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Chemerin is an adipokine which is the product of the RARRES2 gene expressed mainly in adipose tissue. This hormone exerts biological effects via three metabotropic receptors: chemokine like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C chemokine receptor-like 2 (CCRL2). Chemerin exerts pleiotropic effects and participates in the regulation of many processes, such as blood pressure control, immune system regulation, inflammation, and angiogenesis. There is a growing body of evidence to indicate that chemerin is involved in the regulation of reproductive system functions. However, little is known about the regulation of chemerin system (chemerin and its receptors) expression. The authors hypothesized that steroid hormones affect the expression of the chemerin system in the uterus of domestic pigs. Thus, the aim of this study was to investigate the influence of steroids, oestradiol (E

The aim of this study was to evaluate the effect of prostaglandins E2 (PGE2) and F2α (PGF2α) on orexins (OXA and OXB) secretion (ELISA), prepro-orexin (PPO) gene expression and the content of orexin receptors (OX1R, OX2R) mRNA (qPCR) and proteins (Western blot) in porcine endometrial and myometrial tissue slices during early pregnancy (days 10–28) and on days 10–11 of the oestrous cycle. On days 10–11 of pregnancy, prostaglandins (PGs) decreased the expression of the PPO gene and OXR gene and protein in the endometrium. On days 12– 13, PGs increased OXB secretion, PGE2 enhanced OXA secretion, and PGF2α suppressed PPO expression. On days 15–16, both PGs inhibited PPO expression and OXB secretion, and PGF2α increased OXA release. On days 27–28, PGs decreased PPO mRNA and OX1R protein levels, PGE2 decreased OX2R protein content, and PGF2α decreased OXB secretion. On days 10-11 of the cycle PGs increased the expression of PPO mRNA and OX2R protein, whereas PGE2 increased OX1R protein content. The effect of PGs on orexin system expression in the porcine myometrium was dependent on the animal physiological status and the concentrations of specific PGs. The study demonstrated that PGs exert regulatory effects on orexin system expression in the porcine uterus, which suggests that a local regulatory mechanism could be responsible for modulating uterine metabolism. The observed differences in PGs’ influence on orexin system expression could be attributed to changes in the sensitivity of the studied tissues, associated with the phase of the oestrous cycle, the stage of early pregnancy or PGs concentration.

Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment, learning, and memory. Autophagy is a mechanism that allows cells to degrade their unnecessary or dysfunctional components. Autophagosomes appear at dendritic spines in response to plasticity-inducing stimuli. Autophagy defects contribute to altered dendritic spine development, autistic-like behavior in mice, and neurological disease. While several studies explored the involvement of autophagy in synaptic plasticity, the steps preceding autophagosome emergence at the postsynapse remain unknown. Here we show a postsynaptic association of autophagy-related protein 9A (Atg9A), known to be involved in the initial stages of autophagosome formation, with Rab11, a small GTPase that regulates endosomal trafficking. Rab11 activity is necessary for the maintenance of Atg9A-positive structures at dendritic spines. Inhibition of mTOR increased Rab11 and Atg9A interaction and increased the emergence of autophagosomes in dendritic spines when coupled to NMDA receptor stimulation. Dendritic spines with newly formed autophagosomes were more resistant to NMDA-induced morphologic change. These results collectively suggest that autophagy initiation in dendritic spines depends on an activity-dependent Rab11a-Atg9A interaction regulated by mTOR.

