Apelin-13 modulates the endometrial transcriptome of the domestic pig during implantation.

BACKGROUND: The peri-implantation period is a critical time during pregnancy that mostly defines the overall litter size. Most authors agree that the highest percentage of embryo mortality occurs during this time. Despite the brevity of the peri-implantation period, it is the most dynamic part of pregnancy in which the sequential and uninterrupted course of several processes is essential to the animal's reproductive success. Also then, the maternal uterine tissues undergo an intensive remodelling process, and their energy demand dramatically increases. It is believed that apelin, a member of the adipokine family, is involved in the control of female reproductive functions in response to the current metabolic state. The verified herein hypothesis assumed the modulatory effect of apelin on the endometrial tissue transcriptome on days 15 to 16 of gestation (beginning of implantation). RESULTS: The analysis of data obtained during RNA-seq (Illumina HiSeq2500) of endometrial slices treated and untreated with apelin (n = 4 per group) revealed changes in the expression of 68 genes (39 up-regulated and 29 down-regulated in the presence of apelin), assigned to 240 gene ontology terms. We also revealed changes in the frequency of alternative splicing events (397 cases), as well as single nucleotide variants (1,818 cases) in the presence of the adipokine. The identified genes were associated, among others, with the composition of the extracellular matrix, apoptosis, and angiogenesis. CONCLUSIONS: The obtained results indicate a potential role of apelin in the regulation of uterine tissue remodelling during the peri-implantation period.

The Role of Adipokines in the Control of Pituitary Functions.

The pituitary gland is a key endocrine gland in all classes of vertebrates, including mammals. The pituitary gland is an important component of hypothalamus-pituitary-target organ hormonal regulatory axes and forms a functional link between the nervous system and the endocrine system. In response to hypothalamic stimuli, the pituitary gland secretes a number of hormones involved in the regulation of metabolism, stress reactions and environmental adaptation, growth and development, as well as reproductive processes and lactation. In turn, hormones secreted by target organs at the lowest levels of the hormonal regulatory axes regulate the functions of the pituitary gland in the process of hormonal feedback. The pituitary also responds to other peripheral signals, including adipose-tissue-derived factors. These substances are a broad group of peptides known as adipocytokines or adipokines that act as endocrine hormones mainly involved in energy homeostasis. Adipokines, including adiponectin, resistin, apelin, chemerin, visfatin, and irisin, are also expressed in the pituitary gland, and they influence the secretory functions of this gland. This review is an overview of the existing knowledge of the relationship between chosen adipose-derived factors and endocrine functions of the pituitary gland, with an emphasis on the pituitary control of reproductive processes.

Rab11 regulates autophagy at dendritic spines in an mTOR- and NMDA-dependent manner.

Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment and learning and memory. Autophagy is a mechanism that allows the cell to degrade its 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 have explored the involvement of autophagy in synaptic plasticity, the initial steps of the emergence of autophagosomes at the postsynapse remain unknown. Here, we demonstrate a postsynaptic association of autophagy-related protein 9A (Atg9A), known to be involved in the early stages of autophagosome formation, with Rab11, a small GTPase that regulates endosomal trafficking. Rab11 activity was necessary to maintain Atg9A-positive structures at dendritic spines. Inhibition of mTOR increased Rab11 and Atg9A interaction and increased the emergence of LC3 positive vesicles, an autophagosome membrane-associated protein marker, in dendritic spines when coupled to NMDA receptor stimulation. Dendritic spines with newly formed LC3+ vesicles were more resistant to NMDA-induced morphologic change. Rab11 DN overexpression suppressed appearance of LC3+ vesicles. Collectively, these results suggest that initiation of autophagy in dendritic spines depends on neuronal activity and Rab11a-dependent Atg9A interaction that is regulated by mTOR activity.

Expression of visfatin in the ovarian follicles of prepubertal and mature gilts and in vitro effect of gonadotropins, insulin, steroids, and prostaglandins on visfatin levels.

