Bovine Oviduct Research Articles

Spatiotemporal profiling of the bovine oviduct fluid proteome around the time of ovulation

Understanding the composition of the oviduct fluid (OF) is crucial to better comprehend the microenvironment in which sperm capacitation, fertilization and early embryo development take place. Therefore, our aim was to determine the spatiotemporal changes in the OF proteome according to the anatomical region of the oviduct (ampulla vs. isthmus), the proximity of the ovulating ovary (ipsilateral vs. contralateral side) and the peri-ovulatory stage (pre-ovulatory or Pre-ov vs. post-ovulatory or Post-ov). Oviducts from adult cyclic cows were collected at a local slaughterhouse and pools of OF were analyzed by nanoLC-MS/MS and label-free protein quantification (n = 32 OF pools for all region × stage × side conditions). A total of 3760 proteins were identified in the OF, of which 65% were predicted to be potentially secreted. The oviduct region was the major source of variation in protein abundance, followed by the proximity of the ovulating ovary and finally the peri-ovulatory stage. Differentially abundant proteins between regions, stages and sides were involved in a broad variety of biological functions, including protein binding, response to stress, cell-to-cell adhesion, calcium homeostasis and the immune system. This work highlights the dynamic regulation of oviduct secretions and provides new protein candidates for interactions between the maternal environment, the gametes and the early embryo.

Extracellular vesicles improve embryo cryotolerance by maintaining the tight junction integrity during blastocoel re-expansion.

Cryopreservation is a process in which the intact living cells, tissues, or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impacts the survival and quality of the in vitro-produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial cells-derived EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between synthetic oviduct fluid (SOF) and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryo’s ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4%) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVs-supplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (aquaporins), and Na+/K+ ATPase alpha 1. The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs.

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 200nm; 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.

Cystic ovary disease impairs transport speed, smooth muscle contraction, and epithelial ion transport in the bovine oviduct.

Cystic ovary disease (COD) is a common cause of bovine infertility but the impact of this disease on the oviduct is unknown. The aim of this study was to analyze the effects of COD on particle transport speed (PTS), ciliary beat frequency, myosalpinx contraction, and epithelial ion transport. Oviducts were obtained from cows affected by COD and compared with those of healthy, mid-diestrus cows. PTS and CBF were examined using live-cell imaging. Smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that muscarinic receptors are involved in cholinergic signaling in the oviduct and that forskolin-induced cyclic AMP production is involved in active ion transport in the oviductal epithelium. Oviducts from cows with luteal cysts revealed significantly decreased PTS (p = 0.02). Further to that, in the oviducts of COD cows, the cholinergic regulation of smooth muscle contractions and active epithelial ion transport were significantly diminished (p < 0.0001). These results imply that in COD cows, oviductal transport is compromised by decreased fluid flow speed and reduced cholinergic regulation of smooth muscle contraction and ion transport. This knowledge contributes to a more comprehensive understanding of COD supporting the development of novel therapeutic concepts for infertility treatment.

208 Sperm Retention, Storage and Release from the Oviduct: A Story of Sugars, Steroids, and Channels

Abstract Because mating is not always synchronized with ovulation, females from many species store sperm in the female reproductive tract until ovulation and fertilization. This may be done for short periods, a day or two for swine and cattle, or longer periods. Other mammals, such as some species of bats, store sperm for several months. Chickens and turkeys store sperm for 2–4 weeks and queens of some species of insects store sperm for over a decade in specialized structures. How sperm are retained, kept fertile for varying times and released is unclear. We have identified two specific carbohydrate motifs that are abundant in the porcine oviduct that bind and retain sperm in the isthmus. When immobilized, these two glycans lengthen sperm lifespan and suppress the normal increase in intracellular Ca2+ that normally accompanies capacitation. Porcine sperm can be released from oviduct cells and immobilized glycans by progesterone, perhaps of ovarian or cumulus-oocyte complex origin, which activates CatSper, a sperm-specific Ca2+ channel. Progesterone, as well as other compounds that stimulate hyperactivated motility, trigger sperm release, suggesting that hyperactivated motility is sufficient to release porcine sperm from oviduct glycans. We also have found that blocking proteasome-induced sperm protein lysis diminishes the number of sperm released from oviduct glycans. Finally, a transcriptomic approach has identified several groups of genes that are differentially regulated in both bovine and porcine oviducts from estrus animals that are storing sperm compared to oviducts from diestrus animals. This provides clues about how sperm lifespan is extended during storage.