Significance Tuberous Sclerosis Complex (TSC) is a hereditary disease that presents with early brain malformations, childhood epilepsy, and TSC-associated neuropsychiatric disorders (TANDs). Cortical malformations arise in utero and have been linked to childhood epilepsy before. Externally developing zebrafish seem convenient to study elementary neurodevelopment; however, without the in-depth functional analysis, the Tsc2-deficient zebrafish cannot be used for studies of TANDs. Here, we found that Tsc2-deficient zebrafish recapitulated symptoms seen in TSC patients on anatomical and behavioral levels, including aberrant brain morphology, thinning of brain connections, epileptogenesis, and increased anxiety-like behavior, which was rescued by reducing TrkB signaling, revealing a potential drug target. Moreover, we show that commissural thinning cause aberrant regulation of anxiety, providing a link between brain anatomy and emotion., Tuberous Sclerosis Complex (TSC) is a rare genetic disease that manifests with early symptoms, including cortical malformations, childhood epilepsy, and TSC-associated neuropsychiatric disorders (TANDs). Cortical malformations arise during embryonic development and have been linked to childhood epilepsy before, but the underlying mechanisms of this relationship remain insufficiently understood. Zebrafish have emerged as a convenient model to study elementary neurodevelopment; however, without in-depth functional analysis, the Tsc2-deficient zebrafish line cannot be used for studies of TANDs or new drug screening. In this study, we found that the lack of Tsc2 in zebrafish resulted in heterotopias and hyperactivation of the mTorC1 pathway in pallial regions, which are homologous to the mammalian cortex. We observed commissural thinning that was responsible for brain dysconnectivity, recapitulating TSC pathology in human patients. The lack of Tsc2 also delayed axonal development and caused aberrant tract fasciculation, corresponding to the abnormal expression of genes involved in axon navigation. The mutants underwent epileptogenesis that resulted in nonmotor seizures and exhibited increased anxiety-like behavior. We further mapped discrete parameters of locomotor activity to epilepsy-like and anxiety-like behaviors, which were rescued by reducing tyrosine receptor kinase B (TrkB) signaling. Moreover, in contrast to treatment with vigabatrin and rapamycin, TrkB inhibition rescued brain dysconnectivity and anxiety-like behavior. These data reveal that commissural thinning results in the aberrant regulation of anxiety, providing a mechanistic link between brain anatomy and human TANDs. Our findings also implicate TrkB signaling in the complex pathology of TSC and reveal a therapeutic target.

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.

Chemerin, belonging to the adipokine family, exhibits pleiotropic activity. We hypothesised that the adipokine could be involved in the regulation of steroidogenesis in the porcine endometrium. Thus, the aim of this study was to determine the effect of chemerin on the key steroidogenic enzyme proteins’ abundance (Western blot), as well as on P4 and E2 secretion (radioimmunoassay) by the porcine endometrium during early pregnancy and the mid-luteal phase of the oestrous cycle. Moreover, we investigated the hormone impact on Erk and Akt signalling pathway activation (Western blot). Chemerin stimulated E2 production on days 10 to 11 of pregnancy. On days 10 to 11 and 15 to 16 of gestation, and on days 10 to 11 of the cycle, chemerin enhanced the expression of StAR and all steroidogenic enzyme proteins. On days 12 to 13 of pregnancy, chemerin decreased StAR and most of the steroidogenic enzyme proteins’ abundance, whereas the P450C17 abundance was increased. On days 27 to 28 of pregnancy, chemerin increased StAR and P450C17 protein contents and decreased 3βHSD protein amounts. It was noted that the adipokine inhibited Erk1/2 and stimulated Akt phosphorylation. The obtained results indicate that chemerin affected P4 and E2 synthesis through the Erk1/2 and Akt signalling pathways.

Chemerin, belonging to the adipokine family, exhibits pleiotropic activity. We hypothesised that the adipokine could be involved in the regulation of steroidogenesis in the porcine endometrium. Thus, the aim of this study was to determine the effect of chemerin on the key steroidogenic enzyme proteins’ abundance (Western blot), as well as on P4 and E2 secretion (radioimmunoassay) by the porcine endometrium during early pregnancy and the mid-luteal phase of the oestrous cycle. Moreover, we investigated the hormone impact on Erk and Akt signalling pathway activation (Western blot). Chemerin stimulated E2 production on days 10 to 11 of pregnancy. On days 10 to 11 and 15 to 16 of gestation, and on days 10 to 11 of the cycle, chemerin enhanced the expression of StAR and all steroidogenic enzyme proteins. On days 12 to 13 of pregnancy, chemerin decreased StAR and most of the steroidogenic enzyme proteins’ abundance, whereas the P450C17 abundance was increased. On days 27 to 28 of pregnancy, chemerin increased StAR and P450C17 protein contents and decreased 3βHSD protein amounts. It was noted that the adipokine inhibited Erk1/2 and stimulated Akt phosphorylation. The obtained results indicate that chemerin affected P4 and E2 synthesis through the Erk1/2 and Akt signalling pathways.

