The urokinase plasminogen activator system components are regulated by vascular endothelial growth factor D in bovine oviduct.

SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.

Effect of exogenous transforming growth factor ß1 (TGF-ß1) on early bovine embryo development.

SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor ß1 (TGF-ß1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-ß1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-ß receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-ß1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-ß1 treated embryos. These results suggest an early action of exogenous TGF-ß1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-ß signalling during early embryogenesis.

Expression of bone morphogenetic protein receptors in bovine oviductal epithelial cells: Evidence of autocrine BMP signaling.

Members of the transforming growth factor beta (TGF-ß) family, including bone morphogenetic proteins (BMPs), are expressed in the epithelial cells of the mammalian oviduct. These signaling molecules play important roles in development and tissue homeostasis; however, little is known about their function in the mammalian oviduct. In the present study, RT-qPCR was used to analyze the mRNA abundance of BMP type I (BMPR1A, BMPR1B, ACVR1) and type II receptors (BMPR2, ACVR2A, ACVR2B) in the bovine oviduct epithelial cells (BOEC) isolated from ampulla and isthmus at both the follicular (FP) and the luteal (LP) phase of the estrous cycle. Results indicate that mRNAs for all the BMP receptors studied are expressed in the BOEC. Significant mRNA abundance differences were observed for both BMPR1B and ACVR2B when comparing both the ampulla and isthmus regions with the greater abundance at the isthmus. When both FP and LP samples were compared, ACVR2B mRNA showed greater abundance during the LP, with significant differences in the isthmus region. These variations highlight differences between the isthmus and ampulla regions of the oviduct. By means of wound healing assays on BOEC primary cultures, exogenous recombinant human BMP5 induced a significant increase in wound healing at 24h. The observed changes at the mRNA abundance of components of the signaling pathway and the BMP5 effect on oviductal epithelial cells suggest a possible autocrine role for the BMP pathway that could affect epithelial cell functions necessary for normal physiology and reproductive success in BOEC homeostasis.

Segment­ specific expression of MMP/TIMP in the oviduct of llama (Lama glama) and gelatinolytic activity in the oviductal fluid / Expresión segmento específica de MMP/TIMP en el oviducto de llama (Lama glama) y actividad gelatinolítica en el fluido oviductal

Oviductal molecules have the potential to improve the reproductive biotechnologies. In camelids, knowledge and assessment of the oviductal environment are necessary to successfully develop species-specific reproductive technologies, especially because of the camelids reproductive particularities. Among the oviductal factors, the matrix metalloproteinases/tissue inhibitor of matrix metalloproteinases system (MMPs/TIMPs) should be investigated more thoroughly due to their participation in reproductive processes. Consequently, the current study assayed gene and protein expression of MMPs throughout the llama oviduct. MMPs zymogen and active forms in the oviductal fluid were also characterized. MMP2 and MMP9 transcripts were detected in ampulla, isthmus, utero-tubal junction and papilla, being MMP2 and MMP9 2.15 and 1.10 folds higher in papilla than in ampulla, respectively. In addition, differences in immunolocalization of MMP2 and MMP9 between the epithelial mucosa layers of the oviductal segments were observed. The presence of MMPs in the epithelium suggests their secretion into the oviductal lumen. Coincidently, bands of 62 and 94 kDa, corresponding to MMP2 and MMP9 were detected by zymography in the oviductal fluid. Treatment with an exogenous activator (APMA) suggests that they are present as proMMPs. TIMP2 and TIMP1, the specific inhibitors of MMP2 and MMP9, respectively, were expressed in each oviductal segment, indicating a well-regulated control of MMP proteolytic activity in the oviduct. These findings prove that the llama oviduct produces and secretes MMPs into the oviductal lumen, suggesting that these enzymes may have an unknown role in the preparation of the oviductal environment for gametes, fertilization and early embryo development in camelids.

