267 EXPRESSION OF RELAXIN FAMILY PEPTIDE RECEPTORS RXFP1 AND RXFP2 IN PIG PREIMPLANTATION EMBRYOS AND DEVELOPMENTAL EFFECTS OF RELAXIN HORMONE

Abstract

Relaxin is a polypeptide hormone secreted by male and female reproductive tissues to facilitate spermatozoa progression in the female tract and parturition. Relaxin secretions are found in the vicinity of oocytes and embryos, and exert their effects through membrane receptors, which have not yet been described in porcine embryos. Here, we determined the presence of RXFP1 and RXFP2 receptors in porcine gametes and embryo, and evaluated the developmental effects of porcine relaxin (pRLX; Yan et al. 2006 Reproduction 131, 943-950). Cumulus-oocyte complexes (COC) were aspirated from sows ovaries collected at a local abattoir. Homogeneous COC were selected for IVM (44 h) and fertilization (6 to 8 h). Presumptive zygotes were cultured in NCSU-23 + 0.4% BSA for up to 7 days. All procedures were done at 39°C, under 5% CO₂ in a humidified atmosphere. Matured oocytes, BTS-diluted spermatozoa, and embryos were collected for gene expression studies. For developmental studies, COC were matured (experiment 1), or embryos cultured from the zygote stage (experiment 2) in the presence ofpRLX (0, 20, or 40 ng mL^-1). In experiment 3, zygotes derived from oocytes matured in the presence of pRLX (40 ng mL^-1) were cultured with pRLX (20 or 40 ng mL^-1). The pRLX effects were assessed on cleaved embryos and blastocysts recorded on Days 2 and 7 postinsemination, respectively. The total cell numbers of Day-7 blastocysts were also evaluated. All data were analyzed using ANOVA. Gametes and embryos expressed RXFP1 and RXFP2 at both the mRNA and protein level. The amounts of both gene transcripts were higher in mature oocytes (metaphase II) compared with spermatozoa (P < 0.05). The RXFP1/2 mRNA ratios were in favor of RXFP2 in mature oocytes (0.9×), zygotes (0.8 ×), and cleaved embryos (0.8×), and for RXFP1 in spermatozoa (1.1 ×) and blastocysts (1.1 ×). A similar pattern during embryo development was revealed at the protein level, showing a higher RXFP2 fluorescence signal in cleaved embryos and a lower signal in blastocysts compared with RXFP1 protein. In experiment 1, COC exposed to 40 ng mL^-1 pRLX resulted in fewer cleaved embryos (36 ± 4%) compared with controls (42 ± 5%, P < 0.05). Of the 40 ng mL^-1 pRLX-derived cleaved embryos, a greater proportion developed to the blastocyst stage (38 ± 6%; P < 0.05) compared with control and 20 ng mL^-1 pRLX-derived cleaved embryos (26 ± 4% and 17 ± 8%, respectively). In experiment 2, however, 40 ng mL^-1 pRLX induced higher cleavage but lower blastocyst rates (51 ± 5% and 20 ± 4%, respectively) compared with the control group (37 ± 4% and 32 ± 7%, respectively) (P < 0.05). In experiment 3, the exposure of both oocytes and derived embryos did not affect the developmental rates (P > 0.05). Nevertheless, pRLX significantly increased the mean cell number of blastocysts in all experiments (P < 0.05). We concluded that pig embryos express RXFP1 and RXFP2 receptors, which may facilitate a role for pRLX during oocyte maturation and embryo development in the pig.

This work was supported by the USDA-ARS Biophotonics Initiative project# 58-6402-3-0120 and the Mississippi Agricultural and Forestry Experiment Station (MAFES).