Plasma level and expression of visfatin in the porcine hypothalamus during the estrous cycle and early pregnancy.

Visfatin appears to be an energy sensor involved in the regulation of female fertility, which creates a hormonal link integrating the control of energy homeostasis and reproduction. This study evaluates the expression levels of visfatin gene and protein in selected areas of the porcine hypothalamus responsible for gonadotropin-releasing hormone synthesis: the mediobasal hypothalamus (MBH) and preoptic area (POA), and visfatin concentrations in the blood plasma. The tissue samples were harvested from gilts on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle, and on days 10-11, 12-13, 15-16, 27-28 of pregnancy. Visfatin was localized in the cytoplasm and nucleus of cells creating both studied hypothalamic structures. The study demonstrated that visfatin gene and protein expression in MBH and POA depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Blood plasma concentrations of visfatin during the estrous cycle were higher on days 2-3 in relation to other studied phases of the cycle, while during early pregnancy, the highest visfatin contents were observed on days 12-13. This study demonstrated visfatin expression in the porcine hypothalamus and its dependence on the hormonal milieu related to the estrous cycle and early pregnancy.

Orexin B affects the transcriptome of incubated in vitro porcine endometrial explants from the early-implantation period.

This study determined the effect of orexin B (OXB) on the porcine endometrial transcriptome during the embryo attachment phase. Microarray analyses of gene ontology (GO), biological pathways, networks and differentially expressed genes (DEG) were performed. Orexin B influenced the expression of 887 genes (fold change > 1.2; p < .05): 620 genes were up-regulated, and 267 were down-regulated. The analysis of the relationship between DEG revealed that OXB interacts with genes linked with processes such as cell hormone binding, regulation of hormone levels, lipid transport, steroid metabolic processes, the apoptotic signalling pathway and the acute inflammatory response, which are pivotal for reproductive success. Orexin B played a bivalent role in the early-pregnant uterus by limiting the pregnancy outcome, promoting embryo development, suppressing the immune system and, consequently, preventing embryo rejection. These findings suggest that OXB could be responsible for the proper course of gestation by adapting litter size to the metabolic status of the maternal organism.

The influence of orexin B on the transcriptome profile of porcine myometrial explants during early implantation.

This study aimed to determine the effect of orexin B (OXB) on the global expression pattern and the relationships among differentially expressed genes (DE-genes) in the transcriptome of myometrial explants during the early implantation period in the pig (day 15 of pregnancy). The changes in the transcriptome profile of the porcine myometrium were investigated using the Porcine (V2) Two-colour Gene Expression Microarray, 4 × 44. An analysis of the data from the microarray experiment revealed that 1540 DE-genes were affected by OXB, of which 1135 exhibited fold changes (FC) greater than 1.2 (P < 0.05). Among these, 576 genes were up-regulated and 559 genes were down-regulated. Among the affected biological processes in the myometrial tissue, 76 were enhanced and 31 were suppressed. Furthermore, the differential expression of nine genes, related to the regulation of reproductive functions and metabolic homeostasis, was confirmed by quantitative RT-PCR. A functional analysis of the relationships between DE-genes indicated that OXB interacts with the genes involved in the processes such as the inflammatory response, the response to interleukin-6, cytokine receptor activity, the regulation of cell activation, growth factor receptor binding, lipid modification and the steroid metabolic process. An analysis of DE-genes and their functional relationships suggests that OXB could be involved in the mechanisms such as the regulation of cell proliferation and development, inhibition of contractility, regulation of programmed cell death, and the development of blood vessels, all of which facilitate implantation.

The In Vitro Effect of Prostaglandin E2 and F2α on the Chemerin System in the Porcine Endometrium during Gestation.

Chemerin belongs to the group of adipocyte-derived hormones known as adipokines, which are responsible mainly for the control of energy homeostasis. Adipokine exerts its influence through three receptors: Chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C motif chemokine receptor-like 2 (CCRL2). A growing body of evidence indicates that chemerin participates in the regulation of the female reproductive system. According to the literature, the expression of chemerin and its receptors in reproductive structures depends on the local hormonal milieu. The aim of this study was to investigate the in vitro effect of prostaglandins E2 (PGE2) and F2α (PGF2α) on chemerin and chemerin receptor (chemerin system) mRNAs (qPCR) and proteins (ELISA, Western blotting) in endometrial tissue explants collected from early-pregnant gilts. Both PGE2 and PGF2α significantly influenced the expression of the chemerin gene, hormone secretion, and the expression of chemerin receptor genes and proteins. The influence of both prostaglandins on the expression of the chemerin system varied between different stages of gestation. This is the first study to describe the modulatory effect of PGE2 and PGF2α on the expression of the chemerin system in the porcine uterus during early gestation.

