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.

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.

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.

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.

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.

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 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% 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

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.