

Ferroptosis suppressor protein 1 (FSP1) is a glutathione-independent ferroptosis inhibitory factor. FSP1 has been found to play a crucial role in the regulation of mitochondrial function and ferroptosis. However, its function in porcine early embryonic development remains unknown. In the present research, we found that FSP1 was expressed at different stages during porcine early embryo development. Compared with the control condition, inhibition of FSP1 reduced the cleavage rate at 24 h and 48 h and the blastocyst rate at 144 h. In addition, inhibiting FSP1 reduced the blastocyst diameter, total cell number, and proliferation capacity. Further analysis showed that inhibition of FSP1 significantly increased the levels of ferrous ions (Fe2+) and MDA but not GPX4. We also found that inhibition of FSP1 significantly decreased mitochondrial membrane potential and ATP levels, which in turn caused excessive accumulation of ROS and decreased the levels of GSH and the activity of the intracellular antioxidant enzymes SOD and CAT in embryos. In conclusion, FSP1, an important regulator, participates in regulating the development and quality of porcine early embryos. Inhibition of FSP1 impairs blastocyst formation, induces glutathione-independent ferroptosis, and further leads to oxidative stress due to mitochondrial dysfunction, ultimately affecting the developmental competence and impairing the quality of porcine early embryos.

Developmental competence and neonatal outcomes of nonpronuclear zygotes following single vitrified-warmed blastocyst transfers using propensity score matching analysis.

To investigate developmental competence and neonatal outcomes of nonpronuclear (0PN) zygotes following single vitrified-warmed blastocyst transfers (VBT). The clinical, laboratorial and neonatal data of 996 patients with ≤ 38years who underwent blastocyst culture and single VBT were retrospectively analyzed. The pregnancy and neonatal outcomes of VBT were compared between 0PN and 2PN blastocysts using propensity score matching (PSM). Moreover, Day 3 (D3) embryo development and blastocyst formation were compared between 0PN and 2PN zygotes. There were no significant differences in clinical pregnancy rate (CPR), live birth rate (LBR) and neonatal outcomes of VBT between the 0PN and 2PN blastocysts irrespectively of whether PSM was used. However, early abortion rate (EAR) was higher in blastocysts from 0PN D3 embryos > 10 cells (p < 0.05) before PSM. Moreover, the early developmental competence of 0PN zygotes was different from that of 2PN zygotes presenting higher percentages of D3 embryos ≤ 6 cells (p < 0.01) and > 10 cells (p < 0.01), lower available blastocyst formation rate (ABFR) (p < 0.01) and good-quality blastocyst formation rate (GBFR) (p < 0.01) in D3 embryos with 4-6 cells. ABFR and GBFR increased with cell number when compared among embryos with 4-6 cells, 7-10 cells and > 10 cells, irrespectively of 0PN or 2PN embryos. The early developmental competence of 0PN zygotes was different from that of 2PN zygotes, but did not influence pregnancy and neonatal outcomes following VBT. ABFR and GBFR increased with cell number, irrespectively of 0PN or 2PN embryos.

Rapamycin encourages the maintenance of mitochondrial dynamic balance and mitophagy activity for improving developmental competence of blastocysts in porcine embryos in vitro.

Rapamycin induces autophagosome formation and activity during oocyte maturation, improved fertilization ability of matured oocytes, and early embryonic developmental competence. However, potential changes in mitochondrial fission and mitophagy via regulation of autophagy in early porcine embryonic development have not been previously studied. Here, we investigated embryonic developmental ability and quality of porcine embryos 2 days after in vitro fertilization and following treatment with 1 and 10 nM rapamycin. As a results, 1 nM rapamycin exposure significantly improved (p < 0.05) blastocyst developmental competence compared to that in nontreated embryos (nontreated: 26.2 ± 5.7% vs. 1 nM rapamycin: 35.3 ± 5.1%). We observed autophagic (LC3B) and mitochondrial fission protein expression (dynamin-related protein-1 [DRP1] and pDRP1-Ser616) at the cleavage stage of 1 and 10 nM rapamycin-treated porcine embryos, using Western blot and immunofluorescence analyses. Interestingly, 1 nM rapamycin treatment significantly improved autophagy formation, mitochondrial activation, and mitochondrial fission protein levels (p < 0.05; p-DRP1 [Ser616]) at the cleavage stage of porcine embryos. Additionally, mitophagy was significantly increased in blastocysts treated with 1 nM rapamycin. In conclusion, our results suggest that rapamycin promotes blastocyst development ability in porcine embryos through mitochondrial fission, activation, and mitophagy in in vitro culture.