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.

Steroidal Regulation of Oviductal microRNAs Is Associated with microRNA-Processing in Beef Cows.

Information on molecular mechanisms through which sex-steroids regulate oviductal function to support early embryo development is lacking. Here, we hypothesized that the periovulatory endocrine milieu affects the miRNA processing machinery and miRNA expression in bovine oviductal tissues. Growth of the preovulatory follicle was controlled to obtain cows that ovulated a small follicle (SF) and subsequently bore a small corpus luteum (CL; SF-SCL) or a large follicle (LF) and large CL (LF-LCL). These groups differed in the periovulatory plasmatic sex-steroid’s concentrations. Ampulla and isthmus samples were collected on day four of the estrous cycle. Abundance of DROSHA, DICER1, and AGO4 transcripts was greater in the ampulla than the isthmus. In the ampulla, transcription of these genes was greater for the SF-SCL group, while the opposite was observed in the isthmus. The expression of the 88 most abundant miRNAs and 14 miRNAs in the ampulla and 34 miRNAs in isthmus were differentially expressed between LF-LCL and SF-SCL groups. Integration of transcriptomic and miRNA data and molecular pathways enrichment showed that important pathways were inhibited in the SF-SCL group due to miRNA control. In conclusion, the endocrine milieu affects the miRNA expression in the bovine oviduct in a region-specific manner.

Isolation, Characterization, and MicroRNA Analysis of Extracellular Vesicles from Bovine Oviduct and Uterine Fluids.

Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.

58 Reduced nutrient availability during invitro culture improves embryo production and morphological quality and alters metabolic status of bovine embryos

Invitro production (IVP) of embryos is designed to reproduce an environment that resembles the female reproductive tract. However, the system does not perform optimally in terms of quality and embryo production. A major setback lies in the loss of dynamics observed in a static invitro system, which might affect the availability of substrates that reach the embryo. A reduction in the amount of nutrients in media has been used as an approach to improve IVP (Ermisch et al. 2020 Sci. Rep. 10, 9263; https://doi.org/10.1038/s41598-020-66019-4). The present study aimed at describing a defined sequential medium (embryonic culture supplementation, ECS) and to investigate the effect of reducing nutrient availability on embryo production, quality, and metabolism. ECS was developed in our laboratory and is a serum-free, salt-based culture medium supplemented with the amount of energy substrates and amino acids found in bovine oviduct (Ov) and uterus (Ut) fluids as previously described (Hugentobler et al. 2007Mol. Reprod. Dev. 74, 445–454; https://doi.org/10.1002/mrd.20607; Hugentobler et al. 2008Mol. Reprod. Dev. 75, 496–503; https://doi.org/10.1002/mrd.20760). Embryos were cultured according to the following ECS supplementation: ECS100 (supplemented with 8mg mL−1 bovine serum albumin and 100% of the energy substrates and amino acids concentrations of the Ov and Ut fluids) and ECS50 (half dilution of ECS100). Bovine oocytes from abattoir ovaries were submitted to IVP using standard protocols. On Day 0 of invitro culture, presumptive zygotes were randomly divided into groups ECS100-Ov or ECS50-Ov. On Day 4, embryos were respectively transferred to ECS100-Ut and ECS50-Ut. Expanded blastocysts were collected on Day 7 to assess embryo production, morphology (total cell number by Hoescht 33342 staining; inner cell mass and trophectoderm cells by CDX2 immunostaining), and metabolic status (mitochondrial activity and reactive oxygen species content by MitotrackerTM RedCMXRos and CellROXTM Green staining, ThermoFisher Scientific; NADH and FAD+ by autofluorescence). Data were analysed by Student’s t-test (a=4%). Although cleavage rates were similar between ECS50 and ECS100 (78.13±3.73 vs. 79.70±4.18; P=0.788), blastocyst rates were positively influenced by the reduction in concentration (28.88±1.74 vs. 16.73±2.41; P=0.004). This difference likely comes from a blockage at the morula stage in group ECS100, because the conversion from morula to blastocyst was 20% lower in this group (57.73±3.81 vs. 38.15±3.45; P=0.008). In terms of morphology, blastocysts produced in ECS50 had a higher number of cells (152.4±9.61 vs. 118.3±7.22; P=0.036), which is explained by the higher number of trophectoderm cells. Finally, metabolic status was affected by nutrient reduction: embryos from ECS50 had higher mitochondrial activity, reactive oxygen species content (P&amp;lt;0.0001), and lower NADH (P=0.01), suggesting higher oxidative phosphorylation to produce energy, as expected at this stage. In conclusion, ECS is a functional medium, and a reduced nutrient concentration (ECS50) improves embryo production, morphological quality, and metabolic status of blastocysts, suggesting that culture conditions must be adapted to the invitro system rather than resembling invivo conditions. This research was funded by FAPESP (2016/00350-0, 2017/18384-0).

