Examination of relaxin and its receptors expression in pig gametes and embryos

Reproductive Biology and Endocrinology Examination of relaxin and its receptors expression in pig gametes and embryos Jean M Feugang 0 Juan C Rodriguez-Munoz 0 Scott T Willard Ross A Bathgate Peter L Ryan 0 0 Department of Animal & Dairy Sciences, Mississippi State University , 4025 Wise Center, Mississippi State, MS 38762 , USA Background: 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. Methods: 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. Results: 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 westernimmunoblotting 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. Conclusion: All together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond. - Background The inadequate culture conditions greatly limit the production of high quality embryos [1,2]. In vivo, maturing gametes and developing embryos maintain complex interactions with their immediate environments which are rich in a variety of molecules such as relaxin, whose embryotrope effects are not completely understood [3,4]. Relaxin is a small peptide ( 6 kDa) commonly known as a pregnancy hormone in many mammals [5,6], consisting of several members expressed in various tissues across a broad range of mammalian species [6,7]. Consequently, relaxin is found in a variety of body fluids and has pleiotropic actions on numerous tissue targets [6,8]. In female reproductive tissues, relaxin is involved in a range of events such as ovarian follicle growth and ovulation, development of mammary glands, preparation of the uterus and cervix for pregnancy and delivery, while relaxins action in males is mainly limited to the improvement of sperm motility [3,6,8]. These various effects of relaxin are mediated through a family of plasma membrane receptors known as RXFP1, 2, 3 and 4 [9]. Ovarian relaxin or relaxin-2 is the specific ligand of RXFP1 (or LGR7), but also binds with low affinity to RXFP2 (or LGR8), the natural receptor of insulin-like peptide 3 (INSL3) [10]. Different molecular and immunological techniques have been used to detect their expression in mammalian tissues [11-13], including oocytes of primates [11,12] and rats [14]. Despite the high relaxin levels found in follicular fluids and oviduct environment of sows, there are still no reports available on the expression of these receptors in porcine oocytes and embryos [15-20]. This presence of relaxin may suggest its potential roles during oocyte developmental competency acquisition and early embryo development. Indeed, relaxin detection in follicular fluids and granulosa cells has been purposed as a predictor of successful embryo transfer in humans and early pregnancy status in common marmosets [21,22]. From this background, the present study aimed at investigate the possible expressions of relaxin and its receptors RXFP1 and RXFP2 in porcine gametes and cultured embryos using both semi-quantitative and quantitative PCR techniques, western-immunoblotting and in situ immunofluorescence approaches. Methods Chemicals and media Unless otherwise indicated, all chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, USA) for embryo production or Invitrogen Co. (Carlsbag, USA) for gene expression. Relaxin (pRLN) obtained from pregnant sow ovaries was a gift from Dr. C Bagnell laboratory [23]. INSL3 was purchased from Phoenix Pharmaceutics, Inc. (Burlingame, USA), respectively. Ovaries were washed in NaCl (0.9%; w/v) and oocytes and embryos in Hepes-buffered Tyrode Lactate medium supplemented with polyvinyl alcohol (PVA; 0.1%; w/v) and pyruvate (100 M). Cumulus-oocyte complexes 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 g/mL), and fertilized in the modified Tris-buffered medium containing caffeine (2 mM) and BSA-fraction V (0.1%; w/v). Embryos were cultured in NCSU-23 medium supplemented with BSA-FAF (0.4%; w/v). All media contained 10 l/mL penicillin/streptomycin and were pre-incubated at 37C for at least 2 h before use. Cumulus-oocyte complexes (COCs) collection and in vitro embryo production Sow (Yorkshire-Landrace) ovaries were collected at a local abattoir (Southern Quality Meats, Pontotoc, USA) and transported at 37C to the laboratory. Immature COCs were aspirated from medium size follicles (3-6 mm of diameter), washed and in vitro-matured (50-75 COCs per 500 l) for 44 hours. Diluted (in BTS) pools semen of at least two boars were purchased (Prestage Farms; West Point, MS, USA) and high motile spermatozoa were purified by centrifugation through a percoll gradient (90%:45%). Final concentrations of 6 105 spermatozoa/ml were used to fertilize the COCs (= 0 h post-insemination or 0 hpi). After 18 hpi, presumptive zygotes were harvested, mechanically denuded and cultured (1 embryo/1-2 l of culture medium) for up to 6 days (Day 7pi). All incubations took place in a humidified atmosphere of 39C and 5% CO2 in air. In each experimental replicates, groups of oocytes were treated separately as controls for embryo production. The current culture system allowed maturation of (...truncated)