Oviductal extracellular vesicles miRNA cargo varies in response to embryos and their quality.

BACKGROUND: Increasing evidence points to an active role of oviductal extracellular vesicles (oEVs) in the early embryo-maternal dialogue. However, it remains unclear whether oEVs contribute to the recognition of the presence of embryos and their quality in the oviduct. Hence, we examined whether the molecular cargo of oEVs secreted by bovine oviduct epithelial cells (BOEC) differs depending on the presence of good (≥ 8 cells, G) or poor (< 8 cells, P) quality embryos. In addition, differences in RNA profiles between G and P embryos were analyzed in attempt to distinguish oEVs and embryonic EVs cargos. METHODS: For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53 h post fertilization as follows: BOEC with G embryos (BGE); BOEC with P embryos (BPE); G embryos alone (GE); P embryos alone (PE); BOEC alone (B) and medium control (M). After 24 h of co-culture, conditioned media were collected from all groups and EVs were isolated and characterized. MicroRNA profiling of EVs and embryos was performed by small RNA-sequencing. RESULTS: In EVs, 84 miRNAs were identified, with 8 differentially abundant (DA) miRNAs for BGE vs. B and 4 for BPE vs. B (P-value < 0.01). In embryos, 187 miRNAs were identified, with 12 DA miRNAs for BGE vs. BPE, 3 for G vs. P, 8 for BGE vs. GE, and 11 for BPE vs. PE (P-value < 0.01). CONCLUSIONS: These results indicated that oEVs are involved in the oviductal-embryo recognition and pointed to specific miRNAs with signaling and supporting roles during early embryo development.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway.

BACKGROUND: Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-ß pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-ß pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-ß signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Characterization and profiling analysis of bovine oviduct and uterine extracellular vesicles and their miRNA cargo through the estrous cycle.

Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 1010  EVs/ml and 6.0 × 1010  EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development.

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro†.

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2- to 8-cell stage, MNEGA) or major (8- to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 µM AKT-InhIII; 10 µM AKT-InhIV; 10 µM nobiletin; nobiletin + AKT-InhIII; nobiletin + AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors, which infers that nobiletin probably uses another signaling cascade that PI3K/AKT during early embryo development in bovine.

Role of reproductive fluids and extracellular vesicles in embryo–maternal interaction during early pregnancy in cattle.

The coordinated interaction between the developing embryo and the maternal reproductive tract is essential for the establishment and maintenance of pregnancy in mammals. An early cross-talk is established between the oviduct/uterus and the gametes and embryo. This dialogue will shape the microenvironment in which gamete transport, fertilisation, and early embryonic development occur. Due to the small size of the gametes and the early embryo relative to the volume of the oviductal and uterine lumina, collection of tissue and fluid adjacent to these cells is challenging in cattle. Thus, the combination of in vivo and in vitro models seems to be the most appropriate approach to better understand this fine dialogue. In this respect, the aim of this review is to summarise the recent findings in relation to gamete/embryo-maternal interaction during the pre-elongation period.

Culture Medium and Sex Drive Epigenetic Reprogramming in Preimplantation Bovine Embryos.

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25-25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.

Catatonia Associated with Hyponatremia: Case Report and Brief Review of the Literature.

BACKGROUND: Catatonia is a syndrome of altered motor behavior that is mostly associated with general medical, neurologic, mood and schizophrenia-spectrum disorders. The association of newly onset catatonic symptoms with hyponatremia has been rarely reported in the literature. CASE PRESENTATION: We present a rare case of a young female patient with schizophrenia, who presented with catatonic symptoms in the context of hyponatremia due to water intoxication. The symptoms were eliminated with the correction of hyponatremia. There are only a few reports of hyponatremia-associated catatonia in psychiatric and non-psychiatric patients. Sometimes, catatonic symptoms may co-occur with newly onset psychotic symptoms and confusion, suggesting delirium. In several cases, the catatonic symptoms responded to specific treatment with benzodiazepines or electroconvulsive therapy. CONCLUSION: Hyponatremia may induce catatonic symptoms in patients, regardless of underlying mental illness, but this phenomenon is even more relevant in patients with a psychotic or mood disorder, which may itself cause catatonic symptoms. It is important for clinicians not to attribute newly-onset catatonic symptoms to the underlying psychotic or mood disorder without measuring sodium serum levels. The measurement of sodium serum levels may guide treating psychiatrists to refer the patient for further investigation and appropriate treatment.

Asynchrony between the early embryo and the reproductive tract affects subsequent embryo development in cattle.

