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Adipokines play crucial roles in both reproductive and energy metabolic processes. This study aimed to compare the hormonal plasma profile of adiponectin, apelin, vaspin, chemerin, resistin, visfatin, and adipolin, and the expression of their receptors in the anterior pituitary (AP) between normal-weight Large White (LW) and fat Meishan (MS) pigs during different phases of the estrous cycle. We measured adipokine levels in the plasma and assessed their gene expression in the AP. We used Pearson's correlation analysis to examine potential links between adipokines levels, their receptors, and metabolic parameters (body weight; backfat thickness) and reproductive parameters (pituitary weight; age at puberty; levels of gonadotropins, steroid hormones; and gene expression of gonadotropin-releasing hormone receptor and gonadotropins in AP). The plasma levels of the evaluated adipokines fluctuated with phase and breed, except for visfatin and adipolin. Moreover, adipokine expression in AP varied significantly between breeds and estrous cycle phases, except for resistin receptor CAP1. Notably, we observed a positive correlation between plasma levels of adiponectin and its transcript in the AP only in MS pigs. Apelin gene expression correlated negatively with its receptor in MS, while we observed a breed-dependent correlation between chemerin gene expression and its receptor CMKLR1. We identified significant positive or negative correlations between adipokines or their receptor levels in plasma and AP as well as metabolic or reproductive parameters, depending on the breed. In conclusion, we have demonstrated breed-specific and estrous cycle-dependent regulation of adipokines in AP, underscoring their potential impact on metabolic and reproductive processes in swine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Visfatin is an adipokine involved in energy metabolism, insulin resistance, inflammation, and female reproduction. Due to limited data about its action in the human placenta, the aims of our studies included the analysis of visfatin expression and immunolocalization in trophoblast cell lines JEG-3 and BeWo as well as in human placentas from normal and pathological pregnancies. Moreover, we also checked the hormonal regulation of visfatin levels and the molecular mechanism of observed changes in JEG-3 cells. Cell culture and placental fragments collection along with statistical analysis were performed using standard laboratory procedures also described in our previous papers. We demonstrated an increased gene and protein expression of visfatin in JEG-3, BeWo cells, while variable expression in maternal and fetal parts of normal/ pathological pregnancy placentas. In addition, the immunolocalization of visfatin was observed in the cytoplasm of both cell lines, the capillary epithelium of the maternal part and syncytiotrophoblasts of the placental fetal part; in all tested pathologies, the signal was also detected in decidual cells. Furthermore, we demonstrated that hormones: progesterone, estradiol, human chorionic gonadotropin, and insulin increased the visfatin levels in JEG-3 cells with the involvement of specific signaling pathways. Taken together, differences in the expression and localization of visfatin between normal and pathological placentas suggested that visfatin may be a potential marker for the diagnosis of pregnancy disorders. In addition, we found that placental levels of visfatin can be regulated by hormones known to modulate the function of placental cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dawid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The pituitary belongs to the most important endocrine glands involved in regulating reproductive functions. The proper functioning of this gland ensures the undisturbed course of the oestrous cycle and affects the female's reproductive potential. It is believed that visfatin, a hormone belonging to the adipokine family, may regulate reproductive functions in response to the female's metabolic state. Herein we verified the hypothesis that suggests a modulatory effect of visfatin on the anterior pituitary transcriptome during the mid-luteal phase of the oestrous cycle., Results: RNA-seq analysis of the porcine anterior pituitary cells revealed changes in the expression of 202 genes (95 up-regulated and 107 down-regulated in the presence of visfatin, when compared to the non-treated controls), assigned to 318 gene ontology terms. We revealed changes in the frequency of alternative splicing events (235 cases), as well as long noncoding RNA expression (79 cases) in the presence of the adipokine. The identified genes were associated, among others, with reproductive system development, epithelial cell proliferation, positive regulation of cell development, gland morphogenesis and cell chemotaxis., Conclusions: The obtained results indicate a modulatory influence of visfatin on the regulation of the porcine transcriptome and, in consequence, pituitary physiology during the mid-luteal phase of the oestrous cycle., (© 2024. The Author(s).)

