|Feugang, J -|
|Rodriguez-Munoz, Juan -|
|Willard, Scott -|
|Bathgate, Ross -|
|Ryan, P -|
Submitted to: Reproductive Biology and Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 20, 2011
Publication Date: January 20, 2011
Citation: Feugang, J.M., Rodriguez-Munoz, J.C., Willard, S.T., Bathgate, R.A., Ryan, P.L. 2011. Examination of relaxin and its receptors expression in pig gametes and embryos. Reproductive Biology and Endocrinology. 9(1):10. Interpretive Summary: Relaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors in pig oocytes (eggs) and embryos. Our report provides the first evidence of porcine oocytes and pre-implantation embryos containing relaxin receptors at both gene and protein levels. Both cell types do not express relaxin genes but seem to rely on an accumulation of relaxin protein that may occur during oogenesis. Collectively, the data indicate possible roles of the relaxin/receptors systems during oocyte (egg) maturation, embryo development, and beyond. Our study paves the way for further studies to (1) evaluate the specific involvement of each receptor type and (2) determine the effect of relaxin during fertilization and beyond. These additional investigations will bring more insights into the biological roles of relaxin/receptor complexes.
Technical Abstract: Relaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors RXFP1 and RXFP2 in pig gametes and embryos. Immature cumulus-oocyte complexes (COCs) were aspirated from sows' ovaries collected at the abattoir. After in vitro-maturation, COCs were in vitro-fertilized and cultured. For studies, immature and mature COCs were separately collected, and oocytes were freed from their surrounding cumulus cells. Denuded oocytes, cumulus cells, mature boar spermatozoa, zygotes, and embryos (cleaved and blastocysts) were harvested for temporal and spatial gene expression studies. Sections of ovary, granulosa and neonatal porcine uterine cells were also collected to use as controls. Using both semi-quantitative and quantitative PCRs, relaxin transcripts were not detected in all tested samples, while RXFP1 and RXFP2 mRNA were present. Both receptor gene products were found at higher levels in oocytes compared to cumulus cells, irrespective of the maturation time. Cleaved-embryos contained higher levels of RXFP2 mRNA, whereas, blastocysts were characterized by a higher RXFP1 mRNA content. Using western-immunoblotting or in situ immunofluorescence, relaxin and its receptor proteins were detected in all samples. Their fluorescence intensities were consistently more important in mature oocytes than immature ones. The RXFP1 and RXFP2 signal intensities were mostly located in the plasma membrane region, while the relaxin ones appeared homogeneously distributed within the oocytes and embryonic cells. Furthermore, spermatozoa displayed stronger RXFP2 signal than RXFP1 after western-immunoblotting. Collectively, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond.