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

Gene expression is one of the major events occurring during oocyte maturation and early embryo development, and its perturbation in culture system may contribute to the limited production of high quality blastocysts [1‐3]. Numerous strategies such as low oxygen tension during culture, co-culture, and utilization of simple or complex media supplemented with various molecules have been developed to improve embryo culture efficiencies [1, 4‐6]. These molecules such as relaxin are generally found in the vicinity of oocytes and embryos. Relaxin is a peptide hormone found at higher levels in follicular fluids and genital tract secretions of various species [7‐11]. Despite its pleiotropic roles reported in various reproductive and non-reproductive tissues [11‐13], little is still known on embryonic effects of relaxin during early pregnancy.

In livestock, the pig is the primary animal model used for the study of relaxin effects [11, 14, 15] due to the important production of relaxin in both pregnant and non-pregnant pigs. Indeed, pigs exhibit high levels of relaxin in ovarian follicular fluids [8, 14], and its secretions by corpora lutea and uterine cells significantly increase during the post-ovulatory period [11, 14, 16‐18]. Numerous studies have reported various actions of relaxin on pig reproductive tissues such as cervical and uterine tissues, including modulation of embryo implantation during pregnancy in rats [19‐21]. Despite these physiological conditions, it is not known whether relaxin influences oocyte maturation and/or embryo development in the pig.

Relaxin hormone peptide exerts autocrine, endocrine, and paracrine effects through membrane receptors known as RXFP1 and RXFP2 [22‐24]. These receptors have been detected in porcine reproductive tissues [25, 26], including oocytes and embryos [27]. But, their potential activation by relaxin and further implications on oocyte maturation and embryo development are still unknown.

Unless otherwise indicated, all chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA) for embryo production or Invitrogen Co. (Carlsberg, CA, USA) for gene expression. Lyophilised relaxin hormone (pRLN) produced from pregnant sow ovaries was a gift from Dr. C Bagnell [15]. It was dissolved in deionized water (10 μg/ml), aliquoted, and stored as stock solution at -80°C. Pre-warmed solutions of NaCl (0.9%; w/v) and Hepes-buffered Tyrode Lactate solution (Vigro-Bioniche, Pullman, WA, USA) supplemented with polyvinyl alcohol (PVA: 0.1%; w/v) and pyruvate (100 μM) were used to wash the ovaries, and the oocytes and the embryos, respectively. Oocytes were matured in TCM199+L-glutamine medium supplemented with PVA (1%; w/v), glucose (2.8 mM), pyruvate (0.91 mM), cysteamine (0.57 mM), EGF (10 ng/ml), and FSH (0.4 μg/ml) and fertilized in the modified Tris-buffered medium (mTBM) containing caffeine (2 mM) and BSA-fraction V (0.1%; w/v). Embryos were cultured in NCSU-23 medium (Millipore, Billerica, MA, USA) supplemented with BSA-FAF (0.4%; w/v). All media contained 10 μl/ml antibiotics (gentamycin and penicillin G/Streptomycin sulphate) and were pre-incubated for at least 2 h before use. Oocytes and embryos were placed in 4-well dishes (Nunc, Roskilde, Denmark) for incubations under a humidified atmosphere of 39°C and 5% CO₂ in air.

Numerous physiological roles have been attributed to relaxin [10, 11]; but its effects during embryo development are still unknown despite its presence in gamete and embryo environments. Here, we could not detect the expression of relaxin in porcine COCs but found an accumulation of its receptors RXFP1 and RXFP2 mRNA in COCs which appeared to be modulated by exogenous relaxin. In addition, relaxin moderately influenced embryo development but significantly increased the total cell number of blastocysts irrespective of the moment of its addition in the media (oocyte maturation and/or embryo culture).

The present study reports developmental effects of relaxin used at concentrations similar to those found in the literature [29‐31]. Indeed, variable but large amounts of relaxin are found in follicular fluids surrounding porcine oocytes, while pre-implantation embryos are suspected to be exposed to unknown amounts of relaxin during their utero-tubal progression and development (corresponding to the ovarian luteal phase). Beneficial roles of relaxin have been demonstrated during follicle growth and ovulation [7, 14], uterine receptivity and activity process in various species such as pigs [14], primates [32, 33] and rodents [21, 34, 35]. To our knowledge, the current study is the first to report developmental effects of exogenous relaxin during both oocyte maturation and early embryo development.

These effects are results of putative interactions between relaxin and its receptors located on plasma membranes of target cells [22, 24, 36]. In previous studies, we reported the presence of these receptors on porcine oocytes and embryos [27]. Here, we found that the addition of porcine relaxin in a follicular fluid-free maturation medium induces an accumulation of RXFP2 mRNA in COCs. This effect is surprising given that relaxin is the specific ligand of RXFP1, although it can bind with low affinity to RXFP2. This observation suggests a possible role of RXFP2 receptors during mammalian oocyte maturation process in the pig, as also suggested in rhesus macaque [37] and rat [38] oocytes expressing RXFP2 mRNA. In-depth studies of INSL3-RXFP2 complex during oocyte maturation and embryo development will definitely contribute to better characterize the full developmental effects of this complex.

Nevertheless, given the complexity of molecular interactions existing within the follicular fluid, a likely activation of RXFP1 receptors during in vivo oocyte maturation cannot be ruled out; and seems to be highlighted in the current study by the significant increase of RXFP1 mRNA in oocytes matured in the presence of porcine follicular fluid alone. 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. In our study, oocytes were matured in the absence of follicular fluid, and the supplementation of the basal medium with relaxin significantly increased the proportions of oocytes resuming and accomplishing meiotic maturation. This beneficial effect of relaxin could be explained by its interactions with its receptors leading to an inhibition of oocyte meiosis blocking factors such as cyclic adenosine monophosphate (cAMP) as previously suggested [38].

Most interesting, the developmental effects of relaxin suggest both direct and indirect effects on embryos and oocytes, respectively. While embryos were freed of the surrounding cumulus cells after fertilization, oocytes were matured together with their surrounding cumulus cells. Using rodent oocytes, Kawamura et al. have proposed an indirect effect of relaxin on its targets in a paracrine way [38]. Irrespective of the moment of its addition, the increased total cell number of blastocysts appears as the hallmark of relaxin during early embryo development. Indeed, beneficial effects of relaxin were limited at the cleavage stage (high proportions) following oocyte maturation in the presence of relaxin, while any effects were observed on blastocyst formation compared to the control groups.

Developmental effects of relaxin in our study indicate possible perturbations of molecular mechanisms associated with cell cycle, and specifically cell proliferation. 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]. Therefore, we suggest that the increase of blastocyst cell number in our study is probably associated to an increase of GSH in oocytes and/or developing embryos.

In addition, production of GSH induces an imbalance of reduced-oxidative (REDOX) status of cells leading to potential modifications of cellular functions such as cell cycle (incidence of apoptosis) [49]. Therefore, we anticipated an activation of apoptotic-related genes by relaxin. Apoptosis is an important biological process during blastocyst formation of which fine-tuned balances between pro-apoptotic (Bax) and anti-apoptotic (Bcl2-like1) genes are critical for the survival of embryos [50]. Our study revealed that Bcl2-like1/Bax ratios were always in direction of Bcl2-like1 transcripts which is favorable to embryo survival. The presence of relaxin during culture did not significantly affect this ratio which remained comparable between groups.