Previously, we demonstrated the expression of visfatin in porcine reproductive tissues and its effect on pituitary endocrinology. The objective of this study was to examine the visfatin effect on the secretion of steroid (P 4 , E 2 ) and prostaglandin (PGE 2 , PGF 2α ), the mRNA and protein abundance of steroidogenic markers (STAR, CYP11A1, HSD3B, CYP19A1), prostaglandin receptors (PTGER2, PTGFR), insulin receptor (INSR), and activity of kinases (MAPK/ERK1/2, AKT, AMPK) in the porcine corpus luteum. We noted that the visfatin effect strongly depends on the phase of the estrous cycle: on days 2-3 and 14-16 it reduced P 4 , while on days 10-12 it stimulated P 4 . Visfatin increased secretion of E 2 on days 2-3, PGE 2 on days 2-3 and 10-12, reduced PGF 2α release on days 14-16, as well as stimulated the expression of steroidogenic markers on days 10-12 of the estrous cycle. Moreover, visfatin elevated PTGER mRNA expression and decreased its protein level, while we noted the opposite changes for PTGFR. Additionally, visfatin activated ERK1/2, AKT, and AMPK, while reduced INSR phosphorylation. Interestingly, after inhibition of INSR and signalling pathways visfatin action was abolished. These findings suggest a regulatory role of visfatin in the porcine corpus luteum., (© 2024. The Author(s).)

Visfatin (VIS) is a hormone belonging to the adipokines' group secreted mainly by the adipose tissue. VIS plays a crucial role in the control of energy homeostasis, inflammation, cell differentiation, and angiogenesis. VIS expression was confirmed in the hypothalamic-pituitary-gonadal (HPG) axis structures, as well as in the uterus, placenta, and conceptuses. We hypothesised that VIS may affect the abundance of proteins involved in the regulation of key processes occurring in the corpus luteum (CL) during the implantation process in pigs. In the present study, we performed the high-throughput proteomic analysis (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the in vitro influence of VIS (100 ng/mL) on differentially regulated proteins (DRPs) in the porcine luteal cells (LCs) on days 15-16 of pregnancy (implantation period). We have identified 511 DRPs, 276 of them were up-regulated, and 235 down-regulated in the presence of VIS. Revealed DRPs were assigned to 162 gene ontology terms. Western blot analysis of five chosen DRPs, ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), lanosterol 14-α demethylase (CYP51A1), inhibin subunit beta A (INHBA), notch receptor 3 (NOTCH3), and prostaglandin E synthase 2 (mPGES2) confirmed the veracity and accuracy of LC-MS/MS method. We indicated that VIS modulates the expression of proteins connected with the regulation of lipogenesis and cholesterologenesis, and, in consequence, may be involved in the synthesis of steroid hormones, as well as prostaglandins' metabolism. Moreover, we revealed that VIS affects the abundance of protein associated with ovarian cell proliferation, differentiation, and apoptosis, as well as CL new vessel formation and tissue remodelling. Our results suggest important roles for VIS in the regulation of ovarian functions during the peri-implantation period., (© 2024. The Author(s).)

