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.

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.

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.

Effects of cafeteria diet on the jejunum in sedentary and physically trained rats.

OBJECTIVE: The effects of a cafeteria diet on the small intestine were investigated in adult Wistar rats under sedentary conditions and after physical training. METHODS: Parameters including morphometry, enzyme activities, and total myenteric populations in the jejunum were evaluated. RESULTS: The cafeteria diet, characterized as hyperlipidic, produced obese rats, corroborated by increases in the Lee index and the weights of the periepididymal and retroperitoneal adipose tissues (P<0.01). Obesity caused increases in the length of the small intestine, villi height, crypt depth, whole-wall thickness (P<0.05), and the enzymatic activities of alkaline phosphatase, lipase, and sucrase (P<0.01), in addition to a reduction in the number of goblet cells (P<0.05). With reference to the jejunal intrinsic innervations, the total number and area of myenteric neurons was unchanged regardless of the group. Physical training promoted 1) a reduction of the weight in the retroperitoneal and periepididymal adipose tissues (P<0.05) and 2) an increase in the thickness of the muscular layer (P<0.05). CONCLUSION: The cafeteria diet promoted obesity in rodents, leading to alterations in morphometry and enzymatic intestinal parameters, which were partily attenuated by physical training.

Vitamin E (alpha-tocopherol) supplementation in diabetic rats: effects on the proximal colon.

BACKGROUND: Neuropathy is one of the complications caused by diabetes mellitus which is directly related to the gastrointestinal manifestations of the disease. Antioxidant substances, such as vitamin E, may play an important role in the reduction of the neurological damage caused by diabetes mellitus. The aim of the present study was to determine whether vitamin E (alpha-tocopherol) at different concentrations induces any effects on the morphology of the intestinal wall and intrinsic innervation in the proximal colon of diabetic rats. METHODS: Thirty rats (90-day-old) were assigned to the following groups: N (normoglycemic), NE1 (normoglycemic supplemented with vitamin E 0.1%), NE2 (normoglycemic supplemented with vitamin E 2%), D (diabetic), DE1 (diabetic supplemented with vitamin E 0.1%), and DE2 (diabetic supplemented with vitamin E 2%). Animals received vitamin E supplementation for 120 days and were sacrificed when they were 210 days old. The proximal colon of each animal was subjected to histology to study the intestinal wall and goblet cells and processed for whole-mount preparations to morphoquantitatively determine the total myenteric population. RESULTS: Supplementation with vitamin E significantly reduced glycemia and glycated hemoglobin values and preserved the number of myenteric neurons in group DE2, without affecting intestinal area or thickness of the intestinal wall or muscular tunic. CONCLUSION: Vitamin E (2%) influenced the glycemic parameters and had a neuroprotective effect on the total myenteric population, but the morphometric characteristics of the intestinal wall were unaffected.