Bovine Oviduct Epithelial Cells Research Articles

Maternal stress and the early embryonic microenvironment: investigating long-term cortisol effects on bovine oviductal epithelial cells using air–liquid interface culture

The oviduct epithelium is the initial maternal contact site for embryos after fertilization, offering the microenvironment before implantation. This early gestation period is particularly sensitive to stress, which can cause reduced fertility and reproductive disorders in mammals. Nevertheless, the local impact of elevated stress hormones on the oviduct epithelium has received limited attention to date, except for a few reports on polyovulatory species like mice and pigs. In this study, we focused on the effects of chronic maternal stress on cattle, given its association with infertility issues in this monoovulatory species. Bovine oviduct epithelial cells (BOEC) differentiated at the air–liquid interface (ALI) were stimulated with 250 nmol/L cortisol for 1 or 3 weeks. Subsequently, they were assessed for morphology, bioelectrical properties, and gene expression related to oviduct function, glucocorticoid pathway, cortisol metabolism, inflammation, and apoptosis. Results revealed adverse effects of cortisol on epithelium structure, featured by deciliation, vacuole formation, and multilayering. Additionally, cortisol exposure led to an increase in transepithelial potential difference, downregulated mRNA expression of the major glucocorticoid receptor (NR3C1), upregulated the expression of cortisol-responsive genes (FKBP5, TSC22D3), and significant downregulation of oviductal glycoprotein 1 (OVGP1) and steroid receptors PGR and ESR1. The systematic comparison to a similar experiment previously performed by us in porcine oviduct epithelial cells, indicated that bovine cultures were more susceptible to elevated cortisol levels than porcine. The distinct responses between both species are likely linked to their divergence in the cortisol-induced expression changes of HSD11B2, an enzyme controlling the cellular capacity to metabolise cortisol. These findings provide insights into the species-specific reactions and reproductive consequences triggered by maternal stress.

Expression patterns of folate metabolism-related enzymes in the bovine oviduct: estrous cycle-dependent modulation and responsiveness to folic acid

Folate metabolism is required for important biochemical processes that regulate cell functioning, but its role in female reproductive physiology in cattle during peri- and post-conceptional periods has not been thoroughly explored. Previous studies have shown the presence of folate in bovine oviductal fluid, as well as finely regulated gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). Additionally, extracellular folic acid (FA) affects the transcriptional levels of genes important for the functioning of BOECs. However, it remains unknown whether the anatomical and cyclic features inherent to the oviduct affect regulation of folate metabolism. The present study aimed to characterize the gene expression pattern of folate cycle enzymes in BOECs from different anatomical regions during the estrous cycle and to determine the transcriptional response of these genes to increasing concentrations of exogenous FA. A first PCR screening showed the presence of transcripts encoding dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase (MTR) in bovine reproductive tissues (ovary, oviduct and uterus), with expression levels in oviductal tissues comparable to, or even higher than, those detected in ovarian and uterine tissues. Moreover, expression analysis through RT-qPCR in BOECs from the ampulla and isthmus during different stages of the estrous cycle demonstrated that folate metabolism-related enzymes exhibited cycle-dependent variations. In both anatomical regions, DHFR was upregulated during the preovulatory stage, while MTHFR and MTR exhibited increased expression levels during the postovulatory stage. Under in vitro culture conditions, ampullary and isthmic cells were cultured in the presence of 10, 50, and 100 μM FA for 24 h. Under these conditions, isthmus epithelial cells exhibited a unique transcriptional response to exogenous FA, showing a pronounced increase in MTR expression levels. Our results suggest that the expression of folate metabolism-related genes in BOECs is differentially regulated during the estrous cycle and may respond to exogenous levels of folate. This offers a new perspective on the transcriptional regulation of genes associated with the folate cycle in oviductal cells and provides groundwork for future studies on their functional and epigenetic implications within the oviductal microenvironment.

Effect of the estrous cycle on zinc transporter expression in bovine cumulus-oocyte complexes and oviduct epithelial cells.

