Expression of the orexin system in the porcine uterus, conceptus and trophoblast during early pregnancy.

Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.

Expression of adiponectin and its receptors in the porcine hypothalamus during the oestrous cycle.

Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2-3, 10-12, 14-16 and 17-19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14-16, whereas in SME, the most pronounced gene expression was found on days 2-3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17-19 and in POA on days 2-3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14-16. AdipoR1 gene expression in POA was potentiated on days 2-3 and 10-12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2-3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2-14 of the cycle), and they decreased on days 17-19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2-3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2-3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2-3 (p < 0.05), while in POA on days 17-19 and in SME on days 10-12 and 14-16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig's endocrine status related to the oestrous cycle.

Expression of orexin A and B in the porcine hypothalamus during the oestrous cycle.

Orexins A and B are hypothalamic peptides engaged in a variety of physiological functions related to the control of energy homeostasis, sleep and wakefulness. The presence of orexin receptors in the tissues of the hypothalamus-pituitary-gonadal axis indicates that these hormones are also involved in the control of the reproductive system. The aim of this study was to compare the expression levels of prepro-orexin (a precursor of orexins A and B) mRNA in the porcine hypothalamic structures involved in reproductive processes - mediobasal hypothalamus (MBH), preoptic area (POA) and stalk median eminence (SME), during four stages (days 2-3, 10-12, 14-16, 17-19) of the oestrous cycle. In MBH, lower concentrations of PPO mRNA were observed on days 2-3 than in the remaining stages. In POA, the highest mRNA expression of PPO was noted on days 17-19. In SME, the highest concentrations of PPO was observed on days 2-3, and the lowest on days 14-16. We also investigated the intensity of OXA and OXB immunoreactivity and detected both peptides in all examined structures. In MBH, signal intensity for OXA was highest on days 14-16 and lowest on days 17-19. The highest levels of immunoreactivity were noted on days 2-3 and 10-12 in POA, and in SME additionally on days 17-19. OXB immunoreactivity in hypothalamic tissues also changed during the cycle, and the highest signal intensity was reported on days 10-12 in MBH, on days 14-16 in POA, and on days 14-16 and 17-19 in SME. The results of our study indicate that orexins A and B are produced in the porcine hypothalamus and that their concentrations vary subject to the pig's hormonal status. Our findings also suggest that orexins may affect reproductive functions at the highest level of the hypothalamus-pituitary-gonadal axis.

In vitro effects of genistein and daidzein on the activity of adrenocortical steroidogenic enzymes in mature female pigs.

Soy products, commonly used as a protein source in farm animals' diets, contain considerable quantities of non-nutrient constituents such as phytoestrogens. Genistein and daidzein are known to affect the reproductive processes in humans and animals. However, reports concerning phytoestrogens and porcine adrenal steroidogenesis are scarce, and the adrenal mechanism of phytoestrogen action in species other than humans and rodents is poorly recognized. The goal of the present paper was to examine the in vitro effects of genistein and daidzein on the activity of key enzymes for cortisol and corticosterone synthesis in porcine adrenocortical cells harvested during the luteal or follicular phase of the porcine estrous cycle. The cells were treated with genistein or daidzein (10 µM), with or without ACTH (5 nM), in the presence or absence of precursors (1 µM) of cortisol (pregnenolone, P5; progesterone, P4; 17-hydroxyprogesterone, 17OH-P4; or 11-deoxycortisol, 11d-cortisol) or corticosterone: (P5 or P4) synthesis. The supplementation of a medium with P5, P4, 17OH-P4 or 11d-cortisol enabled us to measure the activity of cholesterol side-chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17α-hydroxylase/C17-20 lyase (P450c17) or 21-hydroxylase (P450c21) and 11ß-hydroxylase (P45011ß), respectively. We demonstrated that in sexually mature, cyclic pigs, regardless of the phase of the estrous cycle, phytoestrogens genistein and daidzein suppressed basal and ACTH-stimulated in vitro secretion of cortisol and corticosterone via progesterone synthesis inhibition. This indicates that phytoestrogens specifically inhibit the 3ß-HSD activity in porcine adrenocortical cells. We suggest that genistein and daidzein present in soy products may negatively affect glucocorticoid synthesis of mature gilts by disrupting adrenal steroidogenesis at the 3ß-HSD level.