P-227 Exposure to triclosan modifies oocyte development and embryo metabolism

Abstract Study question Does the endocrine disruptor, triclosan, affect the function of cumulus-oocyte-complexes (COCs), and if so, do these effects persist in early embryo developmental competence and metabolism? Summary answer Triclosan caused COCs to i) increase progesterone secretion, ii) switch from releasing to consuming pyruvate, and iii) produce fewer blastocysts that were iv) metabolically compromised. What is known already Triclosan (TCS) is used widely as an antimicrobial agent in a range of consumer healthcare products, including hand sanitiser, toothpastes, soaps, and shampoos. Its prevalent use has led to accumulation in the environment and human exposure. Indeed, TCS has been identified in a range of bodily fluids. It is classed as an endocrine-disrupting chemical and there are reports of its detrimental impact on the vertebrate reproductive system. However, the direct effects of TCS on mammalian Cumulus-Oocyte-Complexes’ (COCs) function and subsequent development of the embryo remains unknown. Study design, size, duration COCs, isolated from abattoir-derived bovine ovaries on 5 independent occasions, were exposed of two environmentally-relevant doses of TCS (10nM and 1nM) during in vitro maturation. The TCS-exposed COCs were subsequently fertilised in vitro without further TCS addition, and the cleavage and blastocyst rate were recorded on Day 2 and Day 7/Day 8, respectively. The resulting blastocysts were collected and further cultured individually for 24 hours, and depletion/release of key-metabolites quantified. Participants/materials, setting, methods In vitro production was performed for the maturation of the bovine oocytes and the embryo culture. The production of steroid hormones (17b-estradiol and progesterone) during IVM was determined by ELISA. The concentrations of glucose, pyruvate, lactate during IVM and blastocyst formation were quantified in the spent media using microfluorometric assays. Main results and the role of chance TCS at 1nM had a significant impact on steroidogenesis during COCs in vitro maturation; progesterone production was significantly higher compared to control group (4.784 ±1.69 vs 2.85 ±0.83 pg/hour/COCs, p = 0.035; n = 5), although oestrogen release was not affected. In addition, there was a dramatic shift in pyruvate metabolism. Over the course of in vitro maturation, COCs exposed to TCS depleted pyruvate from the culture media regardless of the administrated dose whilst net pyruvate concentration increased in the medium of COCs in the control groups. The cleavage rate of bovine embryos produced from TCS-exposed COCs was not significantly different to control group. However, COCs-exposed to 1nM TCS produced significantly fewer blastocysts (12.22% vs 29.11% n = 5; p = 0.02). Those blastocysts that did originate from COCs treated with TCS depleted significantly more pyruvate from the culture media compared to controls, regardless of TCS dose (19.86 ±10.54 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.011 and 21.41 ±10.08 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.002, TCS-10nM and TCS-1nM compared to Control, respectively). Additionally, while glucose depletion did not differ, the lactate release from blastocysts derived from COCs exposed to 10nM TCS was approximately halved compared to controls (15.20 ±14.59 vs 31.83 ±22.73 pmol/hour/embryo p = 0.0054). Limitations, reasons for caution The origin of the ovaries may be a limitation since they came from animals at different stages of estrous cycle. Furthermore, pre-exposure to TCS prior to animal slaughter cannot be discounted. Finally, these observations were made in an animal model in a laboratory setting which may differ from natural exposure. Wider implications of the findings TCS modifies oocyte maturation, developmental competence, and metabolic activity of resulting embryos. The differences in pyruvate metabolism and steroidogenesis point to altered mitochondrial function. These data indicate that TCS directly modulates oocyte and embryo physiology which may relate to fertility in domestic livestock species and may be extrapolated to humans. Trial registration number not applicable

Cytoplasmic streaming induced by intracytoplasmic spindle translocation contributes to developmental competence through mitochondrial distribution in mouse oocytes

