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

Adiponectin is secreted by the white adipose tissue and is one of the most important hormones that regulate metabolic homeostasis. The expression of adiponectin and adiponectin receptor genes and proteins in reproductive organs, such as the testes, ovaries, and uterus, suggests that adiponectin is also involved in the regulation of reproductive functions. Changes in the expression of adiponectin and adiponectin receptor genes and proteins in the porcine uterus during the estrous cycle and early pregnancy imply that adiponectin activity may be controlled by the local hormonal milieu. Estrone (E1) and estradiol (E2) are the key steroid hormones that regulate reproductive functions, including the early recognition of pregnancy and implantation. We hypothesize that E1 and E2 may regulate the expression of the adiponectin system in a pregnant uterus. The aim of this study was to investigate the influence of E1 and E2 on the expression of adiponectin and its receptor genes and proteins by porcine endometrial and myometrial explants harvested from gilts (n = 5 per group) 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 estrous cycle. The expression of adiponectin and AdipoRs genes was examined with the real-time polymerase chain reaction, adiponectin secretion was evaluated with the ELISA method, and the expression of receptor proteins was determined using the Western blotting method. The results revealed that both E1 and E2 significantly influenced the expression of the adiponectin gene, hormone secretion in vitro, and the expression of AdipoRs genes and proteins. The influence of E1 and E2 on the expression of the adiponectin system varied in the early gestation, during the estrous cycle and between different stages of gestation. The examined steroids had a tissue-specific and a dose-dependent effect. This is the first ever study to describe the modulatory effect of E1 and E2 on the expression of the adiponectin system in the porcine uterus during early gestation.

Adiponectin, orexin A and orexin B concentrations in the serum and uterine luminal fluid during early pregnancy of pigs.

Adiponectin is the most abundant adipose-released protein that circulates in human plasma at high concentrations. The neuropeptides orexin A (OXA, hypocretin-1) and orexin B (OXB, hypocretin-2) are derived from a common precursor peptide, prepro-orexin and are produced mainly by neurons located in the lateral hypothalamus. It has been demonstrated that the peptides such as adiponectin and orexins have an important role in the regulation of energy metabolism and neuroendocrine functions. These hormones appear to be implicated in both normal and disturbed pregnancy. The objectives of this study were to determine adiponectin and orexin concentrations in the plasma and uterine luminal fluid (ULF) of pigs during early gestation and to explore the relationships between hormone concentrations and stages of pregnancy. The greatest plasma concentrations of adiponectin were observed on days 15-16 and 27-28 of pregnancy, and the least concentrations were on days 30-32 of gestation and on days 10-11 of the oestrous cycle. In ULF, adiponectin concentrations were greater on days 15-16 of pregnancy and on days 10-11 of the oestrous cycle than on days 10-11 and days 12-13 of pregnancy. The greatest OXA concentrations in the blood plasma were noted on days 10-16 of gestation, and the least OXA concentrations were on days 27-32 of pregnancy and on days 10-11 of the oestrous cycle. Orexin A concentrations in ULF were greater on days 10-11 of the cycle than throughout pregnancy. Serum OXB concentrations were greatest on days 10-11 and 30-32 of pregnancy, and least on days 12-28 of gestation. The greatest OXB concentrations in ULF were on days 10-13 of gestation, and the least OXB concentrations were on days 15-16 of pregnancy. This is first study to demonstrate the presence of adiponectin and orexins in the serum and ULF during early pregnancy of pigs as well as the relationships between adiponectin and orexin concentrations and the stage of pregnancy. The fluctuations in adiponectin and orexin concentrations in the plasma and ULF suggest that the hormones present in ULF are mostly of local origin and that these hormones participate in the processes that accompany early pregnancy.

Modulation of adiponectin system expression in the porcine uterus during early pregnancy by prostaglandin E2 and F2α.

Studies have demonstrated that adiponectin could be a link between reproductive functions and energy metabolism in animals. The aim of the present study was to investigate the effects of prostaglandin (PG) E2 and PGF2α (10, 50, 100, 250 and 500ngmL-1) on the expression and secretion of adiponectin and its receptor genes and proteins by cultured in vitro porcine endometrial and myometrial tissues on Days 10-28 of pregnancy and Days 10-11 of the oestrous cycle. The gene expression was analysed using the real-time PCR method. Adiponectin protein secretion was determined by ELISA, whereas the receptors proteins content was defined using Western Blot analysis. Both PGE2 and PGF2α modulated the expression of adiponectin system genes and proteins in the uterus during early pregnancy. PGE2 and PGF2α had similar effects on the adiponectin system, which differed between the stages of gestation and between pregnancy and the oestrous cycle. On Days 10-11 of gestation, PGE2 and PGF2α generally increased adiponectin secretion by endometrial and myometrial tissues. Both PGs decreased levels of endometrial adiponectin receptor type 1 (AdipoR1), whereas only PGF2α decreased myometrial levels of AdipoR1. Both PGs increased myometrial adiponectin receptor type 2 (AdipoR2) levels. On Days 12-13 of gestation, PGE2 decreased AdipoR1 concentrations in both tissues and AdipoR2 levels in the endometrium. PGF2α decreased myometrial concentrations of both receptors. On Days 15-16 of gestation, both PGE2 and PGF2α increased concentrations of AdipoR1 and AdipoR2 in the endometrium and myometrium. PGE2 stimulated the secretion of adiponectin in the endometrium, but not in the myometrium. On Days 27-28 of pregnancy, both PGE2 and PGF2α inhibited the expression of AdipoR1 and AdipoR2 in endometrial and myometrial tissues and decreased the secretion of endometrial adiponectin. Both PGE2 and PGF2α had tissue-specific and dose-dependent effects on the adiponectin system.