Phoenixin-14 (PNX-14) regulates energy metabolism via the G protein-coupled receptor 173 (GPR173); elevated plasma levels have been described in patients with polycystic ovary syndrome. The aims were to investigate the ovarian expression of PNX-14/GPR173 and the in vitro effect of PNX-14 on granulosa cells (Gc) function. Transcript and protein levels of PNX-14/GRP173 were analysed by real-time PCR, western blot and immunohistochemistry in the porcine ovarian follicles at days 2-3, 10-12 and 16-18 of the oestrous. For in vitro experiments, Gc were isolated from follicles at days 10-12 of the oestrous (4-6 mm) and PNX-14 at doses 1-1000 nM was added for 24-72 h to determine Gc proliferation. Cell cycle progression, E2 secretion, expression of proliferating cells nuclear antigen, cyclins, mitogen-activated kinase (MAP3/1; ERK1/2), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) were studied. The involvement of these kinases in PNX-14 action on Gc proliferation was analysed using pharmacological inhibitors. Levels of GPR173 were increased in the ovarian follicles with oestrous progression, while only PNX-14 protein was the highest at days 10-12 of the oestrous. Immuno-signal of PNX-14 was detected in Gc and theca cells and oocyte, while GPR173 was mostly in theca. Interestingly, PNX-14 stimulated Gc proliferation, E2 secretion, cell cycle progression and cyclins expression and had a modulatory effect on MAP3/1, AKT and STAT3 activation. Our study suggests that PNX-14 could be an important factor for porcine reproduction by influencing ovarian follicle growth through direct action on Gc function., Competing Interests: Declaration of Competing Interest No conflict of interest exist in the submission of this manuscript, and the manuscript is approved by all authors for publication., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Omentin (ITLN1) is a novel adipokine mainly expressed in the white adipose tissue. It plays a crucial role in the metabolic homeostasis and insulin sensitivity. Our last study documented that ITLN1 levels in the adipose tissue and plasma are lower in fat Meishan (MS) compared to normal weight Large White (LW) pigs. The aim of this study was to investigate transcript and protein concentrations of ITLN1 as well as its immunolocalisation in the ovarian follicles and examine the molecular mechanism involved in the regulation of its expression in response to gonadotropins (FSH, LH) and steroids (P4, T, E2). Ovarian follicles were collected from LW and MS sows on days 2-3, 10-12, and 14-16 of the oestrous. We found the elevated ITLN1 expression in the ovarian follicles and the increase of concentrations in follicular fluid (FF) of LW pigs vs MS pigs; in both breeds of pigs, the levels of ITLN1 increased with the oestrous progression. We noted ITLN1 signals in oocyte, granulosa and theca cells. Gonadotropins and steroids increased ITLN1 levels in the ovarian follicle cells of LW pigs, while in MS pigs, we observed only the stimulatory effect of LH and T. Both extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3'-kinase (PI3K) were involved in the regulation of ITLN1. Our study demonstrated the levels and regulation of ITLN1 in the porcine ovarian follicles through ERK1/2 and PI3K signaling pathways., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Visfatin/NAMPT (VIS), the hormone exerting a pleiotropic effect, is also perceived as an important factor in the regulation of reproductive processes and pregnancy maintenance. Previous studies confirmed its involvement in the control of porcine pituitary and ovary function. In this study, we hypothesized that VIS may affect the global transcriptome of luteal cells and thus regulate the functioning of the ovaries. Illumina's NovaSeq 6000 RNA sequencing was performed to investigate the differentially expressed genes (DEGs) and long non-coding RNAs (DELs) as well as the occurrence of differential alternative splicing events (DASs) in the porcine luteal cells exposed to VIS (100 ng/mL) during the implantation period. The obtained results revealed 170 DEGs (99 up- and 71 downregulated) assigned to 45 functional annotations. Moreover, we revealed 40 DELs, of which 3 were known and 37 were described for the first time. We identified 169 DASs events. The obtained results confirmed a significant effect of VIS on the transcriptome and spliceosome of luteal cells, including the genes involved in the processes crucial for successful implantation and pregnancy maintenance as angiogenesis, steroidogenesis, inflammation, cell development, migration, and proliferation.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000 nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function., (© The Author(s) 2023. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

Reproductive success consists of a sequential events chronology, starting with the ovum fertilization, implantation of the embryo, placentation, and cellular processes like proliferation, apoptosis, angiogenesis, endocrinology, or metabolic changes, which taken together finally conduct the birth of healthy offspring. Currently, many factors are known that affect the regulation and proper maintenance of pregnancy in humans, domestic animals, or rodents. Among the determinants of reproductive success should be distinguished: the maternal microenvironment, genes, and proteins as well as numerous pregnancy hormones that regulate the most important processes and ensure organism homeostasis. It is well known that white adipose tissue, as the largest endocrine gland in our body, participates in the synthesis and secretion of numerous hormones belonging to the adipokine family, which also may regulate the course of pregnancy. Unfortunately, overweight and obesity lead to the expansion of adipose tissue in the body, and its excess in both women and animals contributes to changes in the synthesis and release of adipokines, which in turn translates into dramatic changes during pregnancy, including those taking place in the organ that is crucial for the proper progress of pregnancy, i.e. the placenta. In this chapter, we are summarizing the current knowledge about levels of adipokines and their role in the placenta, taking into account the physiological and pathological conditions of pregnancy, e.g. gestational diabetes mellitus, preeclampsia, or intrauterine growth restriction in humans, domestic animals, and rodents., (Copyright © 2024. Published by Elsevier Inc.)