TrkB is a tyrosine kinase receptor that is activated upon binding to brain-derived neurotrophic factor (BDNF). To date, the search for low-molecular-weight molecules mimicking BDNF’s action has been unsuccessful. Several molecules exerting antidepressive effects in vivo, such as 7,8-DHF, have been suggested to be TrkB agonists. However, more recent publications question this hypothesis. In this study, we developed a set of experimental procedures including the evaluation of direct interactions, dimerization, downstream signaling, and cytoprotection in parallel with physicochemical and ADME methods to verify the pharmacology of 7,8-DHF and other potential reference compounds, and perform screening for novel TrkB agonists. 7,8 DHF bound to TrkB with Kd = 1.3 µM, however, we were not able to observe any other activity against the TrkB receptor in SN56 T48 and differentiated SH-SY5Y cell lines. Moreover, the pharmacokinetic and pharmacodynamic effects of 7,8-DHF at doses of 1 and 50 mg/kg were examined in mice after i.v and oral administration, respectively. The poor pharmacokinetic properties and lack of observed activation of TrkB-dependent signaling in the brain confirmed that 7,8-DHF is not a relevant tool for studying TrkB activation in vivo. The binding profile for 133 molecular targets revealed a significant lack of selectivity of 7,8-DHF, suggesting a distinct functional profile independent of interaction with TrkB. Additionally, a compound library was screened in search of novel low-molecular-weight orthosteric TrkB agonists, however, we were not able to identify reliable drug candidates. Our results suggest that published reference compounds including 7,8-DHF do not activate TrkB, consistent with canonical dogma, which indicates that the reported pharmacological activity of these compounds should be interpreted carefully in a broad functional context.

Adiponectin is thought to be involved in the regulation of metabolic homeostasis and reproductive processes. It also has an important role in the modulation of female reproductive functions, both directly and by affecting the secretory functions of the hypothalamic-pituitary-gonadal axis. The main aim of this study was to determine the effect of adiponectin on global gene expression and on differentially expressed genes (DE-genes) regulated by adiponectin in anterior pituitary (AP) cells of pigs. The changes in the transcriptomic profile of AP cells of pigs were examined using the Porcine (V2) two-colour gene expression microarray, 4 × 44. An analysis of data from the microarray experiment indicated there were 716 DE-genes. A total of 466 genes (220 up-regulated and 246 down-regulated) with fold change greater than 1.2 (P0.05) were subsequently selected for further analysis. Gene ontology was analysed based on a list of DE-genes. A list of biological processes was generated for both up-regulated and down-regulated DE-genes. The products of up-regulated DE-genes were involved in 60 biological processes, whereas for down-regulated products there were 18 processes. An analysis of the interactions between DE-genes indicated that adiponectin interacted with genes that potentially encode intracellular signalling pathways and factors which regulate reproductive functions. Furthermore, nine genes were selected from the list of DE-genes to confirm microarray results by quantitative PCR. The results enhance the knowledge about adiponectin's role in the pituitary functions of pigs and provide valuable insights for further studies into adiponectin's mechanism of action in the pituitary.

In this study, aims were to evaluate orexin A (OXA) effects on mRNA abundance of important enzymes involved in prostaglandin production, such as cyclooxygenase 2 (PTGS2), microsomal PGE

BackgroundVisfatin exists in two forms: the intracellular form which is a rate limiting enzyme engaged in nicotinamide adenine dinucleotide biosynthesis and the extracellular form considered as an adipokine, produced mainly by the adipose tissue. Visfatin could be an energy sensor involved in regulating female fertility, which creates a hormonal link integrating the control of energy homeostasis and reproduction. MethodsThe study compares the expression levels of visfatin gene (quantitative real time PCR) and protein (Western blotting and fluorescent immunohistochemistry) in the 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 (enzyme-linked immunosorbent assay). 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. Differences between groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ResultsDuring the estrous cycle, visfatin mRNA expression in the MBH was higher on days 2-3 and 17-19, while the visfatin protein concentration on days 17-19. During early pregnancy, the most pronounced gene and protein expression in the MBH was found on days 15-16 and 10-11, respectively. In the POA during the estrous cycle, visfatin gene expression was the highest on days 17-19, and the protein level of visfatin on days 14-16. During early pregnancy, the highest expression of visfatin gene in the POA was observed on days 15-16, whereas the protein concentrations on days 27-28. Blood plasma concentrations of visfatin during the estrous cycle were higher on days 2-3 in relation to other studied phases of the cycle. During early pregnancy, the highest visfatin contents in the blood plasma were observed on days 12-13. Visfatin gene and protein expression in MBH and POA, and visfatin plasma concentrations differed during early pregnancy in relation to days 10-12 of the cycle. ConclusionsThis study demonstrated visfatin expression in the porcine hypothalamus and its dependence on hormonal milieu related to the estrous cycle and early pregnancy.

