Activation of AMP-activated protein kinase may not be involved in AICAR- and metformin-mediated meiotic arrest in bovine denuded and cumulus-enclosed oocytes in vitro.

The adenosine monophosphate-activated protein kinase (AMPK) activators, 5'-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) and metformin (MET), inhibit resumption of meiosis in bovine cumulus-enclosed oocytes (CEO) and denuded oocytes (DO). The objectives of this study were to: (1) examine the effects of AMPK inhibitors on bovine oocyte meiosis in vitro; and (2) determine if AICAR or MET activates oocyte and/or cumulus cell AMPK. The AMPK inhibitor compound C (CC; 0.5, 1, 5, and 10 µM) did not reverse the inhibitory effects of AICAR (1 mM) and MET (2 mM) on bovine oocyte meiosis. Additionally, CC (5 and 10 µM) inhibited meiosis (p < 0.05) in CEO and DO cultured for 7 h. Okadaic acid (1 µM) reversed the inhibitory effect of MET (2 mM) and CC (5 µM; p < 0.05) but not of AICAR (1 mM). Phosphorylation of the alpha subunit of AMPK on Thr172 is required for activation. Based on western blot analysis, AICAR, MET and CC did not affect Thr172 phosphorylation levels in DO and oocytes from complexes (p > 0.05). In cumulus cells, Thr172 phosphorylation decreased after 3 h of culture (p < 0.05), regardless of the presence of AMPK modulators in the culture medium. Higher concentrations of AICAR (2 mM) and MET (10 mM) did not affect Thr172 phosphorylation, but phosphorylation on Ser79 of ACC, a substrate of AMPK, was increased in response to MET (p < 0.05). In conclusion, we inferred that the inhibitory effect of AICAR and MET on bovine oocyte meiosis was probably not mediated through activation of AMPK. Moreover, these compounds probably inhibited meiosis through different pathways.

Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate.

The objectives of this study were to compare different methods of evaluating sperm plasmalemma and to determine their relationship with in vitro fertilization rate. A single batch of frozen semen from each of eight beef bulls was used for assessment of sperm viability and for in vitro fertilization. Conventional viability tests included sperm morphology, motility, acrosome integrity, and abnormal DNA condensation. Methods for evaluation of the sperm plasmalemma included eosin/nigrosin (EN) and trypan-blue (TB) vital stains, propidium iodide (PI) in combination with carboxyfluorescein diacetate (CFDA) or SYBR-14 (SYBR) fluorescent vital stains, and the hypoosmotic swelling test (HOST). A total of 133-150 oocytes were fertilized in vitro with sperm from each bull and cleavage rates were determined. There were high correlations between the results obtained with vital stains and good to excellent interclass correlation coefficients of agreement, indicating that these stains provide measures of the same sperm attribute, i.e. plasmalemma integrity. However, the proportions of membrane-intact sperm identified by EN or TB stains were greater (P<0.0001) than identified by CFDA/PI or SYBR/PI fluorescent stains. The results obtained with the HOST had moderate correlations but poor agreement with the results of the vital stains. The proportion of viable sperm identified by the HOST was lower (P<0.05) than the proportion identified by vital stains, indicating that response to the HOST did not depend only on the integrity of the plasmalemma. Although there were significant differences in fertilization rates and sperm viability among bulls, there was no sharp distinction for the results of sperm viability tests from bulls producing different in vitro fertilization rates. Proportions of normal, motile, acrosome-intact, and HOST-responsive sperm were identified as significant predictors of in vitro fertilizing potential; each of these endpoints explained 12-18% of the variation when evaluated separately (linear regression) and 48% when evaluated collectively (stepwise regression). In conclusion, EN and TB stains overestimated the proportion of plasmalemma-intact sperm compared to PI-based fluorescent stains. Vital stains evaluated the morphological integrity of the plasmalemma, whereas the HOST assessed plasmalemma function. In that regard, the HOST was the only plasmalemma evaluation method that significantly contributed to conventional sperm quality tests in predicting in vitro fertilization rate, indicating that the test could be incorporated to the routine of semen analysis.