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We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed

Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Further work showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.

Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine differentially expressed genes (DEGs) (P value ≤0.05 and absolute fold change ≥2) and to provide functional interpretation. A total of 10 743 transcripts were identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Enriched functional categories were detected only in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response-related genes in the oviduct, with marked upregulation of interferon-associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse.

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHVA59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 10 5 /ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 10 7 mean tissue culture infective dose (TCID50)/ml of MHV-A59 and 10 2 TCID 50 /ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer. assisted reproductive technology, embryo transfer, health monitoring, mouse, mouse hepatitis virus, mouse minute virus

Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1–5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LC...

Co-incubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that, while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes, but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 d of exposure to 2.5 mM procaine, irrespective of sperm presence. However, the cleaved oocytes did not develop beyond 8-16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intra-cytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining, that was not observed after other treatments. This alkalinization was followed, after an additional 18 h incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8-16 cell stage.

Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage.

A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes 1 Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium 1 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of ,20 000 mm2 provided more consistent results than did explants with a surface area of .100 000 mm2 . A positive association was found between the loge transformed number of spermatozoa bound to 0.1 mm2 oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was .60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca 2+ ,a nd albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 3 10 6 spermatozoa/ml), during which it transpired that the highest density (per mm 2 ) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a timedependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviductbound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pH i ) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pH i , presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pH i changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during which it transpired that the highest density (per mm(2)) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.

The aim of the present study was to determine whether or not cytopathic (CP) and noncytopathic (NCP) bovine viral diarrhea virus (BVDV) are able to replicate within in vitro-produced embryos and to investigate whether inoculation of embryos with BVDV affects their normal development. Zona pellucida (ZP)-free oocytes, zygotes, 8-cell-stage embryos, morulae, and hatched blastocysts (HB) were incubated for 1 h in 1 ml of Minimal Essential Medium containing 106.00 tissue culture infectious dose (TCID) 50/ml NCP BVDV isolate 22146 or 106.25 TCID5dml CP BVDV strain Oregon C24V. At 0, 12, 24, 36, 48, 60, and 72 h postinoculation (hpi), groups of embryos were collected for virus titration. A small amount of newly produced virus was detected in 8-cell embryos at 60 hpi (10' -8 TCIDso/100 cells), but only for CP BVDV. For ZP-free morulae and HB, maximal intracellular virus titers were, respectively, 101-'47 and 102.33 TCIDd/100 cells at 48 hpi for the CP biotype and 100.64 and 100.84 TCIDso/100 cells at 72 hpi for the NCP biotype. Only an infection with CP BVDV had a significant inhibitory effect on further development of ZP-free morulae. It can be concluded that ZP-free in vitro-produced embryos are permissive to an infection with BVDV, with increasing susceptibility of the embryos in accordance with their developmental stage. In contrast to observations in ZP-free in vitro-produced embryos, no virus replication or signs of embryonic degeneration were detected in ZPintact in vitro-derived embryos.

Preimplantation development in the bovine embryo was examined by relating the occurrence of three morphogenetic processes (compaction, blastulation, and hatching) to the timing of allocation of embryonic cells to the inner cell mass (ICM) or to the trophectoderm (TE). Embryos were collected from 26 cows between Days 4 and 9 postovulation. Compaction started 5 days postovulation at the 32-cell stage. Morulae remained firmly compact until the seventh cell cycle was almost completed. Blastocyst formation started between the 64- and 128-cell stage at Days 6, 7, and 8 postovulation. Hatching was predominant at Day 9 postovulation. ICM and TE cells could successfully be distinguished by differential staining in 107 of 142 embryos (75%). Inner cells could first be detected in 20% of 16-cell embryos. Unexpectedly, it was found that inner cell allocation and compaction were independent processes, since 31% of compacted morulae displayed no ICM. Beyond the 50-cell stage, in vivo compact morulae displayed at least 10 ICM cells, whereas blastocysts with a minimum total cell number of 65 cells displayed at least 23 ICM cells. It can be concluded that the slow in vivo transition from the morula to the blastocyst stage allows sufficient time for allocation of inner cells to the ICM of the embryo.

In the present study, the in vitro interaction of embryos with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by viral antigen detection and by evaluating the expression of virus receptors, namely, poliovirus receptor-related 1 (PVRL1; formerly known as nectin 1) for PRV and sialoadhesin for PRRSV. Embryonic cells of zona pellucida intact embryos incubated with PRV remained negative for viral antigens. Also, no antigen-positive cells could be detected after PRV incubation of protease-treated embryos, since the protease disrupted the expression of PRVL1. However, starting from the five-cell-stage onwards, viral antigen-positive cells were detected after subzonal microinjection of PRV. At this stage, the first foci of PVRL1, also a known cell adhesion molecule, were expressed. At the expanded blastocyst stage, a lining pattern of PVRL1 in the apicolateral border of trophectoderm cells was present, whereas the expression in the inner cell mass was low. Furthermore, PVRL1-specific monoclonal antibody CK41 significantly blocked PRV infection of trophectoderm cells of hatched blastocysts, while the infection of the inner cell mass was only partly inhibited. Viral antigen-positive cells were never detected after PRRSV exposure of preimplantation embryos up to the hatched blastocyst stage. Also, expression of sialoadhesin in these embryonic stages was not detected. We conclude that the use of protease to investigate the virus embryo interaction can lead to misinterpretation of results. Results also show that blastomeres of five-cell embryos up to the hatched blastocysts can become infected with PRV, but there is no risk of a PRRSV infection. embryo, poliovirus receptor-related 1, porcine reproductive and respiratory syndrome virus, pseudorabies virus, receptor, sialoadhesin

A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of20 000 micro m(2) provided more consistent results than did explants with a surface area of100 000 micro m(2). A positive association was found between the log(e) transformed number of spermatozoa bound to 0.1 mm(2) oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

The aim of the present study was to determine whether or not cytopathic (CP) and noncytopathic (NCP) bovine viral diarrhea virus (BVDV) are able to replicate within in vitro-produced embryos and to investigate whether inoculation of embryos with BVDV affects their normal development. Zona pellucida (ZP)-free oocytes, zygotes, 8-cell-stage embryos, morulae, and hatched blastocysts (HB) were incubated for 1 h in 1 ml of Minimal Essential Medium containing 10(6.00) tissue culture infectious dose (TCID)50/ml NCP BVDV isolate 22,146 or 10(6.25) TCID50/ml CP BVDV strain Oregon C24V. At 0, 12, 24, 36, 48, 60, and 72 h postinoculation (hpi), groups of embryos were collected for virus titration. A small amount of newly produced virus was detected in 8-cell embryos at 60 hpi (10(1.8) TCID50/100 cells), but only for CP BVDV. For ZP-free morulae and HB, maximal intracellular virus titers were, respectively, 10(1.47) and 10(2.33) TCID50/100 cells at 48 hpi for the CP biotype and 10(0.64) and 10(0.84) TCID50/100 cells at 72 hpi for the NCP biotype. Only an infection with CP BVDV had a significant inhibitory effect on further development of ZP-free morulae. It can be concluded that ZP-free in vitro-produced embryos are permissive to an infection with BVDV, with increasing susceptibility of the embryos in accordance with their developmental stage. In contrast to observations in ZP-free in vitro-produced embryos, no virus replication or signs of embryonic degeneration were detected in ZP-intact in vitro-derived embryos.