In vitro effects of relaxin on gene expression in porcine cumulus-oocyte complexes and developing embryos

Abstract

BackgroundRelaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig.MethodsImmature cumulus-oocyte complexes (COCs) were obtained from ovarian follicles of sows. In experiment 1, COCs were matured in the presence of 0, 20, or 40 ng relaxin/ml, or 10% (v/v) porcine follicular fluid. In experiment 2, COCs were in vitro matured, fertilized and resulting embryos were cultured in the presence of 0, 20, or 40 ng relaxin/ml. In experiment 3, COCs were matured in the presence of 40 ng relaxin/ml, fertilized and zygotes were cultured as indicated in experiment 2. We evaluated the proportions of matured oocytes in experiment 1, cleaved and blastocysts on Day 2 and Day 7 post insemination in all experiments. The total cell number of blastocysts was also evaluated. In parallel, transcription levels of both relaxin and its receptors (RXFP1 and RXFP2), as well as a pro- (Bax) and anti- (Bcl2-like 1) apoptotic-related genes were determined. All data were analyzed by ANOVA and significant differences were fixed for P < 0.05.ResultsIn experiment 1, relaxin significantly increased the proportions of matured oocytes and cleaved embryos, as well as the expression level of RXFP2 mRNA compared to RXFP1 (P < 0.05). There was no effect on endogenous expression of relaxin and Bcl2-like1/Bax ratios. In all experiments, relaxin did not affect the proportions of blastocysts, but did significantly increase their total cell numbers (P < 0.05). Furthermore, no effect of relaxin was observed on Bcl2-like1/Bax expression ratios, which were similar between groups.ConclusionsExogenous relaxin influences its own receptors expression, improves oocyte nuclear maturation. Its beneficial effect on total cell number of blastocysts appears to be through a Bcl2-like1/Bax-independent mechanism.

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa

“…Freshly collected motile boar spermatozoa were selected as previously [32]. Spermatozoa (2 x 10 8 sperm/ml) were incubated with 0, 1, or 5 nM BRET-QD at 37°C for 30 min.…”

“…Ovaries were collected from post-mortem gilts and oocytes were selected and matured in vitro according to Feugang et al [32]. Matured oocytes were fertilized at a final concentration of 6 × 10 5 spermatozoa/ml with spermatozoa (10 8 ) pre-exposed to 0 or 1 nM BRET-QD diluted in PBS-PVP (1 mg/ml).…”

Regulation of the reproductive cycle and early pregnancy by relaxin family peptides

“…This has been shown for porcine (Feugang et al, 2011a) and primate embryos (Vandevoort et al, 2011), and most recently also for cryopreserved cat oocytes (Luu et al, 2013). The natural source of relaxin for such effects in vivo would either be from the ruptured follicle or the low circulating levels deriving from the new corpus luteum, though both sources would not be considerable.…”

“…It may also in some circumstances activate PI3-kinase in a G i/o -dependent manner involving PKC-zeta (Nguyen and Dessauer, 2005). Within the ovary, RXFP1 relaxin receptors have been identified at the transcript or protein level on granulosa and cumulus cells of pig antral follicles (Feugang et al, 2011a), and possibly also on oocytes themselves (Feugang et al, 2011b). Moreover, treatment with relaxin of porcine cumulus-oocyte-complexes in vitro, whilst appearing to have little effect on oocyte maturation, did appear to positively influence the resulting embryos (Feugang et al, 2011a;Kim et al, 2010).…”

“…Within the ovary, RXFP1 relaxin receptors have been identified at the transcript or protein level on granulosa and cumulus cells of pig antral follicles (Feugang et al, 2011a), and possibly also on oocytes themselves (Feugang et al, 2011b). Moreover, treatment with relaxin of porcine cumulus-oocyte-complexes in vitro, whilst appearing to have little effect on oocyte maturation, did appear to positively influence the resulting embryos (Feugang et al, 2011a;Kim et al, 2010). RXFP1 is also expressed within the corpus luteum of monkeys and cats (Braun et al, 2012;Maseelall et al, 2009), though the precise cellular localization in these tissues has not been ascertained.…”