Las moléculas oviductales tienen el potencial para mejorar las biotecnologías reproductivas. En los camélidos, debido a sus peculiares características reproductivas, el conocimiento del ambiente oviductal constituye una herramienta útil para el desarrollo de tecnologías reproductivas específicas para estas especies. Entre los factores oviductales de interés se encuentran las metaloproteasas de matriz (MMPs) y sus inhibidores específicos (TIMPs), los cuales han sido involucrados en diferentes procesos reproductivos. Por estas razones, en este trabajo se caracterizó la expresión génica y proteica de MMP2 y MMP9 en el oviducto de llama. Además, se analizó la presencia de las formas activas e inactivas (zimógenos) de estas enzimas en el fluido oviductal. Se observó que todos los segmentos oviductales, ámpula, istmo, unión útero-tubal y papila, expresan MMP2 y MMP9, siendo los niveles de expresión de MMP2 y MMP9 más elevados en papila respecto a ámpula; 2,15 y 1,10 veces respectivamente. Asimismo, se observaron diferencias en la distribución de las MMPs a nivel de la mucosa entre los segmentos oviductales. Consecuentemente, bandas con actividad gelatinolítica de 62 y 94 kDa, se detectaron en el fluido oviductal, las cuales corresponderían a las formas inactivas de la MMP2 y la MMP9, respectivamente. Los inhibidores específicos de MMP2 y MMP9; TIMP2 y TIMP1, también se detectaron en los segmentos oviductales, indicando su probable participación en la regulación de la actividad proteolítica de las MMPs en el oviducto de llama. En conjunto, los datos de este trabajo demuestran que el oviducto de la llama produce y secreta MMPs al lumen oviductal; sugiriendo que estas enzimas pueden participar en la preparación del ambiente oviductal para la recepción de los gametos, la fecundación y el desarrollo embrionario temprano en camélidos.

Genistein affects proliferation and migration of bovine oviductal epithelial cells.

Genistein is one of the most abundant isoflavones in soybean. This molecule induces cell cycle arrest and apoptosis in different normal and cancer cells. Genistein has been of considerable interest due to its adverse effects on bovine reproduction, altering estrous cycle, implantation and fetal development and producing subfertility or infertility. The objective of this work was to study the effects of genistein on the expression of selected genes involved in the regulation of cell cycle and apoptosis. Primary cultures of bovine oviductal epithelial cells (BOEC) were treated with different genistein concentrations (0.2, 2 and 10µM) to analyze CYCLIN B1, BCL-2 and BAX gene expression by Real-time RT-PCR. Results showed that genistein down-regulated CYCLIN B1 expression, affecting cell cycle progression, and caused a decrease in the BCL-2/BAX ratio starting at 2µM of genistein. In addition, in order to determine if genistein affects BOEC migration, in vitro wound healing assays were performed. A significant reduction in cell migration after 12h of culture was observed at both 0.2 and 10µM genistein concentrations. Also, in the presence of genistein the percentage of mitotic cells decreased, although apoptotic cells percentages were not affected. These findings indicate that genistein has an inhibitory effect on BOEC proliferation and migration, suggesting that it could influence the normal physiology of the oviductal epithelium.

Expression and localization of urokinase-type plasminogen activator receptor in bovine cumulus-oocyte complexes.

Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the blastocyst stage in the BMP5 group. Moreover, reverse transcription quantitative real-time polymerase chain reaction analysis showed a significant increase in the relative abundance of SOX2 in two-cell stage embryos, ID2 and OCT4 in eight-cell stage embryos, and NANOG and OCT4 in blastocysts derived from BMP5-treated embryos. In conclusion, our results report that early addition of BMP5 to the embryo culture medium had a positive effect on the blastocyst rate and affected the relative expression of BMP target and pluripotency genes, suggesting that BMP5 could play an important role in the preimplantation development of bovine embryos.

Bone morphogenetic proteins in the bovine oviduct: differential expression of BMP-5 in the isthmus during the estrous cycle.

Bone morphogenetic proteins (BMPs) play a crucial role in mammalian reproduction, but little is known about their expression and function in the oviduct, where preimplantation events take place. In the present study, messenger RNA (mRNA) expression of BMPs was examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in bovine oviduct epithelial cells obtained from ampulla and isthmus at different stages of the estrous cycle. Expression of BMP-2, -3, -4, -7, -10 and -15 mRNA was detected in epithelial cells of both anatomic regions, whereas BMP-5 mRNA was specifically expressed in isthmus epithelial cells throughout the estrous cycle. High expression levels for BMP-5 and for BMP-2, -4, and -7 mRNA were observed during the preovulatory stage. Considering the region-specific gene expression of BMP-5, its protein localization in the oviduct and its presence in the oviductal fluid were evaluated by immunohistochemistry and Western blot analysis. BMP-5 protein staining was observed in isthmus sections with a more intense signal in the luminal epithelial cell layer. In addition, a 21 kDa protein corresponding to the BMP-5 mature monomeric form was detected in bovine oviductal fluid throughout the estrous cycle. In conclusion, these results demonstrate that different members of the BMP family are expressed in the bovine oviduct during the estrous cycle, and reveal that BMP-5 is differentially expressed in the isthmus. The expression of this factor in the oviduct epithelium and its presence in the luminal fluid suggest a possible action of BMP-5 as a new autocrine and/or paracrine regulator of the reproductive events that occur in the bovine oviductal environment.

