Nina Smolinska

Expression of adiponectin and adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine uterus during the oestrous cycle

Adiponectin is a hormone secreted primarily by white adipose tissue. Recent studies have shown that adiponectin and its receptors (AdipoR1 and AdipoR2) are expressed in different reproductive tissues, including the ovary and uterus. This newly discovered endocrine system plays an important role in the regulation of reproductive processes. The expression of the adiponectin system in the porcine uterus during the oestrous cycle has not been researched to date. The aim of the present study was to investigate the presence and changes in adiponectin system expression in the porcine uterus on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. The expression of the adiponectin gene was highest on days 14-16 and 2-3 in the endometrium and myometrium, respectively. In the endometrium, the content of AdipoR1 and AdipoR2 mRNAs was highest on days 10-12, whereas significantly higher expression levels of both genes were noted in the myometrium on days 17-19. The highest content of adiponectin and AdipoR1 protein in the endometrium was reported on days 2-3. In the myometrium, the expression levels of both receptor proteins were significantly higher on days 17-19. Adiponectin system proteins were localized in endometrial epithelial glandular cells, luminal epithelial cells and stromal cells as well as in longitudinal and circular muscles of the myometrium. This study demonstrated the presence of adiponectin, AdipoR1 and AdipoR2 genes and proteins in the porcine uterus and the effect of the stage of the oestrous cycle on the expression of the adiponectin system. Our results suggest that locally synthesized adiponectin directly affects uterine functions.

Adiponectin expression in the porcine pituitary during the estrous cycle and its effect on LH and FSH secretion

Female reproductive success is closely associated with nutritional status and energy balance. In this context, adiponectin appears to be a key hormone connecting reproductive system function and metabolism regulation. It is hypothesized that adiponectin expression in the pituitary depends on the phase of the estrous cycle. The effect of adiponectin on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion is also postulated. Changes in the adiponectin gene and protein expression in the porcine anterior (AP) and posterior (NP) pituitaries as well as the effect of in vitro administration of adiponectin on basal and gonadotropin-releasing hormone (GnRH)- and/or insulin-stimulated LH and FSH secretion were investigated on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle. Adiponectin gene was more pronounced on days 2-3 in AP but on days 10-12 in NP. Protein concentration in AP was the highest on days 10-12 and in NP on days 10-12 and 17-19 of the cycle. In vitro, adiponectin did not affect basal LH secretion but increased FSH release by AP cells. Adiponectin administration affected GnRH- and/or insulin-induced LH and FSH output in a manner dependent on the phase of the estrous cycle. In this study we indicated for the first time adiponectin expression in the porcine AP and NP that was dependent on the phase of the estrous cycle. In vitro studies indicated that adiponectin may affect gonadotropin secretion. The above suggests that the studied adipokine may influence female reproductive functions via its effect on LH and FSH secretion by gonadotrophs, but the cellular mechanism of its action remains unknown.

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

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2-3 relative to days 10-12, 14-16 and 17-19 (p<0.05). In NP the OX1R mRNA level was elevated on days 10-12 compared to the remaining stages (p<0.05). OX2R gene expression in AP was the lowest on days 10-12 (p<0.05 compared to days 2-3 and 17-19) and the expression peak occurred on days 17-19 (p<0.05 vs. the all studied stages). In NP the highest (p<0.05) expression of OX2R mRNA was noted on days 17-19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10-12 (p<0.05), whereas in NP it was greatest on days 2-3 and 14-16 (p<0.05 vs. days 10-12 and 17-19). In both cases the lowest OX1R protein expression was observed during follicular phase (p<0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p<0.05) on days 2-3 and 14-16 compared to days 10-12 and 17-19. In NP the lowest (p<0.05) expression of this protein was on days 17-19 and the highest on days 10-12 (p<0.05 compared to days 2-3 and 17-19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.

Expression of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine pituitary during the oestrous cycle.

BackgroundAdiponectin, protein secreted mainly by white adipose tissue, is an important factor linking the regulation of metabolic homeostasis and reproductive processes. The biological activity of the hormone is mediated via two distinct receptors, termed adiponectin receptor 1(AdipoR1) and adiponectin receptor 2 (AdipoR2). The present study analyzed mRNA and protein expression of AdipoR1 and AdipoR2 in the anterior (AP) and posterior (NP) pituitary of cyclic pigs.MethodsThe total of 20 animals was assigned to one of four experimental groups (n = 5 per group) as follows: days 2–3 (early-luteal phase), 10–12 (mid-luteal phase), 14–16 (late-luteal phase), 17–19 (follicular phase) of the oestrous cycle. mRNA and protein expression were analyzed using real-time PCR and Western Blot methods, respectively.ResultsThe lowest AdipoR1 gene expression was detected in AP on days 10–12 relative to days 2–3 and 14–16 (p < 0.05). In NP, AdipoR1 mRNA levels were elevated on days 10–12 and 14–16 (p < 0.05). AdipoR2 gene expression in AP was the lowest on days 10–12, and an expression peak occurred on days 2–3 (p < 0.05). In NP, the lowest (p < 0.05) expression of AdipoR2 mRNA was noted on days 17–19. The AdipoR1 protein content in AP was the lowest on days 17–19 (p < 0.05), while in NP the variations in protein expression levels during the oestrous cycle were negligible. AdipoR2 protein in AP was most abundant on days 10–12, and it reached the lowest level on days 2–3 and 17–19 of the cycle (p < 0.05). The presence of AdipoR2 protein in NP was more pronounced on days 10–12 (p < 0.05).ConclusionsOur study was the first experiment to demonstrate that AdipoR1 and AdipoR2 mRNAs and proteins are present in the porcine pituitary and that adiponectin receptors expression is dependent on endocrine status of the animals.