During the luteal and follicular phases of the estrous cycle, cumulus-oocyte complexes (COC) and oviduct epithelial cells (OEC) undergo notable physiological and morphological changes. Maintaining proper zinc (Zn) homeostasis is crucial in both somatic and germinal mammalian cells. This study aimed to assess the impact of the estrous phase (luteal or follicular) on Zn transporter expression in bovine COC and OEC (BOEC). The expression of Zn transporters Slc39a6 (ZIP6), Slc39a8 (ZIP8), Slc39a14 (ZIP14), Slc30a3 (ZnT3), Slc30a7 (ZnT7), and Slc30a9 (ZnT9) was analyzed in COC and BOEC from cows during the luteal or follicular phases. Gene expression of ZIP6, ZIP14, and ZnT9 was quantified in COC and BOEC. The gene expression in the remaining transporters could not be quantified due to low mRNA levels (ZIP8 and ZnT3 in COC and BOEC; ZnT7 in BOEC) or absence of expression (ZnT7 in COC). In COC, the relative expression (RE) of all three transporters was higher in the luteal phase compared to the follicular phase (P ≤ 0.05). In BOEC, the luteal phase increased the RE of ZIP 6 (P ≤ 0.05), decreased the RE of ZnT9 (P ≤ 0.05), and did not modify the RE of ZIP14 (P > 0.05) compared to the follicular phase. In conclusion, the study reveals differences in the gene expression of ZIP6, ZIP14, and ZnT9 according to the estrous cycle phase in ex vivo samples of bovine COC and OEC.

Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

A bovine oviduct microfluidic platform to evaluate the effects of cisplatin and hormones on cilia beating frequencies

Chemotherapeutics such as cisplatin modulate the function of cells in fallopian tubes (oviducts) reducing the formation of sperm reservoirs that promote gamete interaction during the fertilization process. The mechanisms behind these resulting deficiencies must be better understood to improve fertility for cancer patients. A microfluidic platform was developed by immobilizing a transparent polycarbonate membrane between two poly(dimethyl siloxane) molds to make channels for the growth of a functional monolayer of oviduct cells on one side and medium on the other side. Freshly isolated bovine oviduct epithelial cells were cultured on the membrane with an air-liquid interface after 5 days, and the resulting cells had consistent cilia beating frequencies (CBF) for 12 weeks between 4.92 ± 0.59 Hz and 5.76 ± 0.94 Hz. Incubation with 75 pg ml−1 17-beta-estradiol and 100 ng ml−1 progesterone increased the CBF from 4.62 ± 0.16 Hz to 7.61 ± 0.41 Hz. When incubated with 50 µM of cisplatin, the CBF dropped by 40 ± 19 % without hormones and 52 ± 9 % with hormones, with similarly decreased CBFs. Short 2-h incubations of bovine oviduct cells with cisplatin directly reduced the beating of ciliated cells, illustrating that the short-term effects (hours) of chemotherapeutics should be considered in oncofertility.

Evaluation of the morphological and functional state of cryopreserved sperm of bulls at different periods of long-term storage

