299 LEPTIN ACCELERATES PRONUCLEAR FORMATION FOLLOWING INTRACYTOPLASMIC SPERM INJECTION OF PORCINE OOCYTES: POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE INACTIVATION

Abstract

Although evidence suggests that leptin modulates oocyte maturation in vitro, few studies have examined the direct effect of leptin on cytoplasmic maturation and pronuclear formation following fertilization of porcine oocytes.The aim of this studywas to determine microtubule and microfilament assembly following oocyte maturation, and to assess blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) of leptin-treated oocytes.A symmetrical shape of the meiotic spindle was considered to be the normal microtubule assembly, and a clear, thick microfilament area containing a second metaphase chromatin was considered to be morphologically normal. Significant differences were determined using Tukey's multiple range test with P < 0.05 being considered significant. Addition of 10 ng mL^-1 of leptin to the in vitro maturation (IVM) medium significantly increased the percentage of zygotes that developed to the blastocyst stage (26.1 ± 2.1%; P < 0.05) compared with that of the control (0 ng mL^-1: 16.3 ± 2.8%) or 1 ng mL^-1 (18.6 ± 2.6%) leptin treatment groups following parthenogenetic activation. After IVM for 44 h, the percentage of oocytes with normal microtubules was higher in the leptin (10 ng mL^-1) group than in the control group (89.1 ± 4.3% vs. 78.5 ± 4.0%, respectively; P < 0.05). However, the percentage of normal microfilament assembly was similar in the leptin-treated and control groups (85.6 ± 4.3% and 82.9 ± 5.0%, respectively). In oocytes matured in vitro in the presence of 10 ng mL^-1 of leptin and subsequently induced by parthenogenetic activation via chemical stimulation, there was a significant increase in the formation of 2 pronuclei (2PN: 52.1 ± 5.2%; P < 0.05) at 6 h, compared with the control non-leptin-treated oocytes (40.7 ± 4.0%). Similarly, addition of 10 ng mL^-1 of leptin to the IVM medium also increased 2PN formation at the same time point following ICSI (leptin treatment: 47.9 ± 4.0; control: 36.2 ± 3.8%; P < 0.05). The addition of 10 ng mL^-1 of leptin to the IVM medium decreased the level of phospho-ERK1/2 at 6 and 9 h and was lower in the leptin-treated group compared with the control non-leptin-treated group (P < 0.01). These results suggest that exogenous leptin enhances cytoplasmic maturation and pronuclear formation following fertilization, possibly via the MAPK pathway.