Introduction The corpus luteum CL is a transient

Introduction The corpus luteum (CL) is a transient endocrine gland that differentiates from the thecal and granulosal cells of the ovarian follicle after ovulation. Its formation and limited lifespan in the mammalian ovary is important for fertility, as the CL produces progesterone (P4), the essential steroid hormone required for embryo implantation and maintenance of pregnancies until placental development (Stouffer, 2003; Stouffer et al., 2013). Recent studies, particularly those involving genome and cellular analyses, have increased the understanding of local factors associated with the development, functional lifespan and regression of the CL. There is compelling evidence for interactive functions among metabolic hormones, such as ghrelin, leptin, or resistin, and female reproduction (Rak-Mardyla, 2013; Rak-Mardyła et al., 2013, 2014). Apelin is a regulator of ovarian physiology (Roche et al., 2016, 2017; Rak et al., 2017). Apelin was originally identified in stomach extracts of cattle as the endogenous ligand of the orphan G protein-coupled receptor APJ (Tatemoto et al., 1998). It is derived from a 77-amino-acid preproapelin that is cleaved into a 55-amino-acid fragment and then into shorter forms. This adipokine is involved in a broad range of physiological functions such as fluid homeostasis, regulation of food intake and energy metabolism (Taheri et al., 2002; Bertrand et al., 2015). Moreover, the apelin peptide is a potent angiogenic factor inducing endothelial cell (EC) proliferation, migration, and development of blood vessels in vivo (Kasai et al., 2004, Cox et al., 2006). Apelin was described as a biomarker of several pathologies including diabetes, obesity, cardiovascular disease, endometriosis, cancer and polycystic ovarian syndrome (Rayalam et al., 2011; Perjes et al., 2014; Narayanan et al., 2015; Roche et al., 2016). Amounts of apelin are increased in obese humans compared with lean control individuals (Boucher et al., 2005). The expression of the apelin and its receptor APJ genes has been detected in human, cattle, rhesus monkey and pig ovaries (Shirasuna et al., 2008; Schilffarth et al., 2009; Shimizu et al., 2009; Fuhua and Stouffer, 2012; Roche et al., 2016, 2017; Rak et al., 2017). In humans, the apelin/APJ genes are expressed in different ovarian cells such as granulosal, thecal cells and oocytes (Roche et al., 2016). Schilffarth et al. (2009) demonstrated that in ovaries of cattle apelin/APJ decreased at the end of the luteal phase and decreased during CL regression, suggesting the role of apelin in CL formation and the luteolytic endocrine cascade pathway. These findings were consistent with data of Shirasuna et al. (2008) where it was demonstrated that the expression of both the apelin/APJ genes is isolated to the smooth muscle cells of luteal Decitabine in the CL of cattle, suggesting that the apelin/APJ system may be associated with the vascular function in the CL. Data concerning the role of apelin in the physiology of pigs are limited. In a study of Del Ry et al. (2009), the apelin gene was initially sequenced for Sus scrofa for future applications to molecular biology studies. In a previous study, it was documented that there was gene expression and a direct role of apelin on ovarian follicular cells steroidogenesis and proliferation in pigs (Rak et al., 2017). There are no reports describing the expression of apelin/APJ genes in corpus luteum, particularly as related to actions on progesterone secretion. The aims of the present study, therefore, were to evaluate a) the relative abundances of mRNA and abundance of protein of apelin/APJ during different stages of CL development; b) immunolocalization in CLs; and c) direct in vitro effects of apelin on P4 secretion and 3βHSD level.
Materials and methods
Results
Discussion The results of this study demonstrate, for the first time, relative abundance of mRNA and abundance of protein of both apelin and its receptor APJ at different stages of CL development in pigs. Relative abundance of apelin mRNA and abundance of protein were similar in CL from early and middle luteal phases of the estrous cycle, but were less in the late luteal phase. Relative abundance of APJ mRNA and abundance of protein were similar in CL from early and late luteal phases and was similar in CL during the mid-luteal phase. The differences in the abundance of proteins between individual CL are probably hormonally controlled by steroid hormones secreted by luteal cells, the concentrations of which change during the luteal phases. Immediately after ovulation, P4 secretion is relatively less and is greater in CL2 compared to secretion during CL regression. There are similar fluctuations in testosterone (T) concentrations as those of P4 during the luteal phases. The aromatase inhibitory actions of P4 prevent the conversion of T to estradiol (E2), therefore, the greatest amounts of E2 occur when P4 concentrations are least (Gregoraszczuk, 1992). The greatest secretion of P4 and expression of the APJ gene during the mid-luteal phase of the estrous cycle indicates P4 can affect APJ regulation via an autocrine pathway. This action of P4 is further confirmed by the results of Shimizu et al. (2009) where it was observed that there was an increase in relative abundance of APJ mRNA in ovarian granulosal cells of cattle in vitro after P4 administration. Published data also indicate that relative abundance of apelin mRNA is regulated by other factors, such as luteinizing hormone (LH), insulin-like growth factor 1 (IGF1) or insulin (Boucher et al., 2005; Wei et al., 2005; Shimizu et al., 2009; Roche et al., 2016). Moreover, in a previous study gonadotropins and steroids hormones increased the relative abundance of resistin in ovarian follicles of pigs (Rak et al., 2015). Results from this previous study are consistent with those of other studies where it was reported that, during the luteal phase in cattle, relative abundance of mRNA for apelin/APJ increased from the early to late luteal phase of the estrous cycle, followed by a decrease in regressing CL (Shirasuna et al., 2008; Schilffarth et al., 2009). In addition, relative abundance of apelin/APJ mRNA changed during pregnancy in the CL of cattle, gradually increasing between 1 and 7 months of pregnancy and then there was a marked decrease during the eighth month. In contrast, the relative abundance of APJ mRNA in CL during pregnancy is relatively constant and similar to that during the mid-leuteal phase of the estrous cycle (Schilffarth et al., 2009). Immunohistochemical analysis resulted in the finding that apelin staining was exclusively present in the cytoplasm of both small and large luteal cells with the greatest intensity staining in CL2. In CL3, apelin was present in a few luteal cells of both types. The APJ was localized in both the cytoplasm and membranes of small and large luteal cells. In CL1, there was faint staining of only a few small luteal cells and for cytoplasmic APJ immunostaining. In CL2,the APJ was located in the cell membrane of large luteal cells while it was located in the cytoplasm of small luteal cells. In the large luteal cells, the staining was of moderate intensity. In luteal cells of CL3, the membrane and cytoplasm had moderate APJ immunostaining. Additionally, there was intense APJ staining in the epithelium of blood vessels of CL2 and CL3. Previously published data by Shirasuna et al. (2008) provided evidence that apelin and APJ was present in luteal arteries, indicating the involvement of apelin in CL angiogenesis. In the present study, there were greater amounts of APJ in mid-luteal phase CL, when blood vessel growth is occurring, and this indirectly confirms that apelin is involved in angiogenesis of CL.