In Brief: Adipolin (C1QTNF12) has been described as a regulator of metabolism and is linked with the pathophysiology of PCOS. In this study, for the first time, we show the expression of C1QTNF12 in granulosa cells and its positive effect on porcine granulosa cell proliferation and steroid synthesis., Abstract: Adipolin (C1QTNF12) is a recently discovered adipokine that plays an important role in glucose and insulin level regulation. Previous studies showed its reduced level in serum of women suffering from polycystic ovarian syndrome; however, whether C1QTNF12 regulates ovary function is still unknown. The aim of the study was first to determine the level of C1QTNF12 in the porcine ovarian follicles granulosa cells (Gc) and then its in vitro effect on proliferation and steroidogenesis as well as phosphorylation of several signalling pathways. Our results showed that the expression of C1QTNF12 was dependent on follicle size and was higher at the mRNA and protein level in Gc of small than large follicles from both prepubertal and mature animals. Similar pattern was observed for C1QTNF12 concentration in porcine follicular fluid. Additionally, we observed immunolocalisation of C1QTNF12 in Gc, theca cells and oocytes. We found that C1QTNF12 stimulated porcine Gc proliferation via the activation of protein kinase B (AKT). Moreover, C1QTNF12 enhanced progesterone, testosterone and oestradiol secretion by elevating STAR, CYP11A1, HSD3B and CYP19A1 mRNA expression and by activation of MAP3/1 pathway. Additionally, C1QTNF12 increased pMAP3/1-to-MAP3/1 protein expression ratio and enhanced IGF1-induced pTyr-IGF1Rβ-to-IGFR1β and pMAP3/1-to-MAP3/1 protein ratios. Taken together, C1QTNF12 could act directly on proliferation and steroid synthesis and serve as an important factor in in vivo ovarian follicle function, possibly regulating the course of folliculogenesis.

Visfatin (VIS), also known as nicotinamide phosphoribosyltransferase (NAMPT), is the rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Recently, VIS has been also recognized as an adipokine. Our previous study revealed that VIS is produced in the anterior and posterior lobes of the porcine pituitary. Moreover, the expression and secretion of VIS are dependent on the phase of the estrous cycle and/or the stage of early pregnancy. Based on this, we hypothesized that VIS may regulate porcine pituitary function. This study was conducted on anterior pituitary (AP) glands harvested from pigs during specific phases of the estrous cycle. We have shown the modulatory effect of VIS in vitro on LH and FSH secretion by porcine AP cells (determined by ELISA). VIS was also found to stimulate cell proliferation (determined by Alamar Blue) without affecting apoptosis in these cells (determined using flow cytometry technique). Moreover, it was indicated that VIS may act in porcine AP cells through the INSR, AKT/PI3K, MAPK/ERK1/2, and AMPK signaling pathways (determined by ELISA or Western Blot). This observation was further supported by the finding that simultaneous treatment of cells with VIS and inhibitors of these pathways abolished the observed VIS impact on LH and FSH secretion (determined by ELISA). In addition, our research indicated that VIS affected the mentioned processes in a manner that was dependent on the dose of VIS and/or the phase of the estrous cycle. Thus, these findings suggest that VIS may regulate the functioning of the porcine pituitary gland during the estrous cycle.

Spexin (SPX) is a novel neuropeptide and adipokine negatively correlated with obesity and insulin resistance. A recent study investigated expression and regulatory function of SPX in the hypothalamus and pituitary; however, the effect on ovarian function is still unknown. The aim of this study was to characterize the expression of SPX and its receptors, galanin receptors 2 and 3 (GALR2/3), in the human ovary and to study its in vitro effect on granulosa cells (GC) function. Follicular fluid (FF) and GC were obtained from normal weight and obese healthy and diagnosed with polycystic ovarian syndrome (PCOS) women. Expression of SPX and GALR2/3 in the ovary was studied by qPCR, western blot, and immunohistochemistry. The level of SPX in FF was assessed by enzyme-linked immunosorbent assay. The in vitro effect of recombinant human SPX on GC proliferation, steroidogenesis, and signaling pathways (MAP3/1, STAT3, AKT, PKA) was analyzed. Moreover, GC proliferation and estradiol (E2) secretion were measured with and without an siRNA against GALR2/3 and pharmacological inhibition of the above kinases. The results showed that both the SPX concentration in FF and its gene expression were decreased in GC of obese and PCOS women, while the protein expression of GALR2/3 was increased. We noted that SPX reduced GC proliferation and steroidogenesis; these effects were mediated by GALR2/3 and kinases MAP3/1, AKT, and STAT3 for proliferation or kinases MAP3/1 and PKA for E2 secretion. The obtained data clearly documented that SPX is a novel regulator of human ovarian physiology and possibly plays a role in PCOS pathogenesis., (© The Author(s) 2023. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

Omentin-1 (OMNT1) is an adipokine involved in the regulation of energy metabolism, insulin sensitivity, and reproduction. The present study was the first to investigate the plasma levels and expression of OMNT1 in the anterior pituitary (AP) gland on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle of normal-weight Large White (LW) and fat Meishan (MS) pigs. Next, we determined the effect of GnRH, LH, and FSH on the OMNT1 levels in cultured AP cells. The gene and protein expression of OMNT1 in AP fluctuated during the estrous cycle, with a higher expression in MS than in LW (except on days 10-12). However, plasma levels of OMNT1 were higher in LW than in MS. OMNT1 was localized in somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs. In LW pituitary cells, GnRH and gonadotropins stimulated OMNT1 protein expression (except FSH on days 14-16) and had no effect on OMNT1 levels in the culture medium. In MS pituitary cells, we observed that GnRH and LH increased while FSH decreased OMNT1 protein expression. These findings showed OMNT1 expression and regulation in the porcine AP and suggested that OMNT1 could be a new player modifying the pituitary functions., (© 2023. The Author(s).)

