The activity of three glycosidases (ß-Ν-acetyloglucosaminidase, α-mannosidase, and ß-galactosidase) in the follicular fluid and in the maturation medium affects bovine oocyte maturation.

We studied the role of follicular fluid's (FF) glycosidase (α-mannosidase [α-ΜΑΝ], ß-Ν-acetyloglucosaminidase [NAGASE], ß-galactosidase [ß-GAL]) activity during IVM of bovine oocytes. Oocytes were allocated into two groups according to the follicular size (small follicle [SF]: 2-5 mm, large follicle [LF]: >5-8 mm). In experiment 1, cumulus-oocyte complexes (COCs) quality was evaluated according to morphologic criteria (grades A, B-C, D); oocyte (n = 801) nuclear maturation was assessed after 24 hours of incubation. Bovine embryos were produced in vitro in groups (experiment 2, n = 1503 oocytes) or individually (experiment 3, n = 50 oocytes). More grade-A and -BC COCs were collected from SF and LF groups, respectively (P < 0.05). Maturation rate (experiment 1) and cleavage rate (experiments 2 and 3) were similar in SF and LF groups. Activity of all glycosidases in FF was higher (P < 0.05) in SF group than in LF group, whereas in maturation medium of SF group it was, overall, significantly lower than in that of LF (experiments 2 and 3). In FF of SF group, NAGASE positively associated with grade-A oocytes and negatively with BC oocytes; increased ß-GAL was associated with degenerated oocytes. Cleavage rate in LF group, related negatively to NAGASE and positively to α-MAN in maturation medium. These results indicate that during maturation, COCs release NAGASE and consume ß-GAL, but differences probably exist between individual and group maturation.

Effects of addition of tissue-type plasminogen activator in in vitro fertilization medium on bovine embryo development and quality.

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.