The in vitro effect of orexin a on the porcine myometrial transcriptomic profile during the early-implantation period.

In pigs, early gestation is the most critical period deciding about the reproduction success, and it depends on many processes, involving a significant number of genes and their products. Myometrium was found to be an important source of factors pivotal for a proper course of gestation. The aim of the study was to determine the effect of orexin A (OXA) on the porcine transcriptome, and the determination of relationships among differentially expressed genes (DEG) in the porcine myometrium during implantation using microarray technology. The analyses of gene ontology (GO), DEG assays, biological pathways and networks were performed. OXA affected the expression of 461 genes with fold-change values greater than 1.2 (p < 0.05). The expression of 260 genes were up-regulated and 201 down-regulated in the OXA-treated myometrium. Twelve genes were selected for qPCR validation of differential expression based on their known role in angiogenesis, immune processes, steroid hormone signaling and prostaglandins synthesis. The analysis of relationship between DEG indicated that OXA interacts with genes involved in the inflammatory response, cytokine binding, cytokine activity, interleukin production, leukocyte migration, angiogenesis and embryonic hemopoiesis. The presented results suggest that OXA may play a key role in ensuring optimal conditions for implanting embryos.

The effect of orexin a on the StAR, CYP11A1 and HSD3B1 gene expression, as well as progesterone and androstenedione secretion in the porcine uterus during early pregnancy and the oestrous cycle.

Orexin A (OXA) is primarily known for its involvement in the regulation of feeding behaviour, energy metabolism and sleep/wake cycle. Nevertheless, studies indicate its engagement in the regulation of the porcine reproductive system. Therefore, the aim of this study was to investigate OXA effect (1, 10, 100 nM), in the presence or absence of the selective orexin receptor type 1 antagonist (SB-3348667; 1 µM), on the gene expression of key steroidogenic enzymes: steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B1), as well as on progesterone (P4) and androstenedione (A4) secretion. Endometrial and myometrial tissue explants were collected from gilts 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 (n = 5 per studied period of pregnancy or mid-luteal phase of the oestrous cycle). Gene expression was evaluated by real-time PCR. The level of steroid hormones secreted into the culture medium was examined by radioimmunoassay (RIA). In the present study, in the endometrium, OXA significantly stimulated StAR expression on days 12 to 13, CYP11A1 expression on days 27 to 28 and HSD3B1 expression on days 15 to 16 of pregnancy. Further, in this tissue, OXA decreased StAR mRNA level on days 10 to 11, CYP11A1 mRNA level on days 15 to 16, as well as HSD3B1 mRNA level on days 10 to 11 and 12 to 13 of gestation. Regarding the myometrium, OXA stimulated CYP11A1 gene expression on days 15 to 16 of pregnancy. In this tissue, OXA decreased StAR transcript content on days 15 to 16 and CYP11A1 mRNA level on days 27 to 28. We also demonstrated that OXA alone enhanced P4 secretion in the endometrium on days 10 to 11 and 12 to 13 of gestation. OXA alone has no significant effect on endometrial and myometrial A4 secretion, whereas OXA in combination with OX1R antagonist increased this hormone secretion during all studied stages of pregnancy. Therefore, we can conclude that OXA may affect de novo synthesis and secretion of P4 and A4 in the porcine uterus via participating in the regulation of key steroidogenic enzymes gene expression, as well as modulating steroid hormones secretion during early pregnancy and mid-luteal phase of the oestrous cycle in pigs. However, further research is required to explain the exact role of OXA in the porcine uterus.

Expression of Chemerin and Its Receptors in the Porcine Hypothalamus and Plasma Chemerin Levels during the Oestrous Cycle and Early Pregnancy.

Chemerin (CHEM) may act as an important link integrating energy homeostasis and reproductive functions of females, and its actions are mediated by three receptors: chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C motif chemokine receptor-like 2 (CCRL2). The aim of the current study was to compare the expression of CHEM and its receptor (CHEM system) mRNAs (quantitative real-time PCR) and proteins (Western blotting and fluorescent immunohistochemistry) in the selected areas of the porcine hypothalamus responsible for gonadotropin-releasing hormone production and secretion: the mediobasal hypothalamus, preoptic area and stalk median eminence during the oestrous cycle and early pregnancy. Moreover, plasma CHEM concentrations were determined using ELISA. The expression of CHEM system has been demonstrated in the porcine hypothalamus throughout the luteal phase and follicular phase of the oestrous cycle, and during early pregnancy from days 10 to 28. Plasma CHEM levels and concentrations of transcripts and proteins of CHEM system components in the hypothalamus fluctuated throughout pregnancy and the oestrous cycle. Our study was the first experiment to demonstrate the presence of CHEM system mRNAs and proteins in the porcine hypothalamus and the correlations between the expression levels and physiological hormonal milieu related to the oestrous cycle and early pregnancy.