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 (P 4 ), oestradiol (E 2 ), prostaglandin E 2 (PGE 2 ) and F 2α (PGF 2α ) 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, E 2, and P 4 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Visfatin is a multifunctional protein which, besides the control of energy homeostasis, seems to be also involved in the regulation of female fertility through the influence on the endocrine hypothalamus-pituitary-gonadal axis, including the pituitary. The aim of this study was to investigate the expression of visfatin mRNA and protein in the anterior (AP) and posterior pituitary lobes of the pig during the oestrous cycle and early pregnancy. In AP, we also examined colocalisation of visfatin with pituitary tropic hormones. Moreover, we aimed to evaluate the in vitro effects of GnRH, FSH, LH, and insulin on visfatin protein concentration and secretion in AP cells during the cycle. The study showed that visfatin is present in all types of porcine pituitary endocrine cells and its expression is reliant on stage of the cycle or pregnancy. GnRH, FSH, LH and insulin stimulated visfatin secretion by AP cells on days 17 to 19 of the cycle, while on days 2 to 3 visfatin release was enhanced only by LH. Summarising, visfatin is locally produced in the pituitary in a way dependent on hormonal milieu typical for reproductive status of pigs. Further research is required to clarify the role of visfatin in the pituitary gland., (© 2023. Springer Nature Limited.)

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder with disrupted uterus structure and function. A positive effect of vitamin D 3 (VD 3 ) in female reproduction was observed. Chemerin (RARRES2) and adiponectin (ADIPOQ) are the main adipokines whose levels are altered in PCOS patients. Therefore, the aim of this study was to investigate the impact of VD 3 supplementation on RARRES2 and ADIPOQ levels in the uterus of PCOS rats., Methods: We analyzed the plasma levels and uterine transcript and protein expression of RARRES2 and ADIPOQ and their receptors (CCRL2, CMKLR1, GPR1, and ADIPOR1 and ADIPOR2, respectively) in rats with letrozole-induced PCOS., Results: In control animals, VD 3 did not change plasma levels of both adipokines, while in PCOS rats supplemented with VD 3 , they returned to control levels. The expression of RARRES2 and all investigated receptors increased in the uterus of VD 3 -treated rats; however, the levels of Rarres2 and Gpr1 genes remained unchanged. VD 3 supplementation decreased RARRES2, CMKLR1, and GPR1 but increased CCRL2 level to the control value. In the uterus of VD 3 -treated rats, the transcript and protein levels of ADIPOQ and both receptors ADIPOR1 increased. At the same time, VD 3 supplementation induced an increase in Adipoq , Adipor1 , and Adipor2 gene expression and restored protein levels to control level values., Conclusions: our findings indicate a new mechanism of VD 3 action in the uterine physiology of PCOS rats.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by excessive proliferation of abnormal myeloid precursors accompanied by a differentiation block and inhibition of apoptosis. Increased expression of an anti-apoptotic MCL-1 protein was shown to be critical for the sustained survival and expansion of AML cells. Therefore, herein, we examined the pro-apoptotic and pro-differentiating effects of S63845, a specific inhibitor of MCL-1, in a single-agent treatment and in combination with BCL-2/BCL-XL inhibitor, ABT-737, in two AML cell lines: HL-60 and ML-1. Additionally, we determined whether inhibition of the MAPK pathway had an impact on the sensitivity of AML cells to S63845. To assess AML cells' apoptosis and differentiation, in vitro studies were performed using PrestoBlue assay, Coulter electrical impedance method, flow cytometry, light microscopy and Western blot techniques. S63845 caused a concentration-dependent decrease in the viability of HL-60 and ML-1 cells and increased the percentage of apoptotic cells. Combined treatment with S63845 and ABT-737 or MAPK pathway inhibitor enhanced apoptosis but also induced differentiation of tested cells, as well as altering the expression of the MCL-1 protein. Taken together, our data provide the rationale for further studies regarding the use of MCL-1 inhibitor in combination with other pro-survival protein inhibitors.