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

IL1B triggers inflammatory cytokine production in bovine oviduct epithelial cells and induces neutrophil accumulation via CCL2.

The oviduct is essential for reproduction. We previously showed that oviduct epithelial cells (OECs) isolated from aged cows expressed higher levels of inflammatory cytokines, including interleukin (IL) 1A and IL1B. In addition, aging is associated with tissue dysfunction and cellular senescence via a senescence-associated secretory phenotype (SASP) and immune cell accumulation. We investigated whether IL1A or IL1B causes SASP production, cellular senescence, and inflammatory responses in bovine OECs. The OECs were isolated from bovine oviducts from young (mean 50.3months) and aged cows (mean 157.0months) and cultured. Treatment with IL1A or IL1B induced SASP production (IL8, IL6, TNFA, and CCL2) and mRNA expression of cell adhesion molecules in bovine OECs, but both IL1s did not induce cellular senescence in OECs and migration of polymorphonuclear neutrophils (PMNs). Cultured medium of OECs treated with IL1s, especially IL1B, dramatically induced PMN migration. Treatment with the CCL2 inhibitor, but not IL8 or its receptor CXCR2 inhibitors, significantly reduced immune cell migration in IL1B-treated OEC-cultured medium. Treatment with IL1B increased PMN adhesion to OECs, resulting in further SASP production in OECs due to a PMN-OEC interaction. We suggest that senescence-associated IL1s cause SASP production in bovine OECs and CCL2 induced by IL1B is essential for the migration of immune cells to OECs. Specifically, IL1B regulates PMN migration and adhesion to bovine OECs, and PMNs accelerate inflammatory cytokine production from bovine OECs via a direct interaction. These phenomena may contribute to chronic oviductal inflammation, resulting in subfertility.

Embryo co-culture with bovine amniotic membrane stem cells can enhance the cryo-survival of IVF-derived bovine blastocysts comparable with co-culture with bovine oviduct epithelial cells.

Culture conditions have a profound effect on the quality of in vitro-produced embryos. Co-culturing embryos with somatic cells has some beneficial effects on embryonic development. Considering the ability of stem cells to secrete a broad range of growth factors with different biological activities, we hypothesized that bovine amniotic membrane stem cells (bAMSCs) might be superior to bovine oviduct epithelial cells (BOECs) in supporting embryonic development and enhancing their cryo-survival. Bovine abattoir-derived oocytes were matured and fertilized in vitro. The resultant presumptive zygotes were then cultured up to the blastocyst stage in the following groups: (i) co-culture with bAMSCs, (ii) co-culture with BOECs, and (iii) cell-free culture (Con). Embryos that reached the blastocyst stage were vitrified and warmed, and their post-warming re-expansion, survival and hatching rates were evaluated after 72 h culture. Results showed that the cleavage, blastocyst, and 2 h post-warming re-expansion rates of embryos did not differ between groups. However, their survival rates in BOEC and bAMSC groups were significantly higher compared with the control (72.7, 75.6 and 37.5%, respectively, P < 0.05). In conclusion, our results showed that the cryo-survivability of IVF-derived bovine embryos could be improved through co-culturing with bAMSCs. Moreover, considering the possibility to provide multiple passages from bAMSCs compared with BOECs, due to their stemness properties and their ability to produce growth factors, the use of bAMSCs is a good alternative to BOECs in embryo co-culture systems.

Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3–4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.

Toll-like receptor 2 mediates the immune response of the bovine oviductal ampulla to sperm binding.