To evaluate the developmental competency of mouse metaphase II oocytes and the pattern of mitochondrial positioning through cytoplasmic streaming in mouse metaphase II oocytes. We observed cytoplasmic streaming as movement indicated by fluorescently stained mitochondria using a newly developed method in which the spindle is translocated to the opposite site of the oocyte. This method is termed as intracytoplasmic spindle translocation (ICST). University research laboratory. Female B6D2F1 mice. None. Fresh oocytes, postovulatory-aged oocytes, and oocytes treated with cytochalasin B were classified based on the presence of cytoplasmic streaming induced by ICST. The pattern of redistributed mitochondria and developmental competence caused by parthenogenetic activation were evaluated in oocytes with or without cytoplasmic streaming. Induced cytoplasmic streaming occurred in 84% of the fresh oocytes but not in the postovulatory-aged oocytes and the oocytes treated with cytochalasin B. Abnormal mitochondrial aggregation was observed in oocytes in which cytoplasmic streaming was not induced. Furthermore, the developmental competence was significantly lower in oocytes without cytoplasmic streaming. Cytoplasmic streaming induced by ICST contributes to developmental competence through the redistribution of mitochondria and may be a valuable criterion for predicting early developmental competence in mouse oocytes.

Aflatoxin B1 impairs in vitro early developmental competence of ovine oocytes

Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 μM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 μM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 μM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 μM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 μM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

Supplementation with Niacin during in vitro maturation improves the quality of porcine embryos

Niacin, also known as vitamin B3, has a pivotal role in energy metabolism, cellular signaling cascades regulating gene expression, and apoptosis. However, the effect of Niacin on porcine early embryo developmental competence remains to be elucidated. The present study aimed to assess the effects of Niacin treatment during in vitro maturation (IVM) on the nuclear maturation of porcine oocytes and subsequent development of in vitro embryos. In addition, the expression profiles of selected genes related to lipid metabolism, oxidative stress, and apoptosis were assessed. The IVM medium was supplemented with different concentrations of Niacin (0, 300, 600, and 900 μM). The results showed that a high concentration of Niacin (900 μM) significantly decreased cumulus expansion compared to the other groups (p < 0.05). No significant difference was observed among the experimental groups for nuclear maturation rate. Niacin treatments (300, 600, and 900 μM) during IVM significantly (p < 0.05) enhanced glutathione levels. Treatment with 300 and 600 μM significantly (p < 0.05) lowered the reactive oxygen species levels compared to treatment with 900 μM and the control group. Niacin supplementation to the IVM media significantly improved the cleavage and blastocyst rates compared to the control group. Supplementation with 300 and 600 μM of Niacin significantly increased the total cell number of blastocysts compared to supplementation with 900 μM or the control groups. Cytoplasmic lipid droplets were significantly reduced after 600 μM treatment. Supplementation of Niacin to IVM media positively affected the relative expression of genes related to energy and oxidative status (SIRT1), pro-apoptosis (BAX), anti-apoptosis (BCL2), and lipid metabolism (ACACA and PNPLA2) in cumulus cells and oocytes. Taken together, Niacin supplementation to porcine IVM media improved the developmental competence of early embryos mainly through protection against oxidative stress and its influence on energy metabolism and apoptosis pathways.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Improvement of early developmental competence of postovulatory-aged oocytes using metaphase II spindle injection in mice.

PurposeAssisted reproductive technology (ART) is a widely applied fertility treatment. However, the developmental competence of aged oocytes from women of a late reproductive age is seriously reduced and the aged oocytes often fail in fertilization even when ART is used. To resolve this problem, we examined usefulness of a new method “the metaphase II spindle transfer (MESI)” as ART using mouse oocytes.MethodsThis work was composed of two experiments. First, 24 hours after collection, embryos from oocytes (1‐day‐old oocytes, called postovulatory‐aged oocytes), were observed, after intracytoplasmic sperm injection (ICSI), and it was found that they were not able to reach the blastocyst stage. Next, the metaphase II chromosome‐spindle complexes from 1‐day‐old oocytes were injected into cytoplasts from oocytes just collected, using piezo pulses to generate reconstructed oocytes. This procedure was named metaphase II spindle injection (MESI).ResultsAfter ICSI, embryos from the reconstructed oocytes (32/105), which contained the genes of 1‐day‐old oocytes, were able to develop into the blastocyst stage. The fragmentation rate after ICSI was 28.6%. Thus, the developmental competence of 1‐day‐old oocytes was improved by MESI.ConclusionsThe MESI method has the potential to improve the success rate of infertility treatments for women of a late reproductive age.