Visfatin/NAMPT creates a hormonal link between energy metabolism and female reproduction. A recent study documented visfatin expression in the ovary and its action on follicular cells; however, the expression of visfatin in luteal cells is still unknown. The aim of this study, therefore, was to investigate the transcript and protein expression of visfatin as well as its immunolocalization in the corpus luteum (CL) and to examine the involvement of extracellular signal-regulated kinases (ERK1/2) in the regulation of visfatin level in response to LH, insulin, progesterone (P 4 ), prostaglandin E 2 (PGE 2 ) and F 2α (PGF 2α ). Corpora lutea were harvested from gilts on days 2-3, 10-12 and 14-16 of the estrous cycle and on days 10-11, 12-13, 15-16 and 27-28 of pregnancy. The current study demonstrated that visfatin expression depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Visfatin was immunolocalized to the cytoplasm of small and large luteal cells. Moreover, visfatin protein abundance was increased by P 4 , and decreased by both prostaglandins, while LH and insulin have modulatory effects, depending on the phase of the cycle. Interestingly, LH, P 4 and PGE 2 effects were abolished in response to the inhibition of ERK1/2 kinase. Thus, this study demonstrated that expression of visfatin in the porcine CL is determined by the endocrine status related to the estrous cycle and early pregnancy and by the action of LH, insulin, P 4 and prostaglandins via activation of the ERK1/2 pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) are one of the most prevalent classes of environmental pollutants. Some evidence shows that PAHs could be involved in human obesity. However, little is known about the distribution patterns of PAHs in human adipose tissue (AT) and the role of PAHs on adipogenesis/lipogenesis. The aims of this pilot study were to determine concentrations of 16 PAHs defined as high-priority pollutants in the plasma and adipose tissue of French and Polish bariatric patients, as well as their correlation with body mass index (BMI), plasma and AT adipokines expression levels. We finally investigated the role of naphthalene on cell proliferation, viability, and differentiation in 3T3-L1 preadipocytes. The concentration of most PAHs was similar in the three types of AT and it was significantly higher in AT as compared to plasma, suggesting bioaccumulation. Polish patients had higher PAH levels in AT than French ones. Only the concentration of naphthalene in AT was positively correlated with the BMI and serum or adipose chemerin, adiponectin and resistin expression, in French but not in Polish patients, who had significantly higher BMIs. Moreover, naphthalene exposure increased the cell proliferation of 3T3-L1 preadipocytes and lipogenesis, and increased the expression of genes involved in adipogenesis after cell differentiation. Taken together, PAHs and more particularly naphthalene could be an obesogenic molecule and increase the risk of obesity.

The apelinergic system comprises two peptide ligands, apelin and ELABELA, and their cognate G-protein-coupled receptor, the apelin receptor APJ. Apelin is a peptide that was isolated from bovine stomach extracts; the distribution of the four main active forms, apelin-36, -17, -13, and pyr-apelin-13 differs between tissues. The mature form of ELABELA-32 can be transformed into forms called ELABELA-11 or -21. The biological function of the apelinergic system is multifaceted, and includes the regulation of angiogenesis, body fluid homeostasis, energy metabolism, and functioning of the cardiovascular, nervous, respiratory, digestive, and reproductive systems. This review summarises the mechanism of the apelinergic system in cell apoptosis. Depending on the cell/tissue, the apelinergic system modulates cell apoptosis by activating various signalling pathways, including phosphoinositide 3-kinase (PI3K), extracellular signal-regulated protein kinase (ERK1/2), protein kinase B (AKT), 5'AMP-activated protein kinase(AMPK), and protein kinase A (PKA). Apoptosis is critically important during various developmental processes, and any dysfunction leads to pathological conditions such as cancer, autoimmune diseases, and developmental defects. The purpose of this review is to present data that suggest a significant role of the apelinergic system as a potential agent in various therapies.

