Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus-oocyte complexes: A biological and spectroscopic approach.

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ɛ-aminocaproic acid (ɛ-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ɛ-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.

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 28 days 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.

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 urokinase type plasminogen activator on in vitro bovine oocyte maturation.

This study examines the impacts of the urokinase-type plasminogen activator (uPA) on the in vitro maturation (IVM) of bovine oocytes. Cumulus-oocyte complexes in IVM medium were treated with uPA, amiloride (an uPA inhibitor), dimethyl sulfoxide (DMSO) or left untreated (control group). After 24 h of IVM, oocytes were recovered for testing or were in vitro fertilized and cultured to the blastocyst stage. The factors examined in all groups were: (i) oocyte nuclear maturation (Hoëscht staining); (ii) oocyte cytoplasmic maturation (cortical granules, CGs, distribution assessed by LCA-FITC); (iii) oocyte and cumulus cell (CC) gene expression (RT-qPCR); and (iv) embryo development (cleavage rate and blastocyst yield). Oocytes subjected to uPA treatment showed rates of nuclear maturation and CG distribution patterns similar to controls (P > 0.05), whereas lower rates of oocyte maturation were recorded in the amiloride group (P < 0.05). Both in oocytes and CC, treatment with uPA did not affect the transcription of genes related to apoptosis, cell junctions, cell cycle or serpin protease inhibitors. In contrast, amiloride altered the expression of genes associated with cell junctions, cell cycle, oxidative stress and CC serpins. No differences were observed between the control and uPA group in cleavage rate or in blastocyst yield recorded on Days 7, 8 or 9 post-insemination. However, amiloride led to drastically reduced cleavage rate (28.5% vs 83.2%) and Day 9 embryo production (6.0% vs 21.0%) over the rates recorded for DMSO. These results indicate that the proteolytic activity of uPA is needed for successful oocyte maturation in bovine.

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.

Plasminogen activation and plasmin inhibition during in vitro fertilization in bovine: implications for fertilization parameters and early embryo development.

Components of the plasminogen/plasmin system, known to be present in the oocyte, play a key role in maturation and fertilization. The objective of this study was to examine the effect of plasminogen activation and plasmin inhibition by exogenous supplementation of the IVF medium with streptokinase (SK) or ɛ-aminocaproic acid (ε-ACA), respectively, on fertilization parameters and preimplantation embryo development. After in vitro maturation, bovine cumulus-oocyte complexes (COCs) were inseminated in the presence of SK or ε-ACA. The addition of SK to the IVF medium facilitated the adhesion of the spermatozoa to the zona pellucida without affecting the percentages of monospermy. Cleavage rates and blastocyst yield were similar between the SK and Control groups while they were lower with the ε-ACA treatment. Additionally, we found that the expression levels of embryo quality-related genes (SDHA and DNMT3A) could be modified in blastocysts by the addition of SK or ε-ACA during IVF. The results obtained indicate that supplementation of the IVF medium with SK did not greatly alter the embryonic developmental parameters related to embryo quality in blastocysts. Moreover, we noticed that ε-ACA treatment compromises the success of in vitro embryo development, thus highlighting the importance of the plasminogen/plasmin activity during the early stages of embryogenesis in bovine.

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

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

Biochemical changes in the cytoplasm of bovine oocytes during the in vitro maturation process: a Raman microscopy study.

The sequence and chronology of the main biochemical changes occurring in the cytoplasm of bovine oocytes during the in vitro maturation process were tracked by Raman microscopy applied to cells previously subjected to enzymatic digestion of the zona pellucida. Specific spectral markers for proteins, lipids and carbohydrates were used to evaluate the developmental status of the ooplasm at four different times. Spectral changes revealed that lipid accumulation was dominant during the first six hours of culture while protein content reached the average levels characteristic of mature oocytes within the last four hours of the maturation process. A time-dependent decrease in carbohydrates was also observed. Finally, the carbohydrate-to-protein (P1037/P1002) ratio proved to be sensitive enough to determine the cytoplasmic maturation state of bovine oocytes and promises to be useful in future research aimed at optimizing culture conditions through the promotion of protein accumulation in the ooplasm.

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 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.

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.

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.