The corpus luteum (CL) undergoes rapid changes, and its functional capabilities are influenced by processes such as angiogenesis and apoptosis. According to the literature, chemerin-a protein that participates in the regulation of energy homeostasis and the immune response, may also affect angiogenesis and apoptosis. Therefore, the aim of this study was to investigate the in vitro effect of chemerin on angiogenesis and apoptosis in porcine luteal cells (Lc) during specific phases related to CL physiology. Luteal cells were harvested from gilts during the early-, mid-, and late-luteal phases of the estrous cycle. The cells were preincubated for 48 h and incubated for 24 h with chemerin or a serum-free medium (controls). The abundance of angiogenesis- and apoptosis-related proteins was determined by enzyme-linked immunosorbent assay (ELISA) in spent culture media, or by ELISA and Western blot in protein extracts. The current study demonstrated that chemerin stimulates the production of vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF) by porcine Lc and increases the protein abundance of angiogenic factors' receptors (VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2) in these cells. The study also revealed that chemerin exerts a modulatory effect (stimulatory/inhibitory, depending on the phase of the cycle) on the protein abundance of first apoptosis signal (Fas), Fas ligand, B-cell lymphoma 2, and caspase-3 in porcine Lc. These results imply that chemerin may affect angiogenesis and apoptosis processes in the porcine CL, as evidenced by its modulatory effect of chemerin on the protein abundance of crucial angiogenesis- and apoptosis-related factors, observed in an in vitro study of porcine Lc. Summary sentence Chemerin may affect angiogenesis and apoptosis in the corpus luteum of pigs, as evidenced by the modulatory effect of this hormone on the protein abundance of angiogenesis- and apoptosis-related factors in the in vitro study of porcine luteal cells. Graphical Abstract

International audience; Recent studies have demonstrated that chemerin participates in the regulation of female reproductive function at the level of the ovaries. Due to the lack of data concerning the presence of the chemerin system (chemerin and its receptors: CMKLR1, GPR1, CCRL2) in the ovaries of pigs, one of the most economically important livestock species, the aim of this study was to investigate the expression and localization of chemerin and its receptors in the ovaries of prepubertal and mature gilts. We also aimed to examine the concentrations of chemerin in the follicular fluid of prepubertal and mature animals. In the present study, we have demonstrated the expression patterns of chemerin system components in the porcine follicles of different sizes of prepubertal and mature animals, as well as in corpora lutea of mature gilts during the estrous cycle and early pregnancy. The obtained results suggest that the expression of chemerin system components is influenced by the reproductive stage, cell type, and the hormonal status of gilts (the estrous cycle/pregnancy). We have also presented the localization of the chemerin system components in various ovarian structures, and also showed changes in the concentration of chemerin in the follicular fluid of pigs. The presented findings not only confirm that chemerin is produced locally in the porcine ovary but they also demonstrate that chemerin directly affects ovarian cells, as confirmed by the presence of chemerin receptors in all ovarian structures. Therefore, chemerin appears to be an important intra-ovarian factor that could regulate ovary function in pigs.

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 F2&alpha, (PGF2&alpha, ) 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 PGF2&alpha, 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 PGF2&alpha, on the expression of the chemerin system in the porcine uterus during early gestation.

Chemerin has been shown to participate in the regulation of ovarian steroidogenesis in women, rats, mice and cows. Even though pigs are one of the most economically important livestock species, there is a general lack of data on the effects of chemerin in this species. Therefore, this study aimed to investigate the in vitro effect of chemerin on basal and luteinizing hormone/follicle-stimulating hormone- and/or insulin-induced secretion of progesterone (P4), androstenedione (A4), testosterone (T), estrone (E1) and estradiol (E2) by the porcine ovarian cells during the estrous cycle and early pregnancy. Granulosa (G) and theca interna (Th) cells were collected from gilts during the follicular phase. Luteal cells (Lc) were harvested from pigs during the early-luteal, mid-luteal and late-luteal phases, as well as during the maternal recognition of pregnancy and beginning of implantation. Cells were preincubated for 24 h (G and Th) or 48 h (Lc) and subsequently incubated for 24 h with or without treatments. Then, the concentrations of steroid hormones in the culture media were determined by radioimmunoassay. The results were analyzed by one-way analysis of variance, followed by Duncan’s post hoc test. The study demonstrated that chemerin exerts a modulatory effect on de novo synthesis of steroid hormones in pigs. Chemerin stimulated basal and/or induced secretion of P4 by the porcine Lc during the early-, mid- and late-luteal phases of the estrous cycle, as well as during both studied periods of early pregnancy. Further, chemerin caused an increase in the induced secretion of A4, T and E1 by the porcine Lc during the maternal recognition of pregnancy. Moreover, chemerin inhibited induced secretion of E2 by the porcine Lc during the early-, mid- and late-luteal phases, as well as during the maternal recognition of pregnancy. During the follicular phase, chemerin stimulated basal and induced secretion of P4 and inhibited induced secretion of E2 by the porcine G, as well as decreased induced secretion of A4, and T by the porcine Th. Therefore, chemerin appears to be a modulator of ovarian steroidogenesis in pigs, whereas its varied effects (stimulatory or inhibitory) on the secretion of steroid hormones may be due to the heterogeneity of factors regulating ovarian functions, possible interactions between these factors, and specific processes related to the ovarian physiology during different phases of the estrous cycle/pregnancy. Chemerin may also affect ovarian steroidogenesis in pigs by regulating the expression/activity of steroidogenic enzymes.