The prevalence of adult obesity has risen markedly in recent decades. The endocrine system precisely regulates energy balance, fat abundance and fat deposition. Interestingly, white adipose tissue is an endocrine gland producing adipokines, which regulate whole-body physiology, including energy balance and reproduction. Endocrine disruptor chemicals (EDCs) include natural substances or chemicals that affect the endocrine system by multiple mechanisms and increase the risk of adverse health outcomes. Numerous studies have associated exposure to EDCs with obesity, classifying them as obesogens by their ability to activate different mechanisms, including the differentiation of adipocytes, increasing the storage of triglycerides, or elevating the number of adipocytes. Moreover, in recent years, not only industrial deception and obesity have intensified but also the problem of human infertility. Reproductive functions depend on hormone interactions, the balance of which may be disrupted by various EDCs or obesity. This review gives a brief summary of common EDCs linked with obesity, the mechanisms of their action, and the effect on adipokine levels, reproduction and connected disorders, such as polycystic ovarian syndrome, decrease in sperm motility, preeclampsia, intrauterine growth restriction in females and decrease of sperm motility in males., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spexin (SPX) is a newly identified neuropeptide, a natural ligand for the galanin receptors (GALR) 2/3, which is involved in maintaining physiological functions including female reproduction. One of the most common endocrine disorder in reproductive system is polycystic ovary syndrome (PCOS), however the role of SPX in PCOS is still unknown. The objective of this study was to determine the expression of mRNA and peptide levels of SPX and its receptors GALR2/3 in the hypothalamus and ovary (by real time PCR and Western blot) as well as plasma levels of SPX (ELISA) in letrozole - induced PCOS rats. We observed that SPX plasma level does not change in PCOS rats. In the hypothalamus transcript level of Spx and Galr3 were significantly higher in PCOS rats compared to the control, while mRNA of Galr2 and protein expression of GALR2/3 were lower. Moreover, expression of Spx and Galr2/3 mRNA as well as GALR2/3 peptide production were lower in the ovary of PCOS rats. In summary, while our results did not show differences in plasma SPX levels, we observed tissue-dependent significant differences in the SPX/GALR2/3 levels between PCOS and control rats, what indicates possible new mechanisms of PCOS neuroendocrinology., Competing Interests: Declaration of competing interest The authors have no financial or commercial conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The corpus luteum is a small gland of great importance because its proper functioning determines not only the appropriate course of the estrous/menstrual cycle and embryo implantation, but also the subsequent maintenance of pregnancy. Among the well-known regulators of luteal tissue functions, increasing attention is focused on the role of neuropeptides and adipose tissue hormones-adipokines. Growing evidence points to the expression of these factors in the corpus luteum of women and different animal species, and their involvement in corpus luteum formation, endocrine function, angiogenesis, cells proliferation, apoptosis, and finally, regression. In the present review, we summarize the current knowledge about the expression and role of adipokines, such as adiponectin, leptin, apelin, vaspin, visfatin, chemerin, and neuropeptides like ghrelin, orexins, kisspeptin, and phoenixin in the physiological regulation of the corpus luteum function, as well as their potential involvement in pathologies affecting the luteal cells that disrupt the estrous cycle.

The apelinergic system, which includes the apelin receptor (APJ) as well as its two specific ligands, namely apelin and ELABELA (ELA/APELA/Toddler), have been the subject of many recent studies due to their pleiotropic effects in humans and other animals. Expression of these factors has been investigated in numerous tissues and organs-for example, the lungs, heart, uterus, and ovary. Moreover, a number of studies have been devoted to understanding the role of apelin and the entire apelinergic system in the most important processes in the body, starting from early stages of human life with regulation of placental function and the proper course of pregnancy. Disturbances in the balance of placental processes such as proliferation, apoptosis, angiogenesis, or hormone secretion may lead to specific pregnancy pathologies; therefore, there is a great need to search for substances that would help in their early diagnosis or treatment. A number of studies have indicated that compounds of the apelinergic system could serve this purpose. Hence, in this review, we summarized the most important reports about the role of apelin and the entire apelinergic system in the regulation of placental physiology and pregnancy.

Resistin plays an important role in adipogenesis, obesity, insulin resistance, and reproduction. Previous studies showed resistin action on ovarian follicular cells; however, whether resistin regulates steroid secretion in luteal cells is still unknown. Our aim was first to determine the expression of resistin and its potential receptors (tyrosine kinase-like orphan receptor 1 (ROR1) and toll-like receptor 4 (TLR4)) in the porcine corpus luteum (CL), regulation of its expression, effect on kinases phosphorylation, and luteal steroidogenesis. Our results showed that the expression of resistin and its receptors was dependent on the luteal phase and this was higher at the mRNA level in the late compared with the early and middle luteal phase. At the opposite, resistin protein expression was higher in the middle and late compared with the early luteal phase, while ROR1 and TLR4 expression was highest in the early luteal phase. Additionally, we observed cytoplasmic localisation of resistin, ROR1, and TLR4 in small and large luteal cells. We found that luteinising hormone, progesterone (P4), insulin, and insulin-like growth factor 1 regulated the protein level of resistin, ROR1, and TLR4. Resistin decreased P4 and increased oestradiol (E2) secretion via changes in steroidogenic enzymes expression and via the activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAP3/1), increased the expression of receptors LHCGR and ESR2 and decreased the expression of PGR. Moreover, resistin decreased PKA phosphorylation and enhanced MAP3/1 phosphorylation. Taken together, resistin could act directly on steroid synthesis and serve as an important factor in in vivo luteal cell function.

