Adiponectin affects uterine steroidogenesis during early pregnancy and the oestrous cycle: An in vitro study.

Reproduction in females is an energetically demanding process. We assumed that adiponectin (ADPN), known for its role in energy balance maintenance, is also engaged in the regulation of uterine steroidogenesis in the pig. We determined the impact of ADPN alone or in combination with insulin (INS) on testosterone (T), estrone (E1) and estradiol (E2) secretion by porcine endometrium and myometrium, uterine expression of CYP17A1 and CYP19A3 genes, and endometrial abundance of P450C17 and P450AROM proteins during the peri-implantation period and the oestrous cycle, using radioimmunoassay, qPCR, and Western Blot, respectively. During pregnancy, in the endometrial explants from days 10-11, ADPN decreased CYP17A1 gene expression, P450C17 protein abundance and T secretion, whereas increased E1 secretion. On days 12-13 of pregnancy, ADPN decreased CYP17A1 and CYP19A3 expression, P450C17 and P450AROM protein abundance and E1 secretion, but stimulated T secretion. On days 15-16 of pregnancy, ADPN decreased P450C17 protein accumulation but enhanced CYP19A3 expression and E1 secretion. On days 27-28 of pregnancy, ADPN increased CYP17A1 and CYP19A3 mRNA content and T secretion in this tissue and decreased P450C17 content. ADPN effect on myometrial explants was dependent on stage of gestation or oestrous cycle. Moreover, INS treatment modulated basal and ADPN-affected steroidogenic enzymes gene and protein expression and steroids secretion. The results obtained indicate that ADPN may affect processes required for successful implantation such as steroidogenesis. ADPN and INS were also shown to modulate each other action, which indicates that the proper course of uterine steroidogenesis may be dependent on both hormones' interaction.

The expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses.

Recent research has demonstrated that chemerin may take part in the regulation of reproduction. The aim of this study was to determine the expression of chemerin system - chemerin and its receptors, chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) - in the porcine uterus during the oestrous cycle and early pregnancy, and in trophoblasts and conceptuses by real-time PCR and western blotting. Chemerin concentrations in uterine luminal flushings (ULF) were determined using ELISA test. In the endometrium, the highest expression of chemerin and GPR1 proteins was observed during the mid-luteal phase; CMKLR1, during the late luteal phase; and CCRL2, during the follicular phase of the cycle. In the myometrium, chemerin protein expression was enhanced during the early luteal phase, and chemerin receptor proteins were highly expressed during the follicular phase. In the endometrium of pregnant pigs, the highest expression of chemerin and CCRL2 protein was observed during implantation; CMKLR1, during placentation; and GPR1, during embryo migration. In the myometrium, chemerin and CCRL2 protein expression increased at the end of implantation, and the expression of CMKLR1 and GPR1 protein was enhanced during implantation. In the conceptuses and trophoblasts, the highest expression of chemerin system proteins was observed during placentation, with the exception of GPR1 protein in the trophoblasts. The highest concentrations of the analysed adipokine were observed in ULF during the luteal phase of the cycle and during maternal recognition of pregnancy. This is the first study to demonstrate that the expression of the chemerin system in the porcine uterus, conceptuses and trophoblasts, and chemerin concentrations in ULF are influenced by the hormonal milieu in different stages of the oestrous cycle and in early pregnancy. The present results also suggest that chemerin is implicated in the regulation of reproductive functions in pigs.

The effect of estrone and estradiol on the expression of the orexin/hypocretin system in the porcine uterus during early pregnancy.

Orexin A and B (OXA, OXB) are hypothalamic neuropeptides acting via two receptors, type 1 (OX1R) and 2 (OX2R). Orexins, also known as hypocretins, take part in a common endocrine system regulating metabolism and reproductive functions. Changes in the orexin system expression during the estrous cycle and pregnancy suggest dependence on the local hormonal milieu. Estrogens are the key hormones controlling reproductive functions, including maternal recognition of pregnancy and implantation. We hypothesize that estrogens may affect orexin system expression in the early pregnant uterus. The aim of this study was to investigate the influence of estrogens on prepro-orexin (PPO), OX1R, and OX2R gene expression, OX1R and OX2R protein content in the porcine uterine tissue, as well as OXA and OXB secretion on days 10-11, 12-13, 15-16, and 27-28 of pregnancy and on days 10-12 of the estrous cycle (n = 5 per group). The expression of PPO, OX1R, and OX2R genes was examined using qPCR, OX1R and OX2R protein content was evaluated using western blotting, and orexins secretion was determined with ELISA. This is the first study to describe the influence of estrogens on orexin system expression in the porcine uterus. Obtained results revealed that estrogens significantly affect the expression of orexin system and orexins secretion. The influence of estrogens varied between different stages of early pregnancy and the estrous cycle. The steroids showed a tissue-specific and dose-dependent effect. Our findings suggest that orexins could act as a "molecular switch" for estrogen activation in the processes of endometrial decidualization and rapid uterine enlargement during early pregnancy.

Effect of orexin B on CYP17A1 and CYP19A3 expression and oestradiol, oestrone and testosterone secretion in the porcine uterus during early pregnancy and the oestrous cycle.

