The role of the orexin (hypocretin) system in controlling energy homeostasis, endocrine system, and reproduction in pigs and other living organisms.

Orexins A (OXA) and B (OXB) (hypocretin 1 and 2) are neuropeptides produced in the brain and peripheral tissues. Biological activities of orexins are mediated through activation of two G-protein coupled receptors termed as orexin 1 receptor (OX1R) and orexin 2 receptor (OX1R). Orexin system (OXA, OXB, OX1R, OX2R) was implicated in controlling sleep, energy expenditure, appetite, reproduction as well as metabolism and energy homeostasis. In this review, we summarize the current knowledge regarding the role of the orexin system in controlling porcine physiology. Particularly, we review and discuss evidence indicating that in pig and other living organisms, orexins and their receptors modulate the energy homeostasis, reproduction as well as functions of peripheral tissues including the pancreas, adrenal glands, gastro-intestinal tract and adipose tissue.

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.

In vitro effects of luteinizing hormone, progesterone and oestradiol-17ß on leptin gene expression and leptin secretion by porcine luteal cells obtained in early pregnancy.

Leptin, the product of the ob gene, is involved in the control of ovarian functions. Leptin transcripts and OB proteins were localized in the porcine corpus luteum, but the regulatory mechanism of leptin mRNA expression and leptin secretion in porcine luteal cells remains unexplained. The aim of the present study was to: 1) determine level of leptin and long form of leptin receptor (OB-Rb) transcript/protein in dispersed porcine luteal cells on days 14-16 of pregnancy and 2) examine, in vitro, the effects of luteinizing hormone (LH), 17ß-oestradiol (E2) and progesterone (P4) on leptin gene expression and leptin secretion by those cells. Isolated luteal cells were first precultured (48 hours) and then cultured in the presence or absence of luteinizing hormone (LH) (1; 10; 100 ng/ml), E2 (0.02; 0.2; 2; 20 ng/ml) and P4 (20; 100; 200 ng/ml) for 24 hours. Leptin and OB-Rb transcript/protein were detected in porcine luteal cells by real-time PCR and fluorescence immunocytochemistry (F-ICC), respectively. A higher level of leptin mRNA expressions in the cells was observed in the presence LH (10 ng/ml), E2 (0.02 ng/ml) and P4 (200 ng/ml). The results of an RIA assay revealed increased leptin secretion when the luteal cells were treated with E2 (20 ng/ml) and P4 (200 ng/ml). LH did not affect leptin release by those cells. We conclude: 1) that leptin and OB-Rb genes/proteins are expressed in porcine luteal cells and 2) there is a modulatory effect of: LH, E2 and P4 on leptin mRNA expression as well as E2 and P4 on leptin secretion by those cells obtained in early pregnancy.

Ontogeny of the long form of leptin receptor gene expression in the porcine ovarian follicles.

Leptin is a polypeptide hormone produced predominantly in adipocytes. It has been found to be implicated in the regulation of satiety and energy homeostasis. A role for leptin in reproduction was later suggested by findings that this hormone may be involved in the regulation of the hypothalamic-pituitary-gonadal axis via endocrine, paracrine and/or autocrine pathways. The objective of the study was to investigate the ontogeny of the long isoform of leptin receptor (OB-Rb) gene in porcine ovarian follicles. The expression of OB-Rb gene was detected in porcine primordial, primary, secondary and antral follicles by in situ hybridization. In summary, our data suggest that leptin might have a direct effect on porcine follicles and plays an important role in the follicular development.

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.

Expression of leptin and its receptor genes in the ovarian follicles of cycling and early pregnant pigs.

Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic-pituitary-gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs.

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine hypothalamus during the oestrous cycle.

Orexin A and B are hypothalamic peptides involved in the control of food intake, sleep patterns, autonomic and neuroendocrine systems. The biological actions of orexins are mediated via two G-protein coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). The present study analyzed mRNA and protein expressions of OX1R and OX2R in porcine hypothalamic structures engaged in GnRH production and secretion, preoptic area (POA), mediobasal hypothalamus (MBH), and stalk median eminence (SME) on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. The highest OX1R gene expression in POA was observed on days 17-19 of the cycle. Changes in the mRNA expression in MBH and SME throughout the cycle were negligible. The expression peak of OX2R gene in POA and SME occurred on days 17-19 as well. There were no changes in the gene expression in MBH. OX1R protein content in POA and SME also was the greatest on days 17-19 and OX2R protein expression was most pronounced in MBH and SME during the same phase of the cycle. In conclusion, fluctuation of OX1R and OX2R mRNAs and proteins content in pig hypothalamus dependently on the phase of the oestrous cycle suggests that orexins, through the influence on the hypothalamic-pituitary-ovarian axis, may affect reproductive functions.