Objective. To study the morphological andfunctional state of cryopreserved sperm of bulls during storage for 6 - 27 years at a temperature of - 196 C in liquid nitrogen in a comparative aspect. Methods. The object of the study was cryopreserved semen of bulls stored for 6 - 10 or 20 - 27 years. In the study of the qualitative parameters offrozen- thawed spermatozoa, the method of visual assessment for motility and survival, the method ofers differential staining with trypan blue/Gymza dyes to determine the morphological and functional integrity of cell membranes and the test for the ability to bind to vesicular explants of bovine oviductal epithelial cells in vitro were used. Results. It was found that the extension of the storage period of spermatozoa to 20 - 27years had no significant effect on the functional characteristics o f spermatozoa in terms of motility, survival and absolute survival. The method of differential staining showed that the proportion of live sperm with an intact acrosome in the total distribution did not differ significantly in both studied storage periods and ranged from 61.6 - 63.9 %. The proportion of dead cells also did not differ significantly and was 26.4 % when stored for up to 10 years and 21.7 % when stored for more than 20 years. No significant difference was found in the overall assessment of the percentage of sperm with varying degrees of acrosomal membrane damage. This figure was 22.5 and 26.0 %, respectively. At the same time, when the shelf life of cryopreserved sperm was extended to 20 - 27 years, a significant increase in the percentage of live sperm with a missing acrosome was observed (p<0.01). It was determined that the index of sperm binding to explants of epithelial cells of the oviduct in vitro was significantly higher when storedfor 6-10 years and was 227.9±32.3 against 114.6±19.5 when stored for 20 - 27 years (p<0.05). Conclusions. The use of the traditional method of assessing sperm quality by indicators of sperm motility, survival and absolute sperm survival did not reveal any differences when storing cryopreserved sperm for 6 - 27 years. At the same time, the differential staining method showed a significant increase of 7.2% in the proportion of live sperm with a missing acrosome when the storage period was extended from 6 - 10 to 20 - 27 years. The in vitro test for sperm binding to oviduct epithelial cells showed a significant decrease of almost 2 times in the index of sperm binding to cellular explants during long-term storage. This may indicate a decrease in the ability of such sperm to fertilise. Keywords: bulls, sperm, cryopreservation, long-term storage, differential colouration, oviduct, sperm binding.

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

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. For this purpose, primary BOEC were co-cultured with in vitro produced embryos (IVP) 53h 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 24h 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. 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). 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.

Effect of estrogen and progesterone on intracellular free zinc and zinc transporter expression in bovine oviduct epithelial cells

Zinc (Zn) plays essential roles in numerous cellular processes. However, there is limited understanding of Zn homeostasis within the bovine reproductive system. This study investigated the influence of estradiol (E2) and progesterone (P4) on Zn transporter expression and intracellular free Zn levels in bovine oviduct epithelial cells (BOEC). For this purpose, cells were harvested from slaughtered cows and cultured in vitro. Intracellular Zn concentrations were measured using FluoZin-3AM staining, while real-time polymerase chain reaction assessed Zn transporter gene expression and quantification. Overall, our results confirmed the gene expression of all the evaluated Zn transporters (ZIP6, ZIP8, ZIP14, ZnT3, ZnT7 and ZnT9), denoted and the active role of E2 and P4 in intracellular Zn regulation. Our findings suggest an interaction between Zn, E2 and P4.

Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-β estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

Serum-free spontaneously immortalized bovine oviduct epithelial cell conditioned medium promotes the early development of bovine in vitro fertilized embryos.

Embryonic transfer of bovine blastocysts produced using in vitro fertilization (IVF) is widely used, although the challenge of compromised conception rates remains. Using bovine oviduct epithelial cells (BOEC) to improve embryo culture conditions has attracted attention, particularly since the recent discovery of extracellular vesicles from BOEC. The selection of embryos for transfer has also been the subject of various studies, and a set of evaluation criteria to predict pregnancy success has been suggested, in which the embryos are judged by their kinetics and morphology at the early stages. In the present study, we established a spontaneously immortalized BOEC line (SI-BOEC) and examined the effects of conditioned medium on IVF embryos, focusing on the results of the recommended criteria. A modified KSOM (mKSOM) was used to prepare conditioned media. Presumptive zygotes were cultured in mKSOM (control), SI-BOEC-conditioned medium, mKSOM supplemented with sediment (pellet) collected after the ultracentrifugation of the conditioned medium (mKSOM/sediment), and the supernatant. A significantly higher percentage of embryos satisfied the recommended criteria when grown in the conditioned medium than in the mKSOM. A higher proportion of embryos developed into blastocysts after achieving the four criteria. A similar tendency was observed when grown in mKSOM/sediment compared to mKSOM; however, this was not observed in the supernatant. Vesicles with a size similar to that of exosomes were observed in the sediment. In conclusion, the culture medium conditioned by SI-BOEC promoted the production of bovine blastocysts that satisfied the four evaluation criteria recommended for embryo selection.