Studies on adipokines, substances that are produced in adipose tissue, indicate that they influence both metabolism and reproduction. Chemerin is a novel addition to the adipokine family. It is believed that chemerin receptors are expressed in different structures of the hypothalamic-pituitary-gonadal (HPG) axis, which are crucial for endocrine control of reproductive functions, including the pituitary. The aim of this study was to investigate the expression of chemerin receptors (CMKLR1, GPR1, CCRL2) genes and proteins in the porcine pituitary. The effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways was also investigated. The anterior (AP) and posterior (PP) lobes of the pituitary were examined on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the oestrous cycle and on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy. This is the first study to demonstrate that CMKLR1, GPR1 and CCRL2 are expressed in the porcine AP and PP, which implies that this gland is sensitive to chemerin action. The expression of the studied chemerin receptors fluctuated during different phases of the cycle and early gestation, which could be related to changes in the endocrine status of female pigs. The study also revealed that CMKLR1 and CCRL2 proteins were present in gonadotrophs and thyrotrophs, whereas CCRL2 was also present in somatotrophs, during the cycle and early pregnancy. We observed that chemerin affected MAPK/Erk1/2, Akt and AMPK signalling pathways in the porcine AP. These results suggest that chemerin may participate in the regulation of reproductive functions at the level of the pituitary.

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.

Background Orexin A (OXA) and orexin B (OXB) are hypothalamic-derived peptides that participate in the regulation of energy metabolism, food intake and reproductive function by influencing the hypothalamic-pituitary-ovarian axis. Orexins are also produced in the endometrium, myometrium and placenta, which suggests that they could act as a link between energy metabolism and the reproductive system. Changes in the expression of orexin and the orexin receptor genes and proteins during the oestrous cycle and early gestation in pigs imply that orexin activity may be regulated by local factors within the uterus. The aim of this study was to investigate the influence of progesterone (P4) on the expression of orexin system genes, and proteins in the porcine uterus during early gestation. Gene expression was analyzed by real-time PCR. Adiponectin secretion was determined by ELISA, and the receptors proteins content was defined using western blot analysis. Results In the endometrium, P4 enhanced OXA secretion on days 10 to 11 of gestation and OXB secretion on days 12 to 13. In the myometrium, P4 inhibited the secretion of both orexins on days 15 to 16 and OXB secretion also on days 12 to 13. In the endometrium, P4 inhibited the expression of orexin receptor 1 (OX1R) protein at nearly all times analyzed, whereas the expression of orexin receptor 2 (OX2R) protein was inhibited only on days 15 to 16 of gestation. In the myometrium, P4 stimulated OX1R protein expression on days 12 to 13 and 15 to 16 of gestation and inhibited OX1R protein expression on days 27 to 28. The expression of OX2R protein in the myometrium increased on days 12 to 13 and decreased on days 10 to 11 and 15 to 16. Conclusions The results indicate that P4 could regulate the expression of the orexin system in the porcine uterus during early pregnancy, which suggests the presence of a local feedback loop that could play an important role in the regulation of maternal metabolism during pregnancy. The findings may contribute to the existing knowledge of the mechanisms linking maternal energy metabolism with the regulation of the reproductive system during pregnancy.

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.

Adipokines such as chemerin affect metabolic status and reproductive function in many species. The hypothesis in the present study was that there were chemerin mRNA transcript and protein in the pituitary of pigs and that relative abundances fluctuate during the estrous cycle and early pregnancy. Chemerin is thought to modulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion during the estrous cycle. Changes in the relative abundance of chemerin mRNA transcript and protein in anterior (AP) and posterior (PP) pituitaries of pigs were investigated, for the first time in the present study, during four phases of the estrous cycle and four periods of early pregnancy. Chemerin protein was localized in gonadotrophs, thyrotrophs and somatotrophs during the estrous cycle and early gestation. Chemerin treatments affected both basal, GnRH- and/or insulin-induced LH and FSH production, with there being variations with phase of the estrous cycle when tissues were collected. These findings indicate chemerin may be produced locally in the pituitary and may affect female reproductive function by controlling the release of LH and FSH from AP cells.