The aim of this study was to determine the effect of maternal-embryonic asynchrony in the reproductive tract (oviduct and uterus) on subsequent embryo development in cattle. Fifty Day 1invitro-produced zygotes were transferred endoscopically into the oviduct ipsilateral to the corpus luteum of heifers (n=40) that were either synchronous with the embryos (Day 1 after ovulation) or asynchronous and ahead of the embryo (Day 3 after ovulation). A subset of heifers was killed in a commercial abattoir 3, 6 or 14 days after embryo transfer. Location within the reproductive tract, developmental stage and the quality of embryos were recorded. Transfer of embryos to an advanced (asynchronous) oviduct resulted, on Day 4, in fewer embryos at the expected location (oviduct), and a greater number of degenerated and retarded embryos with a lower total cell number than for embryos in the synchronous group. Similarly, on Day 7, asynchrony led to a greater number of degenerated and retarded embryos compared with the synchronous group. Total embryo cell number was similar among groups. Although Day 15 conceptuses were longer following asynchronous transfer, only 50% of the asynchronous heifers yielded conceptuses, compared with 100% in the synchronous group. In conclusion, asynchrony between the developing embryo and the reproductive tract has a negative effect on embryo development.

Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality.

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 µM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 µM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid.

Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (І) oviduct anatomy and (ІІ) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 µl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3-10, 17 cm, Protean® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (І) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (ІІ) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ protein concentrations. In conclusion, data indicate that the composition of bovine OF is anatomically dynamic and influenced by the presence of an early embryo.

Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo.

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

Predictors of incident hypertension in healthy non-diabetic postmenopausal women with normal renal function.

Introduction: We aimed to investigate the incidence of hypertension and to identify potential risk factors in healthy, non-diabetic recently postmenopausal Greek women with normal renal function.Patients and methods: This retrospective cohort study evaluated 141 recently postmenopausal women at baseline and annually thereafter (follow-up time: 1 to 8 years). Blood samples were obtained, and ultrasound evaluations were performed at baseline. A detailed medical history, anthropometric parameters, blood pressure and cardiovascular risk factors were recorded for every woman at each visit. Incident hypertension was defined as the first occurrence of office systolic or diastolic blood pressure, measured at 2 different visits within 2 months or history of initiation of antihypertensive medication.Results: Incident hypertension was diagnosed in 30 out of 141 women (21.3%). The median time to incident hypertension was 3.5 years. Adiposity, elevated cholesterol and triglyceride levels, insulin resistance and parity were positively associated with incident hypertension. In multivariate analysis, however, obesity and insulin resistance were the only statistically significant variables associated with more than 3-fold and 2-fold respectively increased risk of incident hypertension (HOMA-IR, O.R = 1.988, p-value =.043; obesity, O.R = 3.746, p-value =.019).Conclusion: A significant proportion of women entering the menopause present incident hypertension and this is mostly associated with obesity and insulin resistance.

An approach to study the local embryo effect on gene expression in the bovine oviduct epithelium in vivo.

This study aimed to examine the local embryo effect on the transcriptomic response of the epithelial cells of the oviduct in vivo. Fifteen heifers were synchronized and artificially inseminated to a standing heat. All heifers were slaughtered on Day 2.5 after oestrus. The oviducts from 13 animals were isolated, trimmed free of tissue and divided between ampulla/isthmus. The ipsilateral isthmus was divided into smaller sections (2 cm). Each section was sequentially flushed until the embryo was located (4/13) and then opened and scraped longitudinally to obtain the epithelial cells. Cells were snap-frozen in LN2 for gene expression analysis. All recovered embryos were found at the beginning of the isthmus. The 2 cm sections selected for the transcriptomic analysis were as follows: embryo section (in which the embryo was found); proximal section (through which the embryo had passed); distal section (on the uterine side of the embryo); and contralateral section (section from the contralateral isthmus). The expression pattern of eight genes (STK32A, KERA, QRFPR, MCTP1, PRELP, VAT1L, SOCS3 and CCL20) differentially expressed between the isthmus of pregnant (multiple embryo model) and cyclic heifers were assessed by RT-qPCR. One-way ANOVA and t test was used for statistical analysis. Comparisons between ipsilateral and contralateral oviduct or along the ipsilateral oviduct resulted in no differences for all genes. Despite the failure to detect a site-specific response of a single embryo on the abundance of distinct transcripts in the bovine oviduct in vivo on Day 2.5, the current methodology with proposed modifications would be useful for future studies to examine the local embryo effect.