Bisphenol S Impaired In Vitro Ovine Early Developmental Oocyte Competence

Introduction: Bisphenol A (BPA) is a widespread compound in the plastic industry that is especially used to produce baby bottles, food packaging and metal cans. BPA, an endocrine disruptor, leads to alterations in reproductive function and therefore has been banned from the food industry. Unregulated BPA analogues, particularly Bisphenol S (BPS), have emerged and are now used in the plastic industry. Thus, this study aimed to examine the acute effects of low and environmental doses of BPS on ewe oocyte quality and developmental competence, and its mechanism of action, during in vitro maturation. Methods: Ewe cumulus-oocyte complexes underwent in vitro maturation in the presence or absence of BPS (1 nM, 10 nM, 100 nM, 1 µM or 10 µM). Oocytes were then subjected to in vitro fertilisation and development. Results: 1 µM BPS induced a 12.7% decrease in the cleavage rate (p = 0.004) and a 42.6% decrease in the blastocyst rate (p = 0.017) compared to control. The blastocyst rate reduction was also observed with 10 nM BPS. Furthermore, 10 µM BPS reduced the oocyte maturation rate, and 1 µM BPS decreased cumulus cell progesterone secretion. PR and AMH gene expression were reduced in cumulus cells. BPS induced a 5-fold increase in MAPK 3/1 activation (p = 0.04). Conclusions: BPS impaired ewe oocyte developmental competence. The data suggest that BPS might not be a safe BPA analogue. Further studies are required to elucidate its detailed mechanism of action.

193 Effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle

Unhygienic practices at the time of parturition or AI lead to uterine infections. The uterine infections ultimately result in genetic drain by culling the elite animals. The invivo developmental competence of embryos is compromised in clinically metritic animals. The genetic potential of problematic females could be harvested by invitro embryo production (IVEP). Therefore, the objective of the present study was to evaluate the effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle. This experiment was carried out from December 2017 to April 2018. Ovaries were collected from a local abattoir (Bos indicus; 5- to 8-year-old dairy cattle, body condition score 2.75±0.25, mixed parity). These ovaries (n=982) were divided into two groups: (1) clinically metritic (n=184), and (2) healthy (n=798), based upon the presence or absence of pus in the uterine lumen. Oocytes were aspirated from follicles using an 18G needle attached to a 10-mL syringe. Cumulus-oocyte complexes (COCs) were categorized into A, B, C, and D grades based on the number of layers of cumulus cells and integrity of ooplasm. The oocytes of grades A and B were subsequently transferred in groups (10/group) in four-well plates containing 100-μL droplets. The droplets with oocytes were covered with prewarmed mineral oil and incubated for 24h at 38.5°C, 5% CO2, and 95% relative humidity. The oocytes were evaluated for IVM on the basis of cumulus expansion. Frozen semen was thawed and prepared using the sperm swim-up procedure for each group. Spermatozoa and oocytes were incubated together for a period of 18h. The presumptive zygotes were invitro cultured for 4 days in a CO2 incubator under similar culture conditions. The cleavage rate, 4-cell, and 8-cell stages were recorded on Days 2, 3, and 4 after the day of insemination, respectively. Data on oocyte recovery, oocyte quality, IVM, cleavage rate, and 4-cell and 8-cell stages were analysed by Chi-squared test using SPSS software (version 20; IBM Corp.) for Windows. Results demonstrated that recovery rate was lower (63.8% vs. 71.7%; P&amp;lt;0.05) in clinically metritic compared with healthy cattle. Similarly, oocytes of grade A and B quality were lower (41.0% vs. 51.1%; P&amp;lt;0.05), whereas those of C and D quality were higher (59.0% vs. 48.9%; P&amp;lt;0.05) in clinically metritic compared with the healthy group. Moreover, 4-cell (38.2% vs. 54.8%) and 8-cell stage embryos (11.3% vs. 29.1%), were lower (P&amp;lt;0.05) in the clinically metritic compared with the healthy group, respectively. However, maturation rate and cleavage rate did not differ (P&amp;gt;0.05) between groups. In conclusion, metritis in slaughterhouse ovaries negatively affects oocyte recovery rate, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle.