Transcriptomic Analysis of Porcine Endometrium during Implantation after In Vitro Stimulation by Adiponectin.

Comprehensive understanding of the regulatory mechanism of the implantation process in pigs is crucial for reproductive success. The endometrium plays an important role in regulating the establishment and maintenance of gestation. The goal of the current study was to determine the effect of adiponectin on the global expression pattern of genes and relationships among differentially expressed genes (DE-genes) in the porcine endometrium during implantation using microarrays. Diverse transcriptome analyses including gene ontology (GO), biological pathway, networks, and DE-gene analyses were performed. Adiponectin altered the expression of 1286 genes with fold-change (FC) values greater than 1.2 (p < 0.05). The expression of 560 genes were upregulated and 726 downregulated in the endometrium treated with adiponectin. Thirteen genes were selected for real-time PCR validation of differential expression based on a known role in metabolism, steroid and prostaglandin synthesis, interleukin and growth factor action, and embryo implantation. Functional analysis of the relationship between DE-genes indicated that adiponectin interacts with genes that are involved in the processes of cell proliferation, programmed cell death, steroid and prostaglandin synthesis/metabolism, cytokine production, and cell adhesion that are critical for reproductive success. The presented results suggest that adiponectin signalling may play a key role in the implantation of pig.

In vitro effect of orexin A on the transcriptomic profile of the endometrium during early pregnancy in pigs.

Orexin A belongs to the group of hypothalamic-derived peptides that are involved in a number of processes, such as regulation of energy metabolism, control of food intake and regulation of the reproductive system, by influencing the hypothalamic-pituitary-ovarian axis. Orexin A is also present in the endometrium, myometrium and placenta, which indicates that it may function as an important local regulator of the reproductive functions. The aim of this study was to explore the effects of orexin A on global gene expression in the endometrium of pigs during early gestation, on days 15 to 16 of pregnancy (implantation period). Orexin A altered the expression of 1,242 genes. In this group, 1,104 genes had a fold change greater than 1.2 (P < 0.05). In the group of genes with a fold change that was greater than 1.2, the expression of 457 genes were increased and 647 decreased because of the effects of orexin A. An analysis of the interactions between differentially expressed genes demonstrated that orexin A interacts with genes that potentially encode intracellular signalling pathways and factors regulating reproductive functions in the endometrium. The data from the present study indicate that orexin A affects a number of genes and processes in the endometrium of pregnant pigs and may be regarded as an important regulator of implantation, depending on maternal nutritional status.

The effect of orexin B on steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase gene expression, and progesterone and androstenedione secretion by the porcine uterus during early pregnancy and the estrous cycle.

The aim of this study was to investigate the effect of orexin B (OXB) on progesterone (P4) and androstenedione (A4) secretion by porcine endometrial and myometrial tissue explants and on the expression of key steroidogenic proteins and enzymes involved in steroid production. The hormones secretion and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (HSD3B1) were analyzed on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy and during the luteal phase of the estrous cycle (days 10 to 11). Endometrial and myometrial explants were cultured in vitro in the presence of OXB (1, 10, or 100 nM) and OXB (1, 10, or 100 nM) with 1 µM of JNJ (OX2R antagonist). Gene expression was examined by real-time PCR, and steroid secretion was determined by radioimmunoassay. Orexin B modulated StAR, CYP11A1, HSD3B1 mRNA content depending on the type of uterine tissue, the applied OXB dose, and the stage of pregnancy or the estrous cycle (P < 0.05). Orexin B increased P4 secretion in all stages of early gestation (P < 0.05). Orexin B enhanced the release of A4 on days 12 to 13, 15 to 16, and 27 to 28 of gestation, whereas on days 10 to 11 of early pregnancy, A4 secretion decreased in the endometrium and increased in the myometrium (P < 0.05). These results indicate that OXB affects the expression of key steroidogenic regulators and the secretion of steroid hormones in the porcine uterus during early pregnancy.