Leptin gene and protein expression in the ovary during the oestrous cycle and early pregnancy in pigs.

Leptin, the product of the obese gene, is the hormone originally identified in adipocytes. It is involved in the control of satiety and energy metabolism. More recent observations suggest that leptin plays an important role in reproduction. Leptin mRNA and protein have been found in the human and the murine ovary. However, the expression of leptin in the porcine ovary has not been examined. Therefore, the aim of the present work was to compare the expression levels of porcine leptin mRNA by semiquantitative RT-PCR and in situ hybridization, as well as leptin protein by Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during mid- and late-luteal phase of the oestrous cycle as well as during days 14-16 and 30-32 of pregnancy. Leptin gene and protein expression in CL was increased on days 14-16 of the cycle compared with pregnant animals. Leptin gene expression in OS was higher during the late-luteal phase of the cycle than on days 30-32 after conception. However, comparison of leptin protein expression in OS between days 14-16 of the cycle and days 30-32 of pregnancy indicates a higher protein expression during pregnancy. Moreover, leptin gene expression was higher in porcine CL and OS on days 14-16 of pregnancy in comparison to days 30-32. Contrary to leptin mRNA expression, a higher leptin protein expression was observed on days 30-32 compared with days 14-16 after conception. In summary, the present study provides the first evidence that leptin mRNA and protein occur in porcine ovary and vary during the oestrous cycle and pregnancy. Moreover, the obtained results indicate that also locally synthesized leptin may participate in the control of pig reproduction by exercising its action at the ovarian level.

Expression of leptin and long-form leptin-receptor proteins in porcine hypothalamus during oestrous cycle and pregnancy.

In this study, we examined the levels of leptin and OB-Rb protein expression in the discrete areas of the porcine hypothalamus (mediobasal hypothalamus--MBH, pre-optic area--POA, stalk median eminence--SME) during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16) as well as two stages of pregnancy (days 14-16 and 30-32). The analysis showed that during the cycle, leptin protein expression in MBH was higher in the mid-luteal phase than late-luteal phase. In the case of OB-Rb protein expression, a higher level was observed in MBH during the late-luteal phase in comparison to the mid-luteal phase, whereas in POA and SME the opposite dependence was noticed. In turn, during pregnancy, leptin protein expression in MBH and POA, and OB-Rb protein expression in POA were more pronounced on days 14-16 than on days 30-32. In contrast, leptin protein content in SME as well as OB-Rb protein in MBH and SME was higher on days 30-32 than during the earlier stage of pregnancy. Comparison of leptin and OB-Rb protein expression between the cycle (days 10-12) and pregnancy showed a higher level of leptin and OB-Rb protein contents in POA as well as in SME during pregnancy (on days 14-16 and 30-32, respectively). Yet, OB-Rb protein expression in POA on days 30-32 of pregnancy was lower in comparison to days 10-12 of the cycle. Furthermore, during pregnancy, leptin protein expression in MBH was lower (days 14-16 and 30-32), whereas OB-Rb protein expression in that area of hypothalamus was higher (days 30-32) in comparison to the mid-luteal phase. Our results indicate that both leptin and OB-Rb are synthesized in the porcine hypothalamus and suggest the participation of leptin in auto/paracrine regulation of these brain areas functions, including control of reproduction during the oestrous cycle and early pregnancy.

Leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs.

Leptin is a 16-kDa protein hormone encoded by the obese (ob) gene and acts on receptors in the hypothalamus to regulate food intake and energy balance. The identification of leptin and its receptor mRNAs and proteins in human and mouse endometrium and placental trophoblast has attracted attention to the potential role of leptin in implantation. Thus, the aim of this study was to compare the expression levels of porcine leptin mRNA and protein in endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of pregnancy respondent to the beginning (days 14 - 16) and the end (days 30 - 32) of the implantation process, and in trophoblast during both periods of pregnancy. Leptin gene and protein expression in myometrium, and leptin mRNA expression in endometrium was more pronounced in the mid- and late-luteal phases of the cycle in comparison to studied periods of pregnancy, whereas leptin protein concentration in endometrium was either enhanced on days 30 - 32 of pregnancy in relation to days 14 - 16 of the cycle or there were no changes between pregnancy and luteal phase of the cycle. On days 30 - 32 of pregnancy, expression of the leptin gene in the endometrium, and of the leptin gene and protein in the myometrium was more pronounced in comparison to the earlier stage of pregnancy. Moreover, leptin gene expression in porcine trophoblast increased during the beginning of the implantation process compared to days 30 - 32 of pregnancy, while the protein concentration decreased on days 14 - 16 of pregnancy. In conclusion, the finding of leptin gene and protein expression in porcine endometrium, myometrium and trophoblast indicates that locally synthesised leptin can participate in the control of pig reproduction. The fluctuation of the hormone concentration during pregnancy and changes in its level between pregnancy and the oestrous cycle may indicate leptin's involvement in the implantation process.

