Effect of estrous cycle phases on gene expression in bovine oviduct epithelial cells.

Background and Aim: The oviduct environment is of particular importance because it is the site of fertilization and early embryo development. The oviduct, as a component of the reproductive system, responds to ovarian hormone (estradiol [E2] and progesterone [P4]) stimuli depending on the estrous cycle phase. This study aimed to elucidate the effect of estrous cycle phases (follicular and early and late luteal phases) on gene expression patterns in bovine oviduct epithelial cells (BOECs). Materials and Methods: Oviducts were obtained from healthy slaughterhouse animals, corresponding to ipsilateral ovaries with dominant follicles or corpus luteum during early and late luteal phases. BOECs were recovered from the isthmus (IST) and ampulla (AMP), and the expression patterns of genes related to cytokinesis and mitosis mechanisms (rho-associated coiled-coil containing protein kinase and cellular communication network factor 2 [CCN2]), growth factors (insulin-like growth factor-binding protein 3, epidermal growth factor receptor [EGFR], vascular endothelial growth factor A, and EGFR), antioxidant mechanisms (glutathione peroxidase 4 [GPX4]), apoptosis (B-cell lymphoma 2), complement component (C3), energy metabolism (aldose reductase gene family 1-member b1 [AKRIB1] and solute carrier family 2), hormone receptors (estrogen receptor 1 and luteinizing hormone/choriogonadotropin receptor), and specific glycoproteins (oviductal glycoprotein 1) were analyzed. Results: High P4 levels (late luteal phase) affected the expression of important genes related to antioxidant mechanisms (GPX4), energy metabolism (AKRIB1), growth factors (IGBP3 and EGFR), and cell growth regulation (CCN2) in the AMP. Low P4 levels (early luteal phase) affected the expression of AKR1B1, IGBP3, and CCN2. In addition, estrogen likely had an effect on OVPGP expression in the cattle oviduct. Conclusion: Differential gene expression patterns of BOECs in the AMP during the luteal phase (antioxidant mechanisms, energy metabolism, growth factors, and immunological regulators) and in the IST during the follicular phase (glycoproteins) may influence their renewal and population proportions, modulating the oviduct environment as well as gamete and embryo physiology.

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.

Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro.

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.

Effect of bovine oviductal fluid on development and quality of bovine embryos produced in vitro.

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C-) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days 7-9 after insemination and blastocyst quality was assessed through cryotolerance, differential cell counting of the inner cell mass and trophectoderm, and gene expression. OF was added to the culture medium at concentrations ranging from 0.625% to 25%. The higher OF concentrations (5%, 10% and 25%) had a detrimental effect on embryo development. Lower OF concentrations (1.25% and 0.625%) supported embryo development until Day 9 (27.5%) and produced higher-quality blastocysts, as reflected by their cryotolerance (53.6% and 57.7% survival at 72h, respectively, vs 25.9% in C+) and total cell number (mean (± s.e.m.) 165.1±4.7 and 156.2±4.2, respectively, vs 127.7±4.9 in C- and 143.1±4.9 in C+). Consistent with these data, upregulation of the water channel aquaporin 3 (AQP3) mRNA was observed in blastocysts supplemented with 1.25% OF compared with C- and C+. Serum supplementation resulted in a reduction in the expression of glucose and lipid metabolism-related genes and downregulation of the epigenetic-related genes DNA methyltransferase 3A (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R). In conclusion, in vitro culture with low concentrations of OF has a positive effect on the development and quality of bovine embryos.

Oviductal response to gametes and early embryos in mammals.

The oviduct is a complex and organized thin tubular structure connecting the ovary with the uterus. It is the site of final sperm capacitation, oocyte fertilization and, in most species, the first 3-4days of early embryo development. The oviductal epithelium is made up of ciliary and secretory cells responsible for the secretion of proteins and other factors which contribute to the formation of the oviductal fluid. Despite significant research, most of the pathways and oviductal factors implicated in the crosstalk between gametes/early embryo and the oviduct remain unknown. Therefore, studying the oviductal environment is crucial to improve our understanding of the regulatory mechanisms controlling fertilization and embryo development. In vitro systems are a valuable tool to study in vivo pathways and mechanisms, particularly those in the oviducts which in livestock species are challenging to access. In studies of gamete and embryo interaction with the reproductive tract, oviductal epithelial cells, oviductal fluid and microvesicles co-cultured with gametes/embryos represent the most appropriate in vitro models to mimic the physiological conditions in vivo.

Maternal-embryo interaction in the bovine oviduct: Evidence from in vivo and in vitro studies.

Assisted reproductive technologies have provided a very useful tool for studying early embryonic development. The exchange of signals between the embryo and maternal environment during this period is critical to successful development, but most mechanisms involved remain to be elucidated. Understanding how the mother communicates with gametes and embryos is a major scientific challenge but in vivo studies are difficult to perform, especially in cattle, since they are expensive, the amount of material is limited, and it is not possible to differentiate between the outcome of fertilization and early embryonic death. In addition, the local interactions of the embryo with the maternal epithelium may not be detectable because of the small size of the embryo and the difficulty of identifying its exact position in the oviduct. On the basis of current knowledge gained from in vivo studies, the challenge now is to identify appropriate in vitro models to facilitate the study of early embryo-maternal communication.

Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5) EVs/mL, 1.5x10(5) EVs/mL or 7.5x10(4) EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7-9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

An efficient system to establish biopsy-derived trophoblastic cell lines from bovine embryos.

Trophoblastic cells play a crucial role in implantation and placentogenesis and can be used as a model to provide substantial information on the peri-implantation period. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stocks in the long-term. Our results show that the combination of a monolayer culture system in microdrops on a surface treated with gelatin and the employment of conditioned media from mouse embryonic fibroblasts support the growth of bovine trophoblastic cells lines from an embryo biopsy. Expression profiles of mononucleate- and binucleate-specific genes in established trophoblastic cells lines represented various stages of gestation. Moreover, the ability to expand trophoblastic cell lines for more than 2 yr together with pluripotency-related gene expression patterns revealed certain self-renewal capacity. In summary, we have developed a system to expand in vitro trophoblastic cells from an embryo biopsy that solves the limitations of using amplified DNA from a small number of cells for bovine embryo genotyping and epigenotyping and, on the other hand, facilitates the establishment of trophoblastic cell lines that can be useful as peri-implantation in vitro models.