The influence of adiponectin on the transcriptomic profile of porcine luteal cells.

Reproductive functions are closely related to nutritional status. Recent studies suggest that adiponectin may be a hormonal link between them. Adiponectin is an adipocytokine, abundantly expressed in adipose tissues. It plays a dominant role in lipid and carbohydrate metabolism by stimulating fatty acid oxidation, decreasing plasma triglycerides, and increasing cells' sensitivity to insulin and has direct antiatherosclerotic effects. The hormone is also postulated to play a modulatory role in the regulation of the reproductive system. The aim of this study was to identify differentially expressed genes (DE-genes) in response to adiponectin treatment of porcine luteal ovarian cells. The global expression of genes in the porcine ovary was investigated using the Porcine (V2) Two-color gene expression microarray, 4 × 44 (Agilent, USA). Analysis of the microarray data showed that 701 genes were differentially expressed and 389 genes showed a fold change greater than 1.2 (p < 0.05). Among this number, 186 genes were up-regulated and 203 were down-regulated. The list of DE-genes was used for gene ontology analyses. The biological process list was generated from up-regulated and down-regulated DE-genes. We found that up-regulated products of DE-genes take part in 30 biological processes and down-regulated products in 9. Analysis of the interaction network among DE-genes showed that adiponectin interacts with genes involved in important processes in luteal cells. These results provide a basis for future work describing the detailed interactions and relationships explaining local regulation of adiponectin actions in the ovary of pigs.

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 adiponectin and adiponectin receptors 1 and 2 in the porcine uterus, conceptus, and trophoblast during early pregnancy.

Adiponectin, one of the several adipocytokines secreted mainly by the adipose tissue, plays an important role in regulating energy homeostasis and controls female fertility. Female reproductive functions are closely associated with nutritional status, and adiponectin seems to be an important factor linking the regulation of metabolic homeostasis with reproductive processes. The biological activity of adiponectin is mediated by two distinct receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). The objective of this study was to determine the presence of and changes in the gene and protein expression pattern of adiponectin and its receptors in the porcine uterus during early pregnancy and on Days 10 to 11 of the estrous cycle and in the conceptus and trophoblast. The highest level of adiponectin transcript was observed on Days 15 to 16 of gestation, Days 10 to 11 of the cycle in the endometrium, and Days 15 to 16 of gestation in the myometrium. The highest expression of AdipoR1 and AdipoR2 genes was detected on Days 10 to 11 of gestation in the endometrium, and Days 12 to 13 in the myometrium. The highest content of adiponectin protein was noted on Days 12 to 13 and 30 to 32 of gestation in the endometrium and Days 10 to 11 of the cycle in the myometrium. The expression of adiponectin protein was higher on Days 27 to 28 and 30 to 32 in the conceptuses. AdipoR1 protein content in the myometrium was highest on Days 12 to 13 and 30 to 32. In contrast, in the endometrium, it was more constant. The highest content of AdipoR2 protein was detected on Days 15 to 16 and 30 to 32 of gestation, Days 10 to 11 of the cycle in the endometrium, and Days 10 to 11 of gestation in the myometrium. In the conceptuses, the highest AdipoR1 protein content was observed on Days 15 to 16, and the highest AdipoR2 protein expression was determined on Days 15 to 16 and 27 to 28. In the trophoblasts, AdipoR1 protein content was higher on Days 27 to 28 than on Days 30 to 32, whereas the expression of AdipoR2 was higher on Days 30 to 32. This study demonstrated the presence of adiponectin and its receptors in the uteri, conceptuses, and trophoblasts of pregnant pigs and that the local adiponectin system is dependent on the stage of pregnancy.

Expression of orexins and their precursor in the porcine ovary and the influence of orexins on ovarian steroidogenesis in pigs.

Orexins A and B are hypothalamic neuropeptides associated with homeostasis and the reproductive system. The aim of the study was to compare the expression of the prepro-orexin gene and the intensity of orexins immunoreactivity in the porcine ovary (corpora lutea, granulosa and theca interna cells) during four different stages of the oestrous cycle (days: 2-3, 10-12, 14-16 and 17-19) and to examine the in vitro effect of orexins on the secretion of steroid hormones by porcine luteal, granulosa and theca interna cells. The highest expression of prepro-orexin mRNA was observed in theca interna cells on days 17-19 of the oestrous cycle. The highest content of immunoreactive orexin A was noted in corpora lutea on days 10-12 and the highest level of immunoreactive orexin B on days 14-16 of the cycle. Immunoreactive orexin A concentrations were higher in theca interna cells than in granulosa cells, whereas similar levels of immunoreactive orexin B were observed in both cell types. Under in vitro conditions, at the concentration of 10 nM, orexins A and B inhibited FSH-induced oestradiol secretion by granulosa cells. The obtained results suggest that the pattern of orexin peptide expression in the porcine ovary is related to the animals' hormonal status. Our findings imply that orexins can affect porcine reproductive functions through modulation of ovarian steroidogenesis.

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.

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.

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.

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

BACKGROUND: Adiponectin, 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. METHODS: The 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. RESULTS: The 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). CONCLUSIONS: Our 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.

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.