Adiponectin Expression in the Porcine Ovary during the Oestrous Cycle and Its Effect on Ovarian Steroidogenesis

Adiponectin is an adipose-secreted hormone that regulates energy homeostasis and is also involved in the control of the reproductive system. The goal of the present study was to investigate changes in adiponectin gene and protein expression in porcine ovarian structures during the oestrous cycle and to examine the effects of in vitro administration of adiponectin on basal and gonadotrophin- and/or insulin-induced secretion of ovarian steroid hormones. Both gene and protein expression of adiponectin were enhanced during the luteal phase of the cycle. Adiponectin affected basal secretion of progesterone by luteal cells, oestradiol by granulosa cells, and testosterone by theca interna cells. The gonadotrophin/insulin-induced release of progesterone from granulosa and theca interna cells and the release of oestradiol and androstenedione from theca cells was also modified by adiponectin. In conclusion, the presence of adiponectin mRNA and protein in the porcine ovary coupled with our previous results indicating adiponectin receptors expression suggest that adiponectin may locally affect ovarian functions. The changes in adiponectin expression throughout the oestrous cycle seem to be dependent on the hormonal status of pigs related to the stage of the oestrous cycle. The effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs.

Localization of orexin A and orexin B in the porcine uterus

The presence of orexins and their receptors in gonads indicate that these hormones participate in the control of reproductive functions. The aim of the study was to compare the expression of the prepro-orexin (PPO) gene in porcine endometrium and myometrium and the intensity of OXA- and OXB-immunoreactivity in the following uterine structures: endometrial glandular and luminal epithelium and stroma as well as the myometrial longitudinal and circular muscles during the four stages (days 2-3, 10-12, 14-16, 17-19) of the estrous cycle. The highest expression of PPO mRNA was observed in the endometrium and the myometrium on days 14-16 of the cycle. The expression of the PPO gene on days 2-3 was more pronounced in the myometrium than in the endometrium, whereas on days 17-19 the gene expression was markedly higher in the endometrium. The OXA signal intensity was highest on days 2-3 in the luminal epithelium and on days 2-3 and 10-12 in the stroma. In circular muscles of the myometrium, the highest immunoreactivity was found on days 2-3 and 10-12, while in longitudinal muscles on days 2-3. OXB-immunoreactivity was highest on days 10-12 in longitudinal muscles, on days 17-19 in glandular epithelium and stroma, and on days 10-12 and 14-16 in luminal epithelium. Our results suggest that orexin A and B are produced in the porcine uterus and that their release is dependent on the hormonal status of animals.

Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle

Leptin, the product of the OB gene, is a 16-kDa polypeptide of 146 amino acid residues produced mainly by adipocytes that regulates metabolism and reproduction. The actions of leptin are mediated mainly via the long form of the leptin receptor (OB-Rb). The identification of leptin and OB-Rb mRNAs and proteins in human and mouse endometrium, and placental trophoblast suggests that leptin may be involved in the implantation process. Thus, the aim of this study was to compare the expression levels of porcine OB-Rb mRNA and protein in the endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16, respectively) as well as during two stages of pregnancy respondent to the beginning of the implantation process (days 14-16) and the post-implantation period (days 30-32), and in trophoblast during both periods of pregnancy. OB-Rb gene expression in endometrium during the examined stages of pregnancy and the mid- and late-luteal phases of the cycle was at the same level. In contrast, in myometrium leptin receptor gene expression decreased on days 14-16 of pregnancy compared to both phases of the cycle, and on days 30-32 of pregnancy in relation to late-luteal phase. OB-Rb protein expression in the tissues was lower during the examined stages of pregnancy in comparison to the mid- and late-luteal phases of the cycle. In trophoblast, OB-Rb mRNA and protein expression was higher on days 30-32 than during days 14-16 of pregnancy. In conclusion, our results might suggest that leptin can participate in the control of pig reproduction by exercising its action at the uterine and trophoblast level and have a direct effect on these organ during both the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in tissues of pig reproductive tract strongly suggest that their sensitivity to leptin varies throughout luteal phase of the cycle and early pregnancy.

EXPRESSION OF ADIPONECTIN RECEPTORS 1 AND 2 IN THE OVARY AND CONCENTRATION OF PLASMA ADIPONECTIN DURING THE OESTROUS CYCLE OF THE PIG

The aim of this study was to compare the expression levels of adiponectin receptor 1 and adiponectin receptor 2 mRNAs and proteins in porcine ovaries during four stages (days 2 to 3, 10 to 12, 14 to 16, 17 to 19) of the oestrous cycle and to measure adiponectin plasma concentrations during the same phases of the cycle. Higher mRNA expression of adiponectin receptor 1 was detected in porcine granulosa cells than in corpora lutea and theca cells (P < 0.01). In contrast, higher gene expression of adiponectin receptor 2 occurred in newly developed and mature corpora lutea (P < 0.01). The adiponectin receptor 1 protein content was the highest in corpora lutea isolated on days 2 to 3 of the cycle and was the lowest in theca interna cells (P < 0.01). The profile of adiponectin receptor 2 protein was similar to that of adiponectin receptor 1. Adiponectin plasma concentrations were significantly higher throughout the luteal phase than in the follicular phase (P < 0.01). In conclusion, the presence of adiponectin receptor 1 and adiponectin receptor 2 mRNAs and proteins in the porcine ovary suggests that adiponectin may directly affect ovarian functions through its own specific receptors. The expression of both receptors and adiponectin plasma concentration were dependent on hormonal status related to the stage of the cycle.

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.

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.