Ascorbic acid-cyclodextrin complex alters the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

Ascorbic acid (AC) used as antioxidant in embryo culture is very sensitive and degrades unavoidably in aqueous solution. Methyl-ß-cyclodextrin (CD) improved the stability of AC in solution to elevated temperature, light, humidity and oxidation. The aim of this study was to evaluate the effect of the complex AC-CD during in vitro maturation (IVM) or in vitro culture (IVC) on oocyte developmental competence and subsequent embryo development and quality. AC-CD (100 µM) was added to IVM media, and maturation level and embryo development were examined. Matured oocytes, their cumulus cells and produced blastocysts were snap-frozen for gene expression analysis by RT-qPCR. Besides, in vitro-produced zygotes were cultured with 100 µM of AC-CD and blastocysts were as well snap-frozen for gene expression analysis. A group without AC-CD (control- ) and other with CD (control+ ) were included. No differences were found on maturation, cleavage or blastocyst rates. However, in matured oocytes, AC-CD downregulated BAX, GPX1 and BMP15. In cumulus cells, AC-CD downregulated BAX/BCL2 and GSTA4 while upregulated BCL2 and CYP51A1. The expression of SL2A1, FADS1, PNPLA and MTORC1 was downregulated in blastocysts derived from oocytes matured with AC-CD, while in blastocysts derived from zygote cultured with AC-CD, CYP51A1 and IGF2R were downregulated and PNPLA2 was upregulated. In conclusion, AC-CD in both IVM and IVC media may reduce accumulated fat by increasing lipolysis and suppressing lipogenesis in blastocysts derived from both oocytes and zygotes cultured with AC-CD, suggesting that CD improves the quality of embryos and bioavailability of AC during IVM and IVC.

The oviduct: from sperm selection to the epigenetic landscape of the embryo.

The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed.

Early sex-dependent differences in response to environmental stress.

Developmental plasticity enables the appearance of long-term effects in offspring caused by exposure to environmental stressors during embryonic and foetal life. These long-term effects can be traced to pre- and post-implantation development, and in both cases, the effects are usually sex specific. During preimplantation development, male and female embryos exhibit an extensive transcriptional dimorphism mainly driven by incomplete X chromosome inactivation. These early developmental stages are crucial for the establishment of epigenetic marks that will be conserved throughout development, making it a particularly susceptible period for the appearance of long-term epigenetic-based phenotypes. Later in development, gonadal formation generates hormonal differences between the sexes, and male and female placentae exhibit different responses to environmental stressors. The maternal environment, including hormones and environmental insults during pregnancy, contributes to sex-specific placental development that controls genetic and epigenetic programming during foetal development, regulating sex-specific differences, including sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review summarizes several human and animal studies examining sex-specific responses to environmental stressors during both the periconception period (caused by differences in sex chromosome dosage) and placental development (caused by both sex chromosomes and hormones). The identification of relevant sex-dependent trajectories caused by sex chromosomes and/or sex hormones is essential to define diagnostic markers and prevention/intervention protocols.

Bovine oviductal and uterine fluid support in vitro embryo development.

In order to mimic the maternal oviductal environment, we evaluated the effect of oviductal fluid (OF) and/or uterine fluid (UF) supplementation on in vitro embryo development and quality. In vitro-produced zygotes were cultured with 1.25% OF from Day 1 to Day 4 after insemination (OF group), 1.25% OF from Day 1 to Day 4 followed by 1.25% UF from Day 4 to Day 9 (OF+UF group) or 1.25% UF only from Day 4 to Day 9 (UF group). Control groups were cultured in the presence of synthetic oviduct fluid (SOF) supplemented with 3mgmL-1 bovine serum albumin (BSA) or 5% fetal calf serum (FCS). Supplementation of the culture medium with OF and/or UF (both at 1.25%) supported embryo development (Day 9 blastocyst rate 28.2-30.6%). At 72h after vitrification-warming, the survival of blastocysts from the OF and OF+UF groups was similar to that of blastocysts in the SOF+BSA group (61.0±5.7% and 62.8±6.4% vs 64.8±6.4% respectively), but significantly higher than that of blastocysts from the SOF+FCS group (31.6±4.9%; P<0.001). Blastocysts from the OF group exhibited upregulation of epigenetic genes (i.e. DNA methyltransferase 3α (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R)), compared with expression in the SOF+FCS group (P<0.05). Whereas those from OF+UF and UF groups exhibited downregulation of oxidative stress genes compared to SOF+BSA and OF groups for glutathione peroxidase (GPX1) and to SOF+FCS, SOF+BSA and OF groups for chloride intracellular channel 1 (CLIC1) (P<0.05). In addition, accumulation of reactive oxygen species was lower in blastocysts from the OF, OF+UF and UF groups. In conclusion, the use of low concentrations of OF and UF in in vitro serum-free culture supports embryo development, with OF providing a better control of embryo methylation, whereas UF may have antioxidant activity.