Expression of leptin and long form of leptin receptor genes and proteins in pituitary of cyclic and pregnant pigs.

Leptin is a multifunctional regulator in numerous tissues, including the pituitary. It is not known, whether the porcine pituitary is a source of leptin synthesis and possesses the leptin receptor protein. It is also unknown, if a relationship exists between expression levels of these proteins in the pituitary and physiological status of sows. Therefore, the aim of the study was 1] to examine, by Western-blotting analysis, the expression levels of leptin and the long form of leptin receptor (OB-Rb) in the porcine anterior (AP) and posterior (NP) pituitary gland during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of early pregnancy (days 14 - 16 and 30 - 32); and 2] to localise, using in situ hybridisation method (ISH), the expression of leptin and OB-Rb genes in the pituitary gland in the above mentioned stages of the cycle and pregnancy. Western-blotting analysis showed that leptin protein expression in AP was higher in the late-luteal phase than in the mid-luteal phase, while OB-Rb protein expression in both lobes was higher in the mid-luteal phase. In turn, during pregnancy leptin protein content in AP and OB-Rb protein content in NP were more pronounced on days 14 - 16 than on days 30 - 32. Comparison of leptin and OB-Rb protein expression levels in AP between the mid-luteal phase and two periods of pregnancy showed, respectively, stimulation of leptin protein and inhibition of OB-Rb protein expressions during both examined stages of pregnancy. Taking AP from late-luteal phase as the point of reference, it was revealed stimulation of leptin expression during earlier period of pregnancy, whereas on days 30 - 32 of pregnancy both the hormone and its receptor expressions were diminished. In turn, comparison of leptin and OB-Rb protein expression levels in NP between the late-luteal phase and days 14 - 16 or 30 - 32 of pregnancy showed inhibition of leptin protein expression and stimulation of OB-Rb protein expression during pregnancy. Moreover, ISH studies localised leptin and OB-Rb mRNAs expression in the cells of AP as well as NP tissue during the two stages of the cycle and pregnancy. In conclusion, our findings suggest that leptin is produced within the pituitary in the pig and may participate in auto/paracrine manner in the regulation of this gland function during the luteal phase of the oestrous cycle and early pregnancy.

The expression of short from of leptin receptor gene during early pregnancy in the pig examined by quantitative real time RT-PCR.

The study was conducted to determine gene expression of short form of leptin receptor (OB-Rs) using real time RT-PCR in distinct tissues of the central nervous system (medial basal hypothalamus, preoptic area, stalk median eminence), pituitary and reproductive tract (corpus luteum, ovarian stroma, endometrium, myometrium, and trophoblast) in pigs during luteal phase of the cycle and early gestation. The expression of OB-Rs mRNA in SME did not differ between analyzed stages of the cycle and pregnancy. In anterior pituitary, transcript levels were almost identical in mid- and late-luteal periods, but significantly decreased on 30-32 day of gestation when compared with day 14-16. In posterior pituitary, significantly higher expression was observed in two periods of pregnancy when compared with two stages of luteal phase. In corpus luteum the lowest expression was observed during days 10-12 of the cycle, whereas markedly higher levels were detected in late-luteal stage and gestation. In ovarian stroma the expression of Ob-Rs mRNA was markedly diminished during days 14-16 of the cycle when compared with days: 10-12 of the cycle and 30-32 of pregnancy. The expression of Ob-Rs mRNA in endometrium and myometrium reached the lowest levels on 30-32 day of pregnancy in comparison with earlier stage, 14-16 day. Summarizing, the expression of the short form of leptin receptor mRNA was found in majority of tested tissues including hypothalamus, pituitary and reproductive tract and their levels fluctuated depending on the phase (mid- and late-luteal) of the cycle and the day of pregnancy (early and late stage of implantation).