Transcriptional profiling of embryo cryotolerance.

The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development.

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows.

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle-stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre-ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct-specific glycoprotein 1, heat-shock protein family A member 5, α-l-fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.

Use of pregnancy-associated plasma protein-A during oocyte in vitro maturation increases IGF-1 and affects the transcriptional profile of cumulus cells and embryos from Nelore cows.

Insulin-like growth factor 1 (IGF-1) activity is established by the regulation of IGF binding protein activity, which blocks IGF-1 functions, whereas pregnancy-associated plasma protein-A (PAPP-A) improves IGF-1 bioavailability and facilitates binding to IGF receptors. To further extend our understanding of the effect of exogenous PAPP-A on bovine embryo production, we added this protein during in vitro maturation of cumulus-oocyte complexes (COCs); moreover, we assessed its effects on IGF-1 quantity in the maturation medium, embryonic yield and postwarming survival, blastocyst quality, and transcript abundance. Bovine COCs were matured in a serum-free medium, either with PAPP-A supplementation (100 ng/ml) or without (control). The treatment group produced higher IGF-1 concentrations in the maturation medium; however, showed no difference on cleavage, blastocysts rates, and embryonic survival 3 and 24 hr postcryopreservation. Regarding gene expression, VNN1 was upregulated, whereas AGPAT9, FASN, EGFR, HAS2, and IMPDH1 were downregulated in PAPP-A treated. PAPP-A treated, CPT2, DNMT3A, and TFAM were upregulated, whereas ATF4 and IFITM3 were downregulated. We concluded that although the addition of PAPP-A did not affect embryo yield and blastocyst survival, higher IGF-1 levels may affect embryo competence through differential expression of genes involved in lipid metabolism, oocyte competence, and mitochondrial function.

Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

PURPOSE: Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. METHODS: We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2 h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. RESULTS: In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. CONCLUSIONS: Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

Treatment with cyclic adenosine monophosphate modulators prior to in vitro maturation alters the lipid composition and transcript profile of bovine cumulus-oocyte complexes and blastocysts.

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus-oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2h and 16 transcripts after 24h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

Antioxidant responses and deregulation of epigenetic writers and erasers link oxidative stress and DNA methylation in bovine blastocysts.

Early mammalian embryos derived from in vitro fertilization are exposed to conditions distinct from the native oviduct-uterine environment, including atmospheric oxygen that promotes cellular oxidative stress and alters gene expression. High oxygen partial pressure during embryo development is associated with low pregnancy rates and increased embryonic apoptosis. We investigated how bovine embryos responded to high (20%) or low (5%) oxygen partial pressure during in vitro culture, evaluating levels of reactive oxygen species (ROS) as well as changes in the expression of oxidative stress- and epigenetic-related transcripts and miRNAs in blastocysts. Additionally, we determined the global DNA methylation levels in the resulting embryos. Our data indicated that bovine blastocysts produced in vitro under high oxygen partial pressure possessed elevated ROS abundance and exhibited increased expression of CAT, GLRX2, KEAP1, NFR2, PRDX1, PRDX3, SOD1, TXN, and TXNRD1, versus reduced levels of the oxidative stress-related bta-miR-210. These stressed embryos also presented altered expression of the epigenetic-associated transcripts DNMT3A, H2AFZ, H3F3B, HDAC2, MORF4L2, REST, and PAF1. In addition, we demonstrated that embryos cultured under high oxygen partial pressure have increased global DNA methylation, suggesting that DNA hypermethylation is mediated by the deregulation of epigenetic-related enzymes due to oxidative stress.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

Autophagy is a pro-survival adaptive response to heat shock in bovine cumulus-oocyte complexes.

Autophagy is a physiological mechanism that can be activated under stress conditions. However, the role of autophagy during oocyte maturation has been poorly investigated. Therefore, this study characterized the role of autophagy on developmental competence and gene expression of bovine oocytes exposed to heat shock (HS). Cumulus-oocyte-complexes (COCs) were matured at Control (38.5 °C) and HS (41 °C) temperatures in the presence of 0 and 10 mM 3-methyladenine (3MA; autophagy inhibitor). Western blotting analysis revealed that HS increased autophagy marker LC3-II/LC3-I ratio in oocytes. However, there was no effect of temperature for oocytes matured with 3MA. On cumulus cells, 3MA reduced LC3-II/LC3-I ratio regardless of temperature. Inhibition of autophagy during IVM of heat-shocked oocytes (3MA-41 °C) reduced cleavage and blastocyst rates compared to standard in vitro matured heat-shocked oocytes (IVM-41 °C). Therefore, the magnitude of HS detrimental effects was greater in the presence of autophagy inhibitor. Oocyte maturation under 3MA-41 °C reduced mRNA abundance for genes related to energy metabolism (MTIF3), heat shock response (HSF1), and oocyte maturation (HAS2 and GREM1). In conclusion, autophagy is a stress response induced on heat shocked oocytes. Inhibition of autophagy modulated key functional processes rendering the oocyte more susceptible to the deleterious effects of heat shock.

Can extracellular vesicles from bovine ovarian follicular fluid modulate the in-vitro oocyte meiosis progression similarly to the CNP-NPR2 system?

C-type natriuretic peptide (CNP) and its natriuretic peptide receptors subtype 2 (NPR2) are essential for the maintenance of oocyte meiotic arrest in different species. Extracellular vesicles (EVs) in bovine follicular fluid (FF) are important for cell communication within the ovarian follicle. This study investigated the involvement of EVs from FF of bovine ovarian follicles in the CNP-NPR2 system, first by analyzing the presence of CNP in the EV contents, followed by addition of EVs to in-vitro maturation (IVM) medium, to evaluate the effect on maintenance of oocyte meiosis arrest and improvements in in-vitro embryo production. As expected, CNP was observed in FF and granulosa cells from the ovarian follicles. To the best of our knowledge, this is the first time that CNP has been found in the EV contents. To evaluate the possible effect of EVs on the progression of oocyte meiosis, the IVM was performed under three conditions: CNP and EV supplementation and control condition. Both the CNP and EV treatments inhibited meiosis resumption in the oocyte within 9 h of IVM. CNP treatment increased cGMP levels in cumulus cells within 6 h of IVM compared to the control group, but the EV treatment did not. In contrast, the relative mRNA abundance of adenylate cyclase 3 and 9 (ADCY3 and ADCY9) was upregulated in oocytes after 6 h of IVM under EV treatment compared to the control group, but not under CNP treatment. Last, these treatments in the IVM medium had no significant effect on the in-vitro embryo production. In conclusion, we demonstrated the presence of endogenous CNP in bovine reproductive structures, especially in the EVs from the FF of antral follicles. The presence of CNP in the EVs suggests an important involvement of this cell-communication system in the CNP-NPR2 system. Therefore, we indeed observed that the EVs from FF can modulate the arrest of oocyte meiosis, acting similarly to the CNP-NPR2 system to block the oocyte in the GV state. However, the mechanism of each system might be different; the CNP-NPR2 system seems to be involved in modulating the cGMP levels, while the contents of EVs might be involved in modulating the cAMP levels.