Department of Animal & Dairy Sciences, Mississippi State University, 4025 Wise Center, Mississippi State, MS 38762, USA.
Reprod Biol Endocrinol. 2011 Jan 20;9:10. doi: 10.1186/1477-7827-9-10.
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.
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.
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 western-immunoblotting 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.
All together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond.
松弛素是一种小肽，在许多哺乳动物中也被称为妊娠激素。它由男性和女性组织合成，其分泌物存在于各种体液中，如血浆血清、卵巢卵泡液、子宫输卵管分泌物和许多哺乳动物的精液中，包括猪。然而，松弛素在猪配子和胚胎中的存在和作用仍不清楚。本研究旨在评估松弛素及其受体 RXFP1 和 RXFP2 在猪配子和胚胎中的存在。
从屠宰场采集的母猪卵巢中抽吸未成熟的卵丘-卵母细胞复合物 (COC)。体外成熟后，COC 进行体外受精和培养。为了进行研究，分别收集未成熟和成熟的 COC，并将卵母细胞从周围的卵丘细胞中释放出来。裸卵母细胞、卵丘细胞、成熟公猪精子、受精卵和胚胎（分裂和囊胚）用于时空基因表达研究。还收集了卵巢、颗粒细胞和新生猪子宫细胞的切片作为对照。
使用半定量和定量 PCR，在所有测试样本中均未检测到松弛素转录本，而 RXFP1 和 RXFP2 mRNA 存在。两种受体基因产物在卵母细胞中的表达水平均高于卵丘细胞，无论成熟时间如何。分裂胚胎中含有更高水平的 RXFP2 mRNA，而囊胚则以更高的 RXFP1 mRNA 含量为特征。使用 Western 免疫印迹或原位免疫荧光法，在所有样本中均检测到松弛素及其受体蛋白。它们的荧光强度在成熟卵母细胞中始终比在未成熟卵母细胞中更为重要。RXFP1 和 RXFP2 信号强度主要位于质膜区域，而松弛素信号则均匀分布在卵母细胞和胚胎细胞内。此外，Western 免疫印迹后，精子显示出比 RXFP1 更强的 RXFP2 信号。
综上所述，我们的研究结果表明松弛素及其受体在卵母细胞成熟、早期胚胎发育以及之后的过程中可能发挥作用。
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