84 Characterization of agouti-signalling protein expression within the bovine ovary and early embryo

Abstract

Factors present in the oocyte and surrounding follicular cells aid in the attainment of oocyte competence. Agouti-signalling protein (ASIP) is a known regulator of melanocyte function through binding to melanocortin receptors including MC1R and MC4R. Additionally, ASIP has been classified as an adipokine due to a link with insulin resistance and obesity in humans. In mice, expression is limited to hair follicles where ASIP regulates hair pigmentation. In cattle, however, ASIP mRNA has been detected in a variety of tissues, including adipose, skin, heart, testis, and the ovary. Despite ovarian expression, the role of ASIP in reproduction remains undetermined. Bovine ASIP is a secreted protein consisting of 133 amino acids. The aim of this experiment was to provide a detailed description of the ASIP expression profile within the bovine ovary and during early embryonic development. Reverse transcription PCR (RT-PCR) was conducted to analyse ASIP, MC1R, and MC4R mRNA expression. Samples examined included fetal ovaries from gestational day 90 to 250, adult ovary, fetal testis, adult testis, and 12 somatic tissues including adrenal, cerebral cortex, gut, heart, intestine, kidney, liver, lung, muscle, pituitary, stomach, and thymus. Amplification of ribosomal protein L19 (RPL19) served as a positive control for all samples. Expression of ASIP was detected in the fetal testis, 9 somatic tissues, and the fetal and adult ovary. In the fetal ovary, ASIP was detected as early as 90 days of gestation and continued throughout gestation. Expression of the ASIP receptors, MC1R and MC4R, were detected exclusively in the fetal ovary. To further characterise ASIP expression, quantitative real-time PCR (RT-qPCR) was utilised to examine samples including germinal vesicle and MII oocytes (pool of 10 oocytes), in vitro-produced embryos ranging from the 2-cell to blastocyst stages (pool of 10 embryos), and cumulus and granulosa cells collected from a pool from 5 cumulus–oocyte complexes (COCs) and follicles, respectively. Theca cells from a single follicle were analysed. Samples with cycle threshold values below 35 were considered to express the gene of interest. Of the follicular cells examined, ASIP expression was present in theca, granulosa, and cumulus cells. ASIP expression was detected in both GV and MII oocytes. Early embryonic expression of ASIP was detected in the 2-cell embryo and continued to the blastocyst stage of development. In conclusion, ASIP is present in the bovine adult and fetal ovary, follicular cells including cumulus, granulosa, and theca cells, GV and MII oocytes, and in vitro-produced embryos from the 2-cell to blastocyst stages. Future research will focus on identifying the function of ovarian and early embryonic ASIP in cattle.