PLASMINOGEN ACTIVATOR ACTIVITY IN BUFFALO IN VITRO MATURED OOCYTES AFTER VITRIFICATION-WARMING

Plasminogen activators (PA) are proteolytic enzymes that convert plasminogen into plasmin. Plasmin is involved in physiological processes such as ovulation (Liu 2004 Front. Biosci. 9, 3356-3373), cumulus cell layer expansion (Liu et al. 1986 Endocrinology 119, 1578-1587), oocyte maturation (Dow et al. 2002 Biol. Reprod. 66, 1413-1421) and fertilization (Huarte et al. 1993 Dev. Biol. 157, 539-546). Although the interest is increasing, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of the present study was to evaluate whether exposure to cryoprotectants and the vitrification procedure affect plasminogen activator activity (PAA) in buffalo in vitro-matured oocytes. A total number of 300 cumulus-oocyte complexes over 5 replicates were selected and in vitro-matured. Cumulus-oocyte complexes were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups (20 oocytes/group, over 5 replicates). The control group consisted of fresh in vitro-matured oocytes. In the vitrification group, denuded oocytes were first exposed to 10% ethylene glycol (EG)+10% dimethyl sulfoxide (DMSO) for 3min, then to 20% EG+20% DMSO and 0.5M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25s. Subsequently, oocytes were warmed in a 1.25M sucrose solution for 1min and then in decreasing concentrations of sucrose (0.625M, 0.42M and 0.31M) for 30s each. In order to test cryoprotectant effects, oocytes were simply exposed to the vitrification and warming solutions (toxicity group). Surviving oocytes were extracted by a fine needle, centrifuged at 4000rpm for 10min and the supernatant was mixed with the reaction solution: TRIS-HCl 0.1M, homologous plasminogen, the chromogenic substrate for plasmin S-2251 and incubated at 37degreesC for 30min. The PAA levels were measured by a spectrophotometer (405nm) expressed as Abs/20 oocytes. The data were analysed by the Kruskal-Wallis nonparametric test. Low levels of PAA were detected in the denuded oocytes of the control, toxicity and vitrification groups. No significant differences in mean PAA values were observed among the 3 experimental groups (0.0170.001, 0.0180.002 and 0.0170.001 units, in the control, toxicity and vitrification groups, respectively). In conclusion, cryoprotectants and the vitrification procedure do not affect the proteolytic activity linked to plasmin in in vitro-matured buffalo oocytes. The results show that the vitrification/warming procedure does not exert an effect on in vitro-matured buffalo oocytes in terms of PAA generation, a parameter that plays an important role in fertilization and in vitro embryo development. Further studies are needed to identify factors affecting the efficiency of oocyte cryopreservation

Effect of crocin on antioxidant gene expression, fibrinolytic parameters, redox status and blood biochemistry in nicotinamide-streptozotocin-induced diabetic rats

Background: Diabetes is regarded as an epidemiological threat for the twenty-first century. Phytochemicals with known pharmaceutical properties have gained interest in the field of alleviating secondary complications of diseases. Such a substance is crocin, a basic constituent of saffron (Crocus sativus). The present study aimed at examining the beneficial effects of per os crocin administration on the antioxidant status, blood biochemical profile, hepatic gene expression and plasminogen activator inhibitor-1 activity (PAI-1) in the liver, kidney and plasma (an important marker of pre-diabetic status and major factor of thrombosis in diabetes) of healthy rats, as well as of rats with nicotinamide-streptozotocin-induced diabetes. Results: Diabetes disrupted the oxidation-antioxidation balance, while crocin improved the antioxidant state in the liver by significantly affecting SOD1 gene expression and/or by restoring SOD and total antioxidant capacity (TAC) levels. In the kidney, crocin improved hydrogen peroxide decomposing activity and TAC. In blood, hepatic transaminases ALT and AST decreased significantly, while there was a trend of decrease regarding blood urea nitrogen (BUN) levels. The expression of PAI-1 gene was affected in the liver by the dose of 50 mg kg-1. Conclusions: Crocin treatment contributed in restoring some parameters after diabetes induction, primarily by affecting significantly hepatic transaminases ALT and AST, SOD1 and PAI-1 gene expression and nephric H2O2 decomposing activity. In conclusion, crocin did contribute to the alleviation of some complications of diabetes. © 2020 The Author(s)