278 EFFECTS OF LEPTIN ON IN VITRO DEVELOPMENT AND GENE EXPRESSION IN PORCINE EMBRYOS

Abstract

Recent evidence suggests that leptin is an important signal in female reproduction, including control of ovarian function (Brann et al. 2002 Steroids 67, 95-104). Leptin receptor transcripts are presented in various cell types of humans, rats, and pigs. However, the results have been contradictory on the role of leptin in embryo development. The objective of this study was to determine the effects of different concentrations of leptin during porcine oocyte maturation on subsequent embryo development and expression of genes related to leptin signal transduction or apoptosis. Porcine oocytes were matured for 44 h in TCM-199 with the addition of leptin at 0 (control), 10, 50, 100, and 200 ng/mL (leptin 10, 50, 100, and 200 groups, respectively), and subsequently fertilized with frozen thawed semen (1 × 10⁵ sperm/mL) in mTBM. Presumptive zygotes were then cultured in NCSU-23 medium. Cleavage and blastocyst rates (in seven replicates) were assessed on Days 2 and 7 (the day of IVF was defined as Day 0), respectively. The total cell number in blastocysts was counted and the number of apoptotic cells was determined by TUNEL. Expression of genes encoding leptin receptor (LEPR), cytoplasmic transcription factor (STAT3), pro-apoptotic (Bax), and anti-apoptotic (Bcl2) regulators in blastocysts were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The percentage of MII oocytes (in four replicates) was significantly (P < 0.05) higher in 50 and 100 groups than in the others [80 (216/270) and 84% (236/280) vs. 70 (158/224), 74 (172/230), and 75% (186/248) in the 0, 10, and 200 groups, respectively]. Oocytes matured in the 50 and 100 groups showed a significantly (P < 0.05) greater proportion of cleaved embryos than those in the other groups [82 (368/448) and 84% (380/451) vs. 71 (339/476), 76 (358/470), and 74% (327/441) in 0, 10, and 200 groups, respectively]. Furthermore, the rate of blastocyst formation at Day 7 was significantly (P < 0.05) higher in the leptin 100 group than in the other groups [29% (131/451) vs. 18 (86/476), 23 (109/470), 20 (90/448), and 22% (98/441) in control and leptin 10, 50, and 200 groups, respectively]. Increased cell number and reduced proportion of apoptotic cells were observed in blastocysts originating from the oocytes matured in the presence of 100 ng/mL leptin. Depending on the concentrations used in the maturation medium, leptin increased LEPR, STAT3, and Bcl2 mRNA levels and reduced the Bax mRNA level in blastocysts. The results indicate that addition of leptin at 100 ng/mL during oocyte maturation improved embryonic development with up-regulation of LEPR, STAT3, and Bcl2 genes and suppression of the Bax gene, and thus optimizes the in vitro culture systems for porcine embryos.

This work was supported by Grant No. 1000520040020000 from Biogreen 21, Republic of Korea.