Orexin A (OXA) and B (OXB) are hypothalamic neuropeptides identified as regulators of food intake, energy homoeostasis, sleep-wake cycle and arousal. They also create an integrative link between energy homoeostasis and reproduction. Although their functions in the ovaries and testes have been partially explored, to date, less attention has been focused on the role of the peptides in the uterus. The aim of this study was to investigate the effect of one of orexins - orexin B on oestradiol (E2), oestrone (E1) and testosterone (T) secretion by porcine endometrial and myometrial slices as well as the gene expression of key steroidogenic enzymes responsible for steroid production (CYP17A1, CYP19A3) during the luteal phase of the oestrous cycle (days 10 to 11) and early pregnancy (days 10 to 11, 12 to 13, 15 to 16, 27 to 28). Orexin B suppressed E2 secretion by endometrial slices on days 10 to 11 and 15 to 16 of pregnancy, and days 10 to 11 of the cycle. In the myometrium, OXB inhibited E2 production on days 10 to 11 of pregnancy, whereas on days 12 to 13 it enhanced steroid output. Endometrial E1 release was potentiated by the peptide during all studied periods of the cycle and pregnancy, with the exception of days 12 to 13, when an inhibitory effect was observed. Myometrial secretion of E1 was increased, except on days 27 to 28. Testosterone secretion by endometrial slices was increased on days 12 to 13 and 27 to 28 of pregnancy. On days 10 to 11 of the cycle, T release was stimulated in response to the lowest and decreased under the influence of the highest dose of OXB. In the myometrium, T production was inhibited by OXB on days 10 to 11 of pregnancy and during the corresponding period of the cycle. On days 27 to 28 of pregnancy, T release was potentiated by the lowest dose of OXB. Expression of both genes was modified by OXB depending on the period of pregnancy and the type of examined uterine tissues. Our findings suggest that OXB, through modulation of uterine steroidogenesis, may have a regulatory role in the uterus.

Effect of progesterone on adiponectin system in the porcine uterus during early pregnancy.

The aim of this study was to investigate the influence of progesterone (P4) on adiponectin system genes and protein expression in the endometrium and myometrium during early gestation. Twenty-five gilts were assigned to 1 of 5 groups ( = 5): d 10 to 11 (embryo migration), 12 to 13 (maternal recognition of pregnancy), 15 to 16 (implantation), and 27 to 28 (end of implantation) of pregnancy and d 10 to 11 of the cycle (fully active corpora lutea, corresponding to the corpora lutea activity during gestation). The endometrial and myometrial tissues were cut into 100 mg slices, treated with P4 (10, 100, 1000 nM) and incubated for 24 h. Gene expression was analyzed by the real-time PCR method. Adiponectin secretion was determined by ELISA. Receptor protein content was defined using Western Blot analysis. In the endometrium, on d 10 to 11 of pregnancy, P4 stimulated adiponectin protein secretion. On those days, P4 enhanced adiponectin receptor type 1 () and type 2 () gene expression but inhibited both receptors' protein content. On d 12 to 13 of pregnancy, P4 inhibited adiponectin gene expression. During those period, P4 enhanced gene expression but suppressed both receptors' protein content. On d 15 to 16 of gestation, P4 increased adiponectin gene expression but inhibited the protein secretion. During those days, P4 suppressed gene expression and enhanced AdipoR2 protein content. On d 27 to 28 of gestation, P4 enhanced gene and AdipoR1 protein expression ( < 0.05). In the myometrium, on d 10 to 11 of gestation, P4 increased both receptors' gene expression but suppressed their protein content. On d 12 to 13 of pregnancy, P4 increased adiponectin and genes and AdipoR1 protein expression but decreased AdipoR2 protein content. On d 15 to 16 of gestation, P4 inhibited adiponectin gene expression. On those days, P4 enhanced gene and protein expression. On d 27 to 28 of gestation, P4 decreased adiponectin gene expression. On those days, P4 increased the myometrial AdipoR2 protein concentration and decreased gene protein expression ( < 0.05). Overall, the influence of P4 was found to be tissue specific and dose dependent. Results presented in this study indicate the modulatory effect of P4 on adiponectin system in the porcine uterus during early pregnancy, which may suggest the involvement of this adipokine in the early pregnancy establishment.

Effect of adiponectin on the steroidogenic acute regulatory protein, P450 side chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase gene expression, progesterone and androstenedione production by the porcine uterus during early pregnancy.

Adiponectin and its receptors are expressed in the human and porcine uterus and this endocrine system has important role in the regulation of reproductive processes. The expression of steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (HSD3B1) were observed in the human and porcine uterus during the oestrous cycle and pregnancy. The de novo synthesis of steroids in the uterus might be a crucial factor for effective implantation and maintenance of pregnancy. We hypothesized that adiponectin modulates the expression of key enzymes in the synthesis of the steroids: StAR, P450 side chain cleavage enzyme (CYP11A1) and HSD3B1, as well as progesterone (P4) and androstenedione (A4) secretion by the porcine uterus. Endometrial and myometrial explants harvested from gilts (n = 5) on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and on days 10 to 11 of the oestrous cycle were cultured in vitro in the presence of adiponectin (1, 10 µg/ml), adiponectin with insulin (10 ng/ml) and insulin alone (10 ng/ml). Gene expression was examined by real-time PCR, and the secretion of the steroids was determined by radioimmunoassay. The content of StAR, CYP11A1 and HSD3B1 mRNAs and the secretion of P4 and A4 was modulated by adiponectin in endometrial and myometrial tissue explants during early pregnancy and the oestrous cycle. In this action adiponectin interacted with insulin. Insulin itself also regulated the steroidogenic activity of the porcine uterus. ere we reported, for the first time, the expression of CYP11A1 genes in the porcine endometrium and myometrium. Our novel findings indicate that adiponectin affects basal and insulin-stimulated expression of key steroidogenic genes and production of steroid hormones by the porcine uterus during maternal recognition of pregnancy and implantation.

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.

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary.

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP - on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) - on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous 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.