Expression and localization of nodal in bovine oviduct and uterus during different functional stages of oestrus cycle and pregnancy.

Members of TGF-ß superfamily play a major role in the endometrial changes involved in the establishment and maintenance of pregnancy. Their deregulated expression and action could lead to absolute or partial failure of embryo implantation. Nonetheless, the precise function and mechanism of many of these cytokines remain unclear. Nodal, a transforming growth factor beta (TGF-ß) superfamily member, was characterized in the human and rodent uterus and implicated in the tissue remodeling events during menstruation and embryo implantation. In order to study its possible role in the cattle reproductive process, we have analyzed Nodal expression pattern and localization in the oviduct and uterine horn during the oestrus cycle and early pregnancy (day 20). Nodal was detected both in oviduct and uterus during either the oestrus cycle or pregnancy; however, it shows a differential expression profile in the uterine horn at dioestrus and pregnancy, decreasing 1.5 and 1.4 folds in comparison with oestrus. Nodal immunostaining intensity was observed in stromal and in epithelial cells of the surface and the glandular epithelium. The staining pattern correlates with the RT-qPCR expression profile. This work is the first to evidence the presence of Nodal in the bovine reproductive tract; our data suggest that Nodal is a novel cytokine that would be involved in the remodelling occurring in the endometrium of cattle during the oestrus cycle and in the embryo implantation. The identification of new molecules that participate in endometrium cycling and/or pregnancy may be useful for predicting the ability of the uterine tissue to establish and maintain pregnancy or for detecting the infertility processes. These results highlight Nodal as a possible novel marker of the fertility process, nevertheless further studies should be done to determine its role in the reproductive system.

In vivo and in vitro expression of the plasminogen activators and urokinase type plasminogen activator receptor (u-PAR) in the pig oviduct.

Plasminogen activator activities have previously been reported in oviductal fluid. At present the question was whether the source of these activities is molecules come from blood plasma or if these activators are synthesized by the oviduct. Gene expression and protein synthesis of urokinase type (u-PA) and tissue type (t-PA) occur in different regions of the pig oviduct. Their relative concentrations do not vary between the ampulla and isthmus regions and are similar throughout the estrous cycle. However, while relative amounts of t-PA mRNA were not different between the different stages of the estrous cycle, u-PA mRNA was greater after ovulation (P<0.05). Regarding the function of u-PA, its receptor (u-PAR) was distinguished by immunohistochemistry at the apical region of the epithelial cells and was more noticeable in the isthmus. Expression of u-PA, t-PA, u-PAR and PAI-1 genes in primary oviductal epithelial cell cultures was studied under 17-ß-estradiol (100 pg/ml) and progesterone (100 ng/ml). u-PA mRNA increased in the presence of progesterone (P<0.05), but not by action of 17-ß-estradiol. t-PA, PAI-1 and u-PAR were similar when cultured with the hormones. These results suggest that u-PA could be regulated by progesterone at a transcriptional level, by the balance of their activity for PAI-1 or at the epithelial surface through the binding of u-PAR. In conclusion, plasminogen activation system components might cooperate in the oviductal lumen to control plasmin generation.

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction.

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.

LEFTY2 expression and localization in rat oviduct during early pregnancy.

In mammals, fertilization and preimplantation embryo development occurs in the oviduct. Cross-talk between the developing embryos and the maternal reproductive tract has been described in such a way as to show that the embryos modulate the physiology and gene expression of the oviduct. Different studies have indicated that transforming growth factor beta (TGF-ß) can modulate the oviductal microenvironment and act as an autocrine/paracrine factor on embryo development. LEFTY2, a novel member of the TGF-ß superfamily is involved in the negative regulation of other cytokines in this family such as nodal, activin, BMPs, TGF-ß1 and Vg1. In previous studies, we have reported that LEFTY2 is differentially expressed in the rat oviduct during pregnancy. In this study, we describe the temporal pattern of LEFTY2 in pregnant and non-pregnant rat oviduct by western blotting, which showed higher levels of LEFTY2 on day 4 of pregnancy, a time at which the embryos are ending their journey along the oviduct. The cellular location of LEFTY2 was assessed by immunohistochemistry, which showed immunolabelling in the cytoplasm and at the apical surface of the oviductal epithelial cells. The oviductal fluid also presented a 26 kDa band, which corresponds to the biologically active form of this protein, at the preimplantation period of pregnancy, indicating LEFTY2 secretion to the lumen. As LEFTY2 is expressed at a high level just before the embryos pass to the uterus, its biological effect might be relevant and significant for the preimplantation stage of embryo development in the oviduct. The fact that embryos do not express LEFTY2 at this stage of development supports this hypothesis.

