113 THE DISTRIBUTION OF THE LEPTIN PROTEIN WITHIN BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS MATURED AND FERTILIZED IN VITRO

Abstract

It has recently been documented that leptin regulates processes linked to reproduction including preimplantation development, embryo implantation (trophoblast invasion), and fetal growth. Transcripts for the leptin gene (LEP) and the leptin receptor gene (LEPR) have been identified in ovary, testis, placenta, endometrium, ovarian follicles, and oocytes, and also in mouse, rat, human, and bovine pre-implantation embryos. Moreover, the leptin protein was detected in mouse and human oocytes and embryos, and its localization was polarized. The distribution of regulatory proteins within oocytes and pre-implantation embryos is critical for early mammalian development, such as determination of the animal pole and the establishment of the trophoblast and the inner cell mass cells (ICM). So far there is no published evidence concerning this phenomenon in bovine oocytes and embryos. Therefore, the aim of this work was to analyze the leptin protein distribution within bovine oocytes and preimplantation embryos matured and fertilized (in vitro). The material for this work consisted of oocytes collected from slaughterhouse ovaries and sperm collected from AI bulls. In vitro oocyte maturation and fertilization were carried out according to the method described by Makarevich and Markkula (2002 Biol. Reprod. 66, 386–392). The preliminary experiment of leptin protein localization by immunofluorescent staining included immature and matured oocytes and blastocysts. Oocytes and embryos were fixed in PBS containing 4% paraformaldehyde and reacted with affinity-purified polyclonal rabbit primary antibody directed against leptin (0.1 mg/mL; Ob (Y20), Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); and then exposed to secondary goat-anti-rabbit antibody (1.0 mg/mL; Santa Cruz Biotechnology Inc.)-fluorescein isothiocyanate (FITC) conjugate. Finally, chromatin was visualized by propidium iodide staining (0.5 μg/mL). Slides were examined under a conventional fluorescence microscope (Nikon) and confocal microscope (Zeiss). The preliminary results demonstrate that the distribution of leptin differed between immature and mature oocytes: it was spherical in immature oocytes (a rim beneath the oolemma) whereas it became evenly distributed after maturation. In blastocysts, leptin signals were present in both the trophoblast cells and in the ICM cells. This is in contrast with studies on mouse embryos which showed the presence of the LEP protein in the trophoblast only. Future experiments will include studies of embryos at the 2-cell, 4-cell, 8–16-cell, and morula stages. The present study for the first time shows the pattern of leptin protein distribution within bovine oocytes and preattachment embryos.