Subclinical atherosclerosis and vascular stiffness in premenopausal women: association with NOS3 and CYBA polymorphisms.

Genetic variations of genes encoding the endothelial nitric oxide synthase (eNOS) and the NADH/NADPH oxidase system are related with atherosclerosis in the general population, but their significance in women is not sufficiently assessed. We investigated the potential association between the G894T polymorphism of the NOS3 gene and the C242T polymorphism of the CYBA gene with subclinical vascular disease. Seventy (70) healthy, normally ovulating, premenopausal women were recruited for this study. Venous blood samples were obtained for biochemical/hormonal assessment as well as for genotyping, using real-time PCR. Sonographically assessed indices of vascular structure and function included carotid and femoral intima-media thickness (IMT), flow-mediated dilation (FMD), carotid-femoral pulse-wave velocity (PWV), and augmentation index. The prevalence of wild type, heterozygote, and homozygote genotype was 44.3% (31/70), 54.3% (38/70), and 1.4% (1/70) for the G894T polymorphism and 38.6% (27/70), 31.4% (22/70), and 30.0% (21/70) for the C242T polymorphism, respectively. After multivariable adjustment, the hC242T polymorphism was a predictor of both internal carotid IMT (b-coefficient - 0.119, p = 0.011) and combined-IMT (b-coefficient - 0.061, p = 0.015). Systolic blood pressure, lipids, and hC242T determined values of FMD (b-coefficient - 1.604, p = 0.034). Concerning the NOS3 G894T polymorphism, carriers of the polymorphic variant had higher values of IMT and PWV compared to the wild-type subgroup (carotid bulb-IMT and PWV, heterozygotes/homozygotes vs wild type 0.7 ± 0.2 vs 0.6 ± 0.1 mm; 7.1 ± 0.8 vs 6.6 ± 0.7 m/s; p = 0.048 and p = 0.029, respectively). These differences, however, were rendered non-significant in the multivariable analysis. In healthy premenopausal women, the CYBA C242T polymorphism is an independent determinant of endothelial function and subclinical atherosclerosis of the carotid arteries. The NOS3 G894T polymorphic variant also correlated with atherosclerosis, an association probably mediated by the traditional risk factors for CVD. The relevance of these findings in the clinical setting remains to be elucidated.

Effects of recombinant OVGP1 protein on in vitro bovine embryo development.

Previously, our group demonstrated that recombinant porcine oviductin (pOVGP1) binds to the zona pellucida (ZP) of in vitro-matured (IVM) porcine oocytes with a positive effect on in vitro fertilization (IVF). The fact that pOVGP1 was detected inside IVM oocytes suggested that this protein had a biological role during embryo development. The aim of this study was to evaluate the effects of pOVGP1 on bovine in vitro embryo development. We applied 10 or 50 µg/ml of pOVGP1 during IVF, embryonic in vitro culture (IVC), or both, to evaluate cleavage and embryo development. Blastocyst quality was assessed by analyzing the expression of important developmental genes and the survival rates after vitrification/warming. pOVGP1 was detected in the ZP, perivitelline space, and plasma membrane of blastocysts. No significant differences (P > 0.05) were found in cleavage or blastocyst yield when 10 or 50 µg/ml of pOVGP1 was used during IVF or IVC. However, when 50 µg/ml pOVGP1 was used during IVF + IVC, the number of blastocysts obtained was half that obtained with the control and 10 µg/ml pOVGP1 groups. The survival rates after vitrification/warming of expanded blastocysts cultured with pOVGP1 showed no significant differences between groups (P > 0.05). The use of pOVGP1 during IVF, IVC, or both, increased the relative abundance of mRNA of DSC2, ATF4, AQP3, and DNMT3A, the marker-genes of embryo quality. In conclusion, the use of pOVGP1 during bovine embryo in vitro culture does not affect embryo developmental rates but produces embryos of better quality in terms of the relative abundance of specific genes.

Cultured bovine embryo biopsy conserves methylation marks from original embryo.

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. This work analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cell mass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation is more similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.