339 DIFFERENTIAL MECHANISM OF LEPTIN ACTION IMPROVING NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

Abstract

Leptin is a pleiotrophic peptide that has been implicated in the events associated with oocyte maturation and acquisition of developmental competence. Previous studies indicated that leptin supplementation during oocyte maturation has long-term effects, increasing blastocyst development and reducing the proportion of TUNEL-positive cells per blastocyst. Moreover, blastocysts derived from leptin-treated oocytes showed increased BIRC4 and reduced BAX expression (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). The objective of the current study was to determine the mechanism of leptin action during bovine oocyte maturation. In the first series of experiments cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0, 1, or 10 ng/mL leptin or 10% estrous cow serum (ECS) as positive control. Leptin reduced the proportion of cumulus cells undergoing cell death through apoptosis as determined by TUNEL staining (6.8 ± 0.4, 1.8 ± 0.4, 1.5 ± 0.4, and 6.3 ± 0.5% for 0, 1, or 10 ng/mL leptin or 10% ECS, respectively; P < 0.0001), but had no effect on the frequency of apoptotic oocytes. Nuclear maturation was also enhanced by leptin. The proportion of oocytes with extruded polar bodies (64.0 ± 2.9, 75.7 ± 2.9, 73.8 ± 2.9, and 63.3 ± 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) and the proportion of DAPI-stained metaphase II oocytes (72.9 ± 2.9, 90.5 ± 2.9, 85.8 ± 2.9, and 82.0 ± 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) were increased by 1 and 10 ng/mL leptin. There was no effect of ECS in any of the parameters examined. A second series of experiments tested whether the maturation-promoting activity of leptin was mediated by cumulus cells. Denuded oocytes (DO) and COCs were matured in serum-free medium containing 0 or 10 ng/mL leptin. The percentage of oocytes with extruded polar bodies (COCs: 59.1 ± 3.7% vs. 75.7 ± 3.7%, and DO: 45.5 ± 3.7% vs. 54.8 ± 3.7% for 0 or 10 ng/mL leptin; P < 0.01) was increased by leptin regardless of cumulus cells. Even though leptin did not affect cleavage rate, it increased blastocyst development. The proportion of COCs that developed to the blastocyst stage increased from 22.3 ± 4.6% in the control group to 35.2 ± 4.1% in the leptin-treated group. On the other hand, DO matured without cumulus cells did not acquire developmental competency, and this was not reversed by leptin supplementation (1.0 ± 5.0% vs. 1.0 ± 4.7% for 0 or 10 ng/mL leptin; P < 0.05; leptin × oocyte interaction P < 0.05). In conclusion, leptin enhanced oocyte maturation by acting directly in the oocyte and indirectly through cumulus cells. This suggests that leptin modulates the release of cumulus-derived factors secreted in the oocyte through gap junction coupling and in the extracellular environment.