Recent studies have demonstrated that visfatin participates in the regulation of female reproduction. Due to the lack of data concerning the level of visfatin in the ovarian follicles of pigs, one of the most economically important livestock species, the aim of this study was to investigate the expression and localisation of visfatin and the follicular fluid concentration in the ovarian follicles of prepubertal and mature gilts. We also aimed to examine the in vitro effects of gonadotropins (LH, FSH), insulin, progesterone (P4), oestradiol (E2), prostaglandin E2 (PGE2) and F2α (PGF2α) on visfatin levels. In the present study, we have demonstrated that visfatin expression is dependent on the maturity of the animals and the stage of ovarian follicle development. Visfatin signal was detected in individual follicular compartments from primordial to antral follicles and even in atretic follicles. We have shown that the expression of visfatin in granulosa cells was higher than in theca cells. The level of visfatin is upregulated by LH, FSH, E2, and P4 and downregulated by insulin, while prostaglandins have modulatory effects, dependent on the dose and type of ovarian follicular cells. To summarise, our research has shown that visfatin is widely expressed in the ovarian follicles of prepubertal and mature pigs, and its expression is regulated by gonadotropins, insulin, steroids, and prostaglandins, suggesting that visfatin appears to be an important intra-ovarian factor that could regulate porcine ovarian follicular function.

The effect of oestradiol and progesterone on chemerin system expression in the porcine endometrium during early pregnancy and the mid-luteal phase of the oestrous cycle.

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 (E2), and progesterone (P4), on the gene (qPCR) and protein (Western blot) expression of chemerin receptors, RARRES2 gene expression as well as chemerin secretion (ELISA) by endometrial slices during early pregnancy (days 10 to 11, 12 to 13, 15 to 16, and 27 to 28) and the mid-luteal phase of the oestrous cycle (days 10 to 11). Both steroid hormones modulated the expression of chemerin receptor genes and proteins, as well as the abundance of RARRES2 mRNA transcripts. The study also demonstrated that E2 increased chemerin protein secretion by endometrial cells during the maternal recognition of pregnancy (days 12 to 13) and decreased its release during embryo implantation (days 15 to 16). P4 had no significant effect on chemerin levels during the studied periods of gestation and the oestrous cycle. These observations suggest that the effects of E2 and P4 on the chemerin system are dependent on the period of gestation or the oestrous cycle. Our results support the hypothesis that the hormonal environment could modulate the expression of the chemerin system in the endometrium.

Effects of orexin A on PTGS2, PTGES, CBR1 and PGFS mRNA transcript abundances and prostaglandin E2 and F2α concentrations in culture medium of pig uterine explants collected during early gestation and the estrogenic cycle.

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 PGE2 synthase-1 (PTGES), PGF2α synthase (PGFS) and carbonyl reductase 1 (CBR1), as well as prostaglandin E2 (PGE2) and F2α (PGF2α) culture medium concentrations for endometrial and myometrial explants. Tissues were collected from gilts during specific phases of the estrogenic cycle or early gestational period. There were greater concentrations of PGE2 with OXA treatments of endometrial tissues collected on days 12-13 and 27-28, as well as PGF2α on days 10-11 of the gestational period. The PGF2α concentrations were less in tissues collected on days 27-28 of the gestational period. The OXA treatments resulted in lesser concentrations of PGE2 from myometrial tissues collected on days 10-11 and greater PGF2α on days 10-11 of the gestational period and 10-11 of the estrogenic cycle. Effects of OXA may occur due to actions at PTGS2, PTGES, PGFS and CBR1 genes because mRNA abundances for proteins encoded by these genes were affected by OXA. Results indicate there is an OXA effect on mRNA abundances and prostaglandin culture medium concentrations of uterine tissue collected at different stages of the gestational period or estrogenic cycle using different doses of OXA. It, therefore, is concluded OXA may affect de novo synthesis and secretion of PGE2 and PGF2α in the uterus of pigs.

Chemerin Affects P4 and E2 Synthesis in the Porcine Endometrium during Early Pregnancy.

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 3ßHSD 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.

Do Small Molecules Activate the TrkB Receptor in the Same Manner as BDNF? Limitations of Published TrkB Low Molecular Agonists and Screening for Novel TrkB Orthosteric Agonists.

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.

Chemerin as a modulator of angiogenesis and apoptosis processes in the corpus luteum of pigs: an in vitro study†.

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.

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.

Chemerin as a modulator of ovarian steroidogenesis in pigs: an in vitro study.

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.

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.

Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs during the estrous cycle and early pregnancy and associations with LH and FSH secretion during the estrous cycle.

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.

Expression of chemerin receptors CMKLR1, GPR1 and CCRL2 in the porcine pituitary during the oestrous cycle and early pregnancy and the effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways.

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.

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.

Expression of chemerin and its receptors in the ovaries of prepubertal and mature gilts.

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.

TrkB hyperactivity contributes to brain dysconnectivity, epileptogenesis, and anxiety in zebrafish model of Tuberous Sclerosis Complex.

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.

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.

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.