88 Co-culture with bovine oviduct epithelial cells during in vitro culture affects the bioenergetic profile of bovine expanded blastocysts

88 Co-culture with bovine oviduct epithelial cells during in vitro culture affects the bioenergetic profile of bovine expanded blastocysts

Spermatozoa, acts as an external cue and alters the cargo and production of the extracellular vesicles derived from oviductal epithelial cells in vitro

The oviduct provides optimum physiological and biochemical milieu essential for successful fertilization, early embryo development and facilitates functional maturation of spermatozoa. A study has revealed that spermatozoa alters the gene expression in bovine oviductal epithelial cells (BOECs) remotely via bio-active particles, thus acting as a cue to the oviduct prior to their arrival. However, very little attention has been paid to the question of whether spermatozoa could alter the cargo of extracellular vesicles (EVs) derived from BOECs. Therefore, the aim of this study was to investigate the alterations in small non-coding RNAs in EVs cargo derived from BOECs when incubated with spermatozoa in contact and non-contact co-culture models. After 4 h of incubation the EVs were isolated from the conditioned media, followed by small non-coding sequencing of the BOEC derived EVs. Our results revealed that EVs from both co-culture models contained distinct cargo in form of miRNA, fragmented mRNA versus control. The pathway enrichment analysis revealed that EV miRNA from direct co-culture were involved in the biological processes associated with phagocytosis, macroautophagy, placenta development, cellular responses to TNF and FGF. The mRNA fragments also varied within the different groups and mapped to the exonic regions of the transcriptome providing vital insights regarding the changes in cellular transcriptome on the arrival of spermatozoa. The findings of this study suggest that spermatozoa, in contact as well as remotely, alter the EV cargo of female reproductive tract epithelial cells which might be playing an essential role in pre and post-fertilization events.Graphical abstract

Evaluation of sperm integrin α5β1 as a potential marker of fertility in humans.

Sperm selection for assisted reproduction techniques is generally based on basic parameters, while key aspects of sperm competence and its journey from the deposition site to the fertilization site are overlooked. Consequently, identifying molecular markers in spermatozoa that can efficiently predict the fertility of a semen sample could be of great interest, particularly in cases of idiopathic male infertility. When spermatozoa reach the female reproductive tract, it provides to them the cellular and molecular microenvironment needed to acquire fertilizing ability. In this sense, considering the role that integrin α5β1 of spermatozoa plays in reproduction-related events, we investigated the correlation between the subcellular localization of sperm integrin α5β1 and early embryo development outcome after in vitro fertilization (IVF) procedures in human. Twenty-four semen samples from normozoospermic men and metaphase II (MII) oocytes from healthy women aged under 38 years, from couples who underwent IVF cycles, were used in this work. Sperm α5β1 localization was evaluated by immunofluorescence assay using an antibody against integrin α5 subunit. Integrin α5β1 was mainly localized in the sperm acrosomal region (45.33±7.89%) or the equatorial segment (30.12±7.43%). The early embryo development rate (data obtained from the Fertility Center) correlated positively with the localization of α5β1 in the acrosomal region (number of usable embryos / inseminated oocytes: ρ = 0.75; p<0.01 and number of usable embryos/total number of two pronuclear zygotes: ρ = 0.80; p<0.01). However, this correlation was not significant when the equatorial segment mark was evaluated. In addition, human sperm released from co-culture with bovine oviductal epithelial cells (BOEC) showed a significant enrichment in the acrosomal localization pattern of α5β1 compared to those sperm that were not co-cultured with BOEC (85.20±5.35% vs 35.00±17.09%, respectively, p<0.05). In conclusion, the evaluation of sperm integrin α5β1 immunolocalization could be a useful tool to select sperm with fertilizing ability from human semen samples before IVF procedures.

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 oocyte developmental competence and gene expression following co-culturing with ampullary cells: An experimental study.