179 Bisphenol S affects invitro early developmental oocyte competence in ewes

In the plastics industry, bisphenol S (BPS) replaces sisphenol A reported to be an oestrogen mimetic endocrine disruptor damaging oocyte meiosis and maturation (Machtinger 2014 Reprod. Biomed. Online 29, 404). Studies on fish and rodents reported that BPS affects reproduction similarly to BPA (Uzumcu 2007 Reprod. Toxicol. 23, 337; Giulivo 2016 Environ. Res. 151, 251; Ullah 2016 Chemosphere 152, 353). Bisphenol S is detected in human urine at nanomolar concentrations (Liao 2012 Environ. Sci. Technol 46, 6860) and in some laboratory supplies (tips and tubes; unpublished data). Therefore, in this study, we assessed effects of BPS at low doses during invitro maturation (IVM) on oocyte developmental competence in ewes. Cumulus-oocyte complexes (COC) collected from ovine follicles &amp;gt;2mm underwent 24-h IVM, in the absence or presence of BPS at 1, 10, and 100 nM and 1 and 10 µM (Sigma Chemical Co.). Nuclear oocyte maturation rate was evaluated by MII oocyte count after chromatin Hoechst staining [n=3 replicates (R), 1159 oocytes]. At 6h of IVM, BPS effects on mRNA expression of oestrogen (E2) and progesterone (P4) receptors in cumulus cells (CC) were assessed by real-time quantitative PCR. After 24h of IVM, The effect of BPS on P4 level was assessed in spent medium by enzyme-linked immunosorbent assay (n=6 R, 40 COC/condition). Transcript expression level and P4 concentration were analysed using nonparametric one-way ANOVA, with Tukey post hoc test (Rcmdr, R version 3.5.3). After 24h of IVM, matured COC underwent IVF and invitro culture (IVC) for 7 days. Cleavage and blastocyst rates were assessed on Days 2 and 7, respectively, after IVF (8 experiments, 300 COC/condition). Data were analysed using logistic regression and linear model (R version 3.5.3). Our results showed a decreased oocyte maturation rate with 10 µM BPS (76.6%, n=171; P=0.0008) compared with control (88%, n=152), with no effect on cell viability. The concentration of P4 decreased with 1 µM BPS (0.02ngmL−1 per COC) compared with control (0.034ngmL−1 per COC; P&amp;lt;0.001). At 6h IVM, BPS had no significant effect on oestrogen receptors (ESR1, ESR2, GPER) transcripts in CC but 10 nM BPS decreased mRNA expression of P4 receptor (PR) (0.00647±0.00145; P=0,005) compared with control (0.01165±0.00196). Within fertilized COC, 1 µM BPS decreased cleavage rate (47.6%, n=152) compared with control (54.6%; P=0.004). Among cleaved embryos, blastocyst rate decreased to 14.2% and 12.5% with 10 nM and 1 µM BPS respectively (n=26, P=0.046; and n=19, P=0.017), compared with control (21.8%, n=44). Bisphenol S at a low dose during ovine IVM reduced COC P4 secretion, PR transcript in CC, and cleavage and blastocyst rates. Our data suggest that BPS at an environmental dose (10 nM) negatively affects early developmental oocyte competence. Studies are ongoing to investigate the effect of BPS on Day 6 embryo cell number and on the ERK1/2 signalling pathway in oocyte. This study was supported by INRA, Region Val-de-Loire (BEMOL) and the French National Research Agency (ANR-18-CE34-0011-01 MAMBO). We thank A. Arnould and T. Delpuech for ovine ovary collection and Endocrinology and Phenotyping laboratory for P4 assay.