Cloning and sequence analysis of Bufo arenarum oviductin cDNA and detection of its orthologous gene expression in the mouse female reproductive tract.

The glycoprotein envelope surrounding the Bufo arenarum egg exists in different functional forms. Conversion between types involves proteolysis of specific envelope glycoproteins. When the egg is released from the ovary, the envelope cannot be penetrated by sperm. Conversion to a penetrable state occurs during passage through the pars recta portion of the oviduct, where oviductin, a serine protease with trypsin-like substrate specificity, hydrolyzes two kinds of envelope glycoproteins: gp84 and gp55. The nucleotide sequence of a 3203 bp B. arenarum oviductin cDNA was obtained. Deduced amino acid sequence showed a complete open reading frame encoding 980 amino acids. B. arenarum oviductin is a multi-domain protein with a protease domain at the N-terminal region followed by two CUB domains and toward the C-terminal region another protease domain, which lacked an active histidine site, and one CUB domain. Expression of ovochymase 2, the mammalian orthologous of amphibian oviductin, was assayed in mouse female reproductive tract. Ovochymase 2 mRNA was unnoticeable in the mouse oviduct but expression was remarkable in the uterus. Phylogenetic relationship between oviductin and ovochymase 2 opens the possibility to understand the role of this enzyme in mammalian reproduction.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

Rat Lefty2/EBAF gene: isolation and expression analysis in the oviduct during the early pregnancy stage.

It has been demonstrated, by RNA Arbitrarily Primed Polymerase Chain Reaction (RAP-PCR), that the endometrial bleeding associated factor (ebaf or lefty2) is expressed in rat oviduct. In this work we isolated and sequenced the full-length lefty2 cDNA from Rattus norvegicus oviducts and described its expression level in this organ during the estrous cycle and early pregnancy stage. The coding deduced sequence (CDS) codifies a 40.91 kDa protein with a highly conserved TGF-beta functional domain. RT-PCR semiquantitative analysis indicated that oviduct cells transcribe lefty2 among different stages of the estrous cycle with the maximum expression at diestrus phase. The highest expression of lefty2 was at the 4(th) day after mating (five folds respect to day one), just when the embryos have completed their transit through the oviduct. The lefty2 expression declined rapidly thereafter and the levels of their transcripts in the oviduct remained low until 7(th) days after mating.

Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs.

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.

Plasminogen detection in oocytes and plasminogen activator activities in the porcine oviduct during the estrous cycle.

Plasminogen activators (PAs) are highly specific serine proteases that convert the extracellular zymogen plasminogen into the active proteinase plasmin. Plasminogen-dependent proteolytic activity was detected by zymography both in the tissue membrane fraction of oviducts and in the oviductal flushing obtained at the preovulatory (Pre-Ov), postovulatory (Post-Ov) and mid-luteal (Mid-L) stages of the estrous cycle. A main proteolytic band, with a relative mobility similar to a human melanoma cell tissue-type plasminogen activator (t-PA), was found in all samples. Two additional components were observed in Pre-Ov and Post-Ov oviductal flushing but not in the tissue membrane fraction. In the oviductal flushing the PA activity was significantly higher in the Post-Ov stage than in the Pre-Ov one. Both urokinase-type plasminogen activator (u-PA, 50 kDa) and t-PA (72 kDa) were detected by Western blot; they showed differences in their relative concentration between Post-Ov and Pre-Ov oviductal flushing. The main PA substrate, plasminogen, was detected by indirect immunofluorescence in the cumulus cell extracellular matrix (ECM) and oocyte zona pellucida (ZP). In denuded oocytes, plasminogen was also detected on the surface of the plasma membrane. It is possible that oviductal PAs may act on the plasminogen present in the cumulus cell ECM and ZP; consequently, the generated plasmin could be involved in the rebuilding or degradation of these oocyte structures during fertilization or early development.

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100 per cent identical to a rat DNA sequence (Accession No. NW_047400)downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100 per cent identical to a rat DNA sequence (Accession No. NW_047400)downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process. (AU)

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct.

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100% identical to a rat DNA sequence (Accession No. NW_047400) downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.