BackgroundThere is no sufficient information on the impact of bovine ampullary oviductal epithelial cells (BAOECs) on in vitro oocyte maturation competence and gene expression.ObjectiveThis study aimed to examine the oocyte developmental competence following co-culturing with a monolayer of fresh and frozen-thawed ampullary cells.Materials and MethodsBovine cumulus-oocyte complexes (COCs) were distributed into three groups: control group; where in COCs were cultured in cell-free media for 24 hr and FML and FTML groups in which the COCs were cultured in maturation media for 18 hr and then transferred into a media containing fresh and frozen-thawed BAOECs monolayer, respectively (BAOECs were extracted from the oviducts of slaughtered cattle and were then cultured freshly or frozen-thawed) for a further 6 hr. After 24 hr, the expanded COCs were evaluated for nuclear maturation, fertilization rate, and gene expression (GDF9, StAR, CASP3, and FSHr).ResultsNuclear maturation rate in the FTML group was significantly higher than the control group (p = 0.02). The fertilization rate of FTML group was significantly higher than the control and FML groups (p = 0.05 and p = 0.03, respectively). In terms of gene expression, GDF9 were upregulated in the presence of the BAOECs during the last 6 hr of the in vitro maturation (p 0.001). Furthermore, the expression of the StAR gene in the FTML group was higher than the other groups (p = 0.02).ConclusionAmpullary cells co-culturing (especially frozen-thawed cells) for in vitro maturation of bovine oocytes yields encourages the results and demonstrates the beneficial effect of co-culture on gene expression and developmental competence.

Porcine oocyte preincubation in oviductal fluid flush before in vitro fertilization in the presence of oviductal epithelial cells improves monospermic zygote production.

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

The growth hormone and insulin-like growth factor 1 axis in cattle during the peri-ovulatory period activates the synthesis and release of oviductal contraction related substances.

Growth hormone (GH) and insulin-like growth factor 1 (IGF1) are crucial for female reproductive functions. The cyclic regulation of the local GH/IGF1 axis in the oviduct and its involvement in oviductal contraction in cattle has not been investigated. Thus, the messenger RNA (mRNA) expression for GH receptor (GHR), IGF1, IGF1 receptor (IGF1R) in the whole oviducts, as well as in cultured bovine oviductal epithelial cells (BOECs) were evaluated. The GHR, IGF1, and IGF1R mRNA expression was significantly higher during postovulatory phase. The luteinizing hormone (LH), estradiol-17β (E2), and LH + E2 treatments significantly increased GHR and IGF1 mRNA expression in cultured BOECs. Further, GH and combination of GH with LH and E2 upregulated IGF1 mRNA expression in the BOECs. Moreover, IGF1 + LH and combined IGF1 + LH + E2 treatments significantly increased prostaglandin synthesis cascade enzyme mRNA expression in the BOECs. An ex vivo microdialysis assay revealed that GH and IGF1 induced the release of oviductal contraction related prostaglandins, endothelin-1, and angiotensin II in follicular and postovulatory phases. Together, the findings strongly suggest that the presence of the active GH/IGF1 axis during the peri-ovulatory period, regulating the local system for the release of oviductal contraction related substances, which may provide the optimal oviductal environment for gametes and early embryo.

A long-term in vitro culture of bovine epithelial cells on collagen rafts.

In the present work, we established and characterized a 3D functional polarized primary bovine oviduct epithelial cells (BOECs) culture on free-floating type I collagen hydrogels (rafts) at an air-liquid interface (ALI). Intercellular junctions, ultrastructural cellular morphology and the expression of the OVGP1 closely recapitulated those of the in vivo epithelium lining. These morphological and physiological epithelial cell features were maintained under standard DMEM/F12 with 10% foetal bovine serum culture medium for at least 28days of ALI culture. The versatility of the BOECs raft cultures should allow testing of toxicity compounds, in vitro evaluation of physiological or pathological oviductal states, and the study of epithelial-mesenchymal interactions that are critical for the maintenance of oviductal homeostasis.

Oviduct as a sensor of embryo quality: deciphering the extracellular vesicle (EV)-mediated embryo-maternal dialogue

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. KEY MESSAGES: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.

Effect of Estrous Cycle Phases on Gene Expression of Bovine Oviduct Epithelial Cells

Effect of Estrous Cycle Phases on Gene Expression of Bovine Oviduct Epithelial Cells