Influence of N-acetyl-L-cysteine against bisphenol a on the maturation of mouse oocytes and embryo development: in vitro study

BackgroundBisphenol A (BPA), an endocrine disruptor, is a widely used chemical that has adverse effects on animal development and reproduction. The current research aimed to evaluate the effect of BPA on the in vitro maturation (IVM) and subsequent embryo development of mouse oocytes following in vitro fertilization (IVF).MethodsIVM was performed in the presence of different concentrations (0, 20, 50, or 100 μg/mL) of BPA. Nuclear maturation, IVF efficiency and embryonic development were determined. The levels of reactive oxygen species (ROS) and glutathione (GSH) in the BPA (50 μg/mL) group were evaluated. We explored the ability of N-acetyl-L-cysteine (NAC) in the IVM medium to rescue the BPA-induced damage by examining changes in nuclear maturation, IVF rate, blastocyst formation, ROS levels and GSH content.ResultsCompared with the control, BPA (50 μg/mL) supplementation during oocyte IVM significantly inhibited nuclear maturation and decreased fertilization and blastocyst formation rates. In addition, BPA exposure increased ROS levels and decreased GSH content in oocytes. The addition of NAC weakened the BPA-induced suppression of nuclear maturation, relieved the BPA-induced downregulation of the fertilization and blastocyst formation rates, and mitigated the increased ROS levels and decreased GSH content.ConclusionBPA affects mouse oocyte maturation and subsequent early embryonic developmental competence following IVF by increasing intracytoplasmic oxidative stress in mature oocytes. NAC can reduce these harmful effects to a certain extent.

RNA-Binding Protein IGF2BP2/IMP2 is a Critical Maternal Activator in Early Zygotic Genome Activation.

A number of genes involved in zygotic genome activation (ZGA) have been identified, but the RNA‐binding maternal factors that are directly related to ZGA in mice remain unclear. The present study shows that maternal deletion of Igf 2bp2 (also commonly known as Imp2) in mouse embryos causes early embryonic developmental arrest in vitro at the 2‐cell‐stage. Transcriptomics and proteomics analyses of 2‐cell‐stage embryos in mice reveal that deletion of IMP2 downregulates the expression of Ccar1 and Rps14, both of which are required for early embryonic developmental competence. IGF2, a target of IMP2, when added in culture media, increases the proportion of wild‐type embryos that develop successfully to the blastocyst stage: from 29% in untreated controls to 65% (50 × 10−9 m IGF2). Furthermore, in an experiment related to embryo transfer, foster mothers receiving IGF2‐treated embryos deliver more pups per female than females who receive untreated control embryos. In clinically derived human oocytes, the addition of IGF2 to the culture media significantly enhances the proportion of embryos that develop successfully. Collectively, the findings demonstrate that IMP2 is essential for the regulation and activation of genes known to be involved in ZGA and reveal the potential embryonic development‐related utility of IGF2 for animal biotechnology and for assisted reproduction in humans.

Lipid content, mitochondrial activity and early embryo development in oocyte collected from crossbred cows (bos taurus indicus)

The objective of this research was to evaluate the effect of phenotypic predominance on lipid content, mitochondrial activity and early developmental competence as indicators of oocyte quality. Cumulus-oocyte complexes (COCs) were recovered through follicular aspiration, and underwent in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of presumptive zygotes. Lipid content and mitochondrial activity in immature and IVM oocytes were determined. A maturation rate of 80.6% and 69.3% was found for oocytes predominantly B. indicus and predominantly B. taurus, respectively. Total fertilization rate was 27.6%; 26.1% for predominantly B. indicus oocytes and 29% for predominantly B. taurus oocytes. A total of 55.5% and 57.5% of cleaved embryos after 48 and 72 h post-insemination (hpi) in predominantly B. indicus group were observed, respectively. As for the predominantly B. taurus group, 48.6% and 60.4% of cleaved embryos were found after 48 and 72 hpi, respectively. In both groups, immature oocytes showed a greater amount of small lipidic droplets (p &lt;0.0001); IVM decreased the number of small lipid droplets (p &lt; 0.0001) and increased the number of medium and large lipid droplets (p &lt; 0.0001). Predominantly B. indicus oocytes had a greater number of small and medium-sized lipid droplets, while there were no significant differences in large lipid droplets. IVM oocytes had higher mitochondrial activity than immature oocytes group (p &lt; 0.05) without any effect of phenotypic predominance on this parameter. Assessment of lipid content was not a predictive factor of oocyte quality in crossbred cows.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.