Effects of Recombinant Relaxin on In Vitro Maturation of Porcine Oocytes

Abstract

ABSTRACT. Relaxin, a member of the insulin superfamily, has diverse functions in both reproductive and nonreproductive tissues. The aim of the present study was to evaluate the effects of recombinant relaxin on the in vitro maturation of porcine oocytes and their subsequent embryonic development following in vitro fertilization. Three concentrations of relaxin (1, 10, and 100 ng/ml) were used in the in vitro maturation (IVM) medium [TCM supplemented with 10% (v/v) porcine follicular fluid, 10 ng/ml of epidermal growth factor, 4 IU/ml of pregnant mare serum gonadotropin, and (only for the first 22 hr) 4 IU/ml of human chorionic gonadotropin]. Relaxin was used during the entire IVM period. Nuclear maturation of oocytes was examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) for 5 min and mounted on a glass slide. The glutathione (GSH) content in oocytes, an important indicator of cytoplasmic maturity, was measured using a micro-glutathione assay. Cryopreserved boar semen was used for in vitro fertilization. Embryos were cultured in modified NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate. Although nuclear maturation of oocytes did not vary, the GSH content in oocytes was significantly higher when cultured with 1 ng/ml (7.9 pmol/oocyte) and 10 ng/ml (8.47 pmol/oocyte) compared to a control group. However, no additional beneficial effect was observed when 100 ng/ml of relaxin was added to the IVM medium. A significantly higher rate of blastocyst formation was observed with 10 ng of relaxin (32.4%) compared to the control (14.4%) or 100 ng of relaxin (21.4%). No difference between 1 ng and 10 ng was observed in terms of the blastocyst production rate. The inner cell mass cell numbers in relaxin-treated groups were significantly higher than control, and trophectoderm cell number was the highest in the 10 ng relaxin group. Relaxin (10 ng/ml) can be supplemented in IVM medium to support the maturation of porcine oocytes. Intensive attempts have been made to establish a reliable in vitro maturation (IVM) system for porcine oocytes. Accordingly, higher oocyte maturation rate is well documented in pigs [19], and production of both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) offspring using in vitro matured oocytes has also been reported [2,14]. However, embryos produced by either IVF or SCNT using in vitro-matured oocytes have resulted in a much lower pregnancy rate compared to the in vivo system. Oocyte quality influences the developmental competence of embryos and the establishment and maintenance of pregnancy following embryo transfer to recipients [11]. During IVM, oocytes progressively acquire developmental competence, which involves both cytoplasmic and nuclear maturation. This is a highly coordinated process that includes morphological, ultrastructural, and transcriptional changes in the cytoplasmic and nuclear compartments of the oocyte. However, the media used for IVM and supplements added in the medium are known to affect both maturation a...

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

“…The existence of such interactions may also explain our contradictory results with a recent study reporting a lack of relaxin effect on nuclear maturation of pig oocytes [39]. These authors tested the effect of recombinant human relaxin added in maturation medium already containing porcine follicular fluid (10%) and whose high relaxin contents [8,40] and beneficial effect on oocyte maturation [41,42] could have interfered with their results.…”

“…A recent study has demonstrated a positive effect of relaxin on glutathione (GSH) synthesis in in vitro matured porcine oocytes [39]. Indeed, GSH is a routine indicator or oocyte cytoplasmic maturation [43], whose beneficial effects on blastocyst formation and quality (hatching and cell number) are well-documented [44-48].…”

Effect of lanosterol on thein vitromaturation in semi-defined culture system of prepubertal ewe oocytes

“…The addition of oestrus serum also supported high maturation rates in IVM of ewe oocytes (Rao et al, 2002;Succu et al, 2007). In addition, the research into supplementing recombinant hormones such as leptin, insulin-like growth factor, and vascular endothelial growth factor in IVM system has served to improve development of in vitro maturation in several species (Moreira et al, 2002;Boelhauve et al, 2005;Cao et al, 2009;Kim et al, 2010). However, developmental competence of in vitro matured oocytes from prepubertal animals has been reported to be lower than oocytes derived from adult animals (Pinyopummintr & Bavister, 1995;O'Brien et al, 1996;Ledda et al, 1997;Gandolfi et al, 2000;Leoni et al, 2007).…”

“…However, developmental competence of in vitro matured oocytes from prepubertal animals has been reported to be lower than oocytes derived from adult animals (Pinyopummintr & Bavister, 1995;O'Brien et al, 1996;Ledda et al, 1997;Gandolfi et al, 2000;Leoni et al, 2007). In addition, the researches supplementing recombinant hormones such as leptin, insulin-like growth factor, and vascular endothelial growth factor in IVM system have been used to improve development of in vitro maturation in several species (Moreira et al, 2002;Boelhauve et al, 2005;Cao et al, 2009;Kim et al, 2010). In addition, the research into supplementing recombinant hormones such as leptin, insulin-like growth factor, and vascular endothelial growth factor in IVM system has served to improve development of in vitro maturation in several species (Moreira et al, 2002;Boelhauve et al, 2005;Cao et al, 2009;Kim et al, 2010).…”

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

“…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.…”