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.

Previous studies demonstrated that cells inhibit protein synthesis as a compensatory mechanism for mitochondrial dysfunction. Protein synthesis can be attenuated by 1) the inhibition of mTOR kinase, which results in a decrease in the phosphorylation of S6K1 and 4E-BP1 proteins, and 2) an increase in the phosphorylation of eIF2α protein. The present study investigated both of these pathways under conditions of short-term acute and long-term mitochondrial stress. Short-term responses were triggered in mammalian cells by treatment with menadione, antimycin A, or CCCP. Long-term mitochondrial stress was induced by prolonged treatment with menadione or rotenone and expression of genetic alterations, such as knocking down the MIA40 oxidoreductase or knocking out NDUFA11 protein. Short-term menadione, antimycin A, or CCCP cell treatment led to the inhibition of protein synthesis, accompanied by a decrease in mTOR kinase activity, an increase in the phosphorylation of eIF2α (Ser51), and an increase in the level of ATF4 transcription factor. Conversely, long-term stress led to a decrease in eIF2α (Ser51) phosphorylation and ATF4 expression and to an increase in S6K1 (Thr389) phosphorylation. Thus, under long-term mitochondrial stress, cells trigger long-lasting adaptive responses for protection against excessive inhibition of protein synthesis.

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 3β-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.

The aim of this study was to investigate the effect of orexin B (OXB) on progesterone (P(4)) and androstenedione (A(4)) 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 3β-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 P(4) secretion in all stages of early gestation (P < 0.05). Orexin B enhanced the release of A(4) on days 12 to 13, 15 to 16, and 27 to 28 of gestation, whereas on days 10 to 11 of early pregnancy, A(4) 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.

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.

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 aim of this study was to investigate whether the promoters of progesterone receptor isoform A (PGRA) and B (PGRB) are methylated and to determine the percentage of methylation occurring for each isoform. Genomic DNA was isolated from the corpora lutea (CL) and endometrial slices from cows on Days 2–5, 6–10, 11–16 and 17–20 of the oestrous cycle. DNA was bisulphite-converted and amplified using methyl-specific polymerase chain reaction (PCR) with primers that detect both methylated and unmethylated sequences. The determination of the percentage of the methylation was performed using HpaII and MspI restriction enzymes. Methyl-specific PCR showed partial methylation of PGRA and PGRB promoters in the CL and endometrium during the oestrous cycle. Methylation for PGRA was between 15 and 17% and for PGRB was in the range of 6 to 7.7% during the oestrous cycle in the CL. In the endometrium, the methylation for PGRA was between 6 and 7.3% and for PGRB was between 3 and 4.8% during the oestrous cycle. The data obtained indicate that the higher promoter methylation of the PGRA isoform could be a mechanism for regulation of PGRA inhibitory activity against PGRB and, in this way, methylation may influence the regulation of progesterone action in the CL and endometrium.

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.

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 &lt, 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.

A new fast method for determination of mobile zinc fractions in soil is proposed in this work. The three-stage modified BCR procedure used for fractionation of zinc in soil was accelerated by using ultrasounds. The working parameters of an ultrasound probe, a power and a time of sonication, were optimized in order to acquire the content of analyte in soil extracts obtained by ultrasound-assisted sequential extraction (USE) consistent with that obtained by conventional modified Community Bureau of Reference (BCR) procedure. The content of zinc in extracts was determined by flame atomic absorption spectrometry. The developed USE procedure allowed for shortening the total extraction time from 48 h to 27 min in comparison to conventional modified BCR procedure. The method was fully validated, and the uncertainty budget was evaluated. The trueness and reproducibility of the developed method was confirmed by analysis of certified reference material of lake sediment BCR-701. The applicability of the procedure for fast, low costs and reliable determination of mobile zinc fraction in soil, which may be useful for assessing of anthropogenic impacts on natural resources and environmental monitoring purposes, was proved by analysis of different types of soil collected from Podlaskie Province (Poland). Electronic supplementary material The online version of this article (doi:10.1007/s10661-015-5020-6) contains supplementary material, which is available to authorized users.