We previously reported that sperm binding to cultured bovine oviduct epithelial cells induces an anti-inflammatory immune response. Now we have developed a differentiated explant model to focus on the oviductal ampulla, where fertilization occurs, and to study the effect of sperm capacitation on the immune response. We used heparin to stimulate bovine sperm capacitation. Fluorescence imaging showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide-labeled sperm pretreated with (Hep(+) ) or without (Hep(-) ) heparin rapidly attached to the explant ciliated epithelium in similar numbers. However, only Hep(+) sperm upregulated explant messenger RNA (mRNA) transcription of TLR2, IL8, TGFB1, and PGES, without changes in TNFA and IL-10 expression, while Hep(-) sperm only upregulated PGES. The responses were primarily anti-inflammatory, with a greater response produced by Hep(+) sperm, which also produced a substantial increase in TLR2 protein expression in the epithelium. The addition of TLR1/2 (toll-like receptor 1/2) antagonist to the Hep(+) and (Hep(-) ) sperm-explant coincubations reduced sperm attachment to the epithelium and inhibited TLR2 protein expression and some of the Hep(+) sperm-induced mRNA transcription. Our observations suggest that the ampullar epithelium immunologically reacts more strongly to sperm that have undergone heparin stimulation of capacitation. This anti-inflammatory response could serve to protect capacitated sperm as they approach the oocyte in the ampulla.

The Effects of Polyampholyte on Vitrification Process for cryopreservation of Bovine Oviduct Epithelial Cell

The Effects of Polyampholyte on Vitrification Process for cryopreservation of Bovine Oviduct Epithelial Cell

Local sex steroid hormone milieu in the bovine oviduct ipsilateral and contralateral to preovulatory follicle or corpus luteum during the periovulatory phase

Estradiol-17β (E2) and progesterone (P4) regulate oviductal functions, providing a suitable environment for the transport and maturation of gametes, fertilization, and embryonic development. In addition to the E2 and P4 nuclear receptors, estrogen receptor (ESR) α and β, nuclear progesterone receptor (PGR), nongenomic mechanisms through G protein-coupled estrogen receptor (GPER1), and progesterone receptor membrane component (PGRMC) 1 and 2 mediate E2 and P4 actions. This study aimed to characterize the local endocrine environment of the oviduct by examining the oviductal E2 and P4 concentrations and their receptors' mRNA expression during the periovulatory phase. The bovine oviducts were collected in a slaughterhouse and the days postovulation were estimated according to state of the ovaries and the uterus. Samples of the ampulla and isthmus ipsilateral and contralateral to the preovulatory follicle or corpus luteum were collected on Days 19 to 21, Days 0 to 1, Days 2 to 4, and Days 5 to 7 of the estrous cycle. The effects of the estrous cycle phase and oviductal region (ampulla and isthmus) and side (ipsilateral and contralateral) were analyzed by 3-way ANOVA. Moreover, to clarify the regulatory mechanisms of the mRNA expression of hormone receptors, the effects of E2 and P4 on mRNA expression in the oviduct were examined by multiple linear regression. The oviductal endocrine milieu on Days 19 to 21 was characterized by an E2-dominant environment with high E2 and low P4, high ESR1 and PGR mRNA expression, and low ESR2, GPER1, and PGRMC2 mRNA expression, whereas the corresponding on Days 0 to 1 was characterized by the endocrine milieu without hormone dominance. The environment on Days 2 to 4 and Day 5 to 7 was characterized by opposite tendency of oviductal hormone concentrations and their receptors' mRNA expression to Days 19 to 21. Additionally, the ipsilateral oviduct had the more P4-dominant endocrine milieu, with lower E2 and higher P4 concentrations, and different expression of ESR1/2, GPER1, PGR, and PGRMC2 mRNA when compared with the contralateral oviduct on Days 2 to 4 and Days 5 to 7, except for PGRMC1. Although oviductal E2 and P4 influenced the mRNA expression of ESR1/2, GPER1, PGR, and PGRMC1/2, their effects were different between regions and sides. In summary, the oviductal endocrine milieu varies according to the estrous cycle phase and the oviductal region and side, which may be involved in the estrous cycle phase-specific and oviductal region-specific and side-specific functions.

Impact of extracellular folic acid levels on oviductal gene expression

Folate plays a specific role as methyl donor for nucleotide synthesis and genomic methylation patterns, which in turn are important epigenetic determinants in gene expression. Previous studies have revealed the presence of folate in bovine oviductal fluid as well as the existence of a fine-tuned regulation of the gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). However, the functional implications of folate in the oviduct remain unknown. The present study aimed to assess the effect of folic acid (FA) on expression levels of selected genes that potentially respond to the folate status in in vitro BOECs. To obtain an insight into the optimization of a culture system for assays, gene expression of folate receptors and transporters was compared between BOECs grown in monolayers and in suspension. The results showed that BOECs from isthmus and ampulla in suspension culture better preserved the region-dependent gene expression profile than in monolayers. Subsequently, BOECs from both anatomical regions were separately cultured in suspension for 24 h assaying different FA concentrations: I) TCM-199 (control); II) TCM-199 + 1 μM FA (similar to the oviduct concentration); III) TCM-199 + 10 μM FA and IV) TCM-199 + 100 μM FA. Expression analysis of genes related to important cellular processes including folate transport, DNA methylation, cell-cell interaction, antioxidant activity and signaling pathways was performed in BOECs using RT-qPCR. Our data demonstrated that addition of 1 μM FA did not affect mRNA levels of most genes analyzed. In contrast, BOECs cultured with 10 μM FA exhibited increased mRNA expression levels of genes involved in folate intake, DNA methylation and antioxidant protection. It is worth noting that at 100 μM FA, transcriptional response in BOECs mainly resulted in decreased mRNA levels of the majority of the genes assayed. Interestingly, cytotoxicity analysis showed a similar LDH activity in the culture media of the experimental groups, indicating that cell integrity was not affected by the FA concentrations assayed. In conclusion, our findings suggest that folate can affect BOECs, promoting changes in gene activity in a framework of functional readjustments in response to environmental conditions.

Sperm‐Driven Micromotors Moving in Oviduct Fluid and Viscoelastic Media

Biohybrid micromotors propelled by motile cells are fascinating entities for autonomous biomedical operations on the microscale. Their operation under physiological conditions, including highly viscous environments, is an essential prerequisite to be translated to in vivo settings. In this work, a sperm-driven microswimmer, referred to as a spermbot, is demonstrated to operate in oviduct fluid in vitro. The viscoelastic properties of bovine oviduct fluid (BOF), one of the fluids that sperm cells encounter on their way to the oocyte, are first characterized using passive microrheology. This allows to design an artificial oviduct fluid to match the rheological properties of oviduct fluid for further experiments. Sperm motion is analyzed and it is confirmed that kinetic parameters match in real and artificial oviduct fluids, respectively. It is demonstrated that sperm cells can efficiently couple to magnetic microtubes and propel them forward in media of different viscosities and in BOF. The flagellar beat pattern of coupled as well as of free sperm cells is investigated, revealing an alteration on the regular flagellar beat, presenting an on-off behavior caused by the additional load of the microtube. Finally, a new microcap design is proposed to improve the overall performance of the spermbot in complex biofluids.

Involvement of activin signal pathway in cyclic apoptosis of the oviductal isthmic epithelium in cows

Activin (ACV) A induces various cellular functions via activin receptor type 2 (ACVR2A/2B)-activin receptor-like kinase (ALK) 4 -Smad 2/3 pathway. Although the production of ACVA is indicated in bovine oviducts, its role on the oviduct is unclear. Oviductal isthmus needs to change its function rapidly at peri-fertilization, however, the mechanism is unknown. This study was aimed to clarify the role of ACVA in the morphological changes of oviductal isthmus in cows. First, mRNA expressions of INHBA (ACVA component) and its receptors (ALK4, ACVR2A and ACVR2B) in the isthmic tissues were examined throughout the estrous cycle. INHBA was the highest, however, ACVR2A was the lowest on the day of ovulation, suggesting reduced ACV signal transduction in the isthmus just after ovulation. Proteins of ACVRs and Smad2/3 were clearly detected in the cultured epithelial cells. It is known that ACVA regulates cellular apoptosis. Our data showed that the number of cleaved caspase-3-positive epithelial cells was largest at 2–3 days after ovulation in the isthmus. Interestingly, our study demonstrated that follistatin (ACV/TGFB/BMP inhibitor) significantly decreased the BCL2/BAX ratio in the cultured isthmic epithelial cells. To clarify which ALK pathway is involved in the regulation of BCL2/BAX ratio, the effects of SB431542 (ACV signaling (ALK4) and TGFB signaling (ALK5) inhibitor), SB525334 (ALK5 inhibitor) and LDN193189 (BMP signaling (ALK2/3) inhibitor) were investigated in the next study. The results showed that only SB431542 significantly decreased BCL2/BAX and the others had no effects. These results suggest that decreased ACVA-ACVR2A-ALK4 signal at the post-ovulation induces cyclic apoptosis of isthmic epithelial cells in bovine oviducts.

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.