Proper functioning of the body depends on hormonal homeostasis. White adipose tissue is now known as an endocrine organ due to the secretion of multiple molecules called adipokines. These proteins exert direct effects on whole body functions, including lipid metabolism, angiogenesis, inflammation, and reproduction, whereas changes in their level are linked with pathological events, such as infertility, diabetes, and increased food intake. Vaspin-visceral adipose tissue-derived serine protease inhibitor, or SERPINA12 according to serpin nomenclature, is an adipokine discovered in 2005 that is connected to the development of insulin resistance, obesity, and inflammation. A significantly higher amount of vaspin was observed in obese patients. The objective of this review was to summarize the latest findings about vaspin expression and action in endocrine tissues, such as the hypothalamus, pituitary gland, adipose tissue, thyroid, ovary, placenta, and testis, as well as discuss the link between vaspin and pathologies connected with hormonal imbalance.

Adipose tissue secretes multiple hormones termed adipokines, which are important regulators of many processes. There are four types of evidence supporting an association between adipokines and female fertility which are effects that occur: centrally at the pituitary; peripherally and locally at the ovary and reproductive tract; directly on the oocyte/embryo and during pregnancy. In this review, there was a focus on the description of adipokines (leptin, apelin, resistin, chemerin, adiponectin, vaspin and visfatin) on ovarian cell proliferation, cell cycle progression and apoptosis in comparison to effects on human and domestic animal ovaries including pigs, cattle and chickens. Knowledge about molecules which regulate the balance between proliferation and apoptosis so that these processes are optimal for ovarian function is essential for understanding the physiology and reducing the incidence of infertility. Furthermore, oogenesis, folliculogenesis, oocyte loss/selection and atresia are important processes for optimal ovarian physiological functions. There, however, is ovulation from only a few follicles, while the majority undergo atresia that is induced by apoptosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Previously, we demonstrated the expression of apelin and G-protein-coupled receptor APJ in human placenta cell lines as well as its direct action on placenta cell proliferation and endocrinology. The objective of this study was to examine the effect of apelin on placenta apoptosis in BeWo cells and villous explants from the human third trimester of pregnancy. The BeWo cells and villous explants were incubated with apelin (2 and 20 ng/mL) alone or with staurosporine for 24 to 72 h. First, we analysed the dose- and time-dependent effect of apelin on the expression of apoptotic factors on the mRNA level by real-time PCR and on the protein level using Western blot. Next, we checked caspase 3 and 7 activity by Caspase-Glo 3/7, DNA fragmentation by the Cell Death Detection ELISA kit and oxygen consumption by the MitoXpress-Xtra Oxygen Consumption assay. We found that apelin increased the expression of pro-survival and decreased proapoptotic factors on mRNA and protein levels in both BeWo cells and villous explants. Additionally, apelin inhibited caspase 3 and 7 activity and DNA fragmentation in staurosporine-induced apoptosis as also attenuated oxidative stress by increasing extracellular oxygen consumption. The antiapoptotic effect of apelin in BeWo cells was mediated by the APJ receptor and mitogen-activated protein kinase (ERK1/2/MAP3/1) and protein kinase B (AKT). The obtained results showed the antiapoptotic effect of apelin on trophoblast cells, suggesting its participation in the development of the placenta.

Background: The meaning of senescence for tissue physiological and pathological conditions is poorly known. Based on initial reports especially proteins and mechanisms that regulate this process are necessary to be determinate., Methods: The main aim of the study was to investigate the presence of senescent cells in canine testicular tissue (mixed breed testes; n = 60) in relation to adiponectin signaling. In detail, new information on the senescence cell number, as well as senescence and adiponectin signaling mechanisms in cryptorchid and germ cell tumor testes were provided with the use of immunohistochemical and colorimetric analyses., Results: Comparison of immunohistochemical results, in cryptorchid and tumor testes revealed increased number of senescent cells (p16 and γH2AX markers). Increased expression of adiponectin and adiponectin receptor 1, as well as extracellular signal-activated kinase (ERK1/2) in pathological testes were detected. In addition, decreased cholesterol and increased testosterone levels in tumor testis were found., Conclusion: The present study is the first to demonstrate the presence as well as the differences that exist in senecent cell number in mixed breed dog testes with cryptorchidism and germ cell tumor. Altered expression of adiponectin signaling and ERK1/2 signaling pathways together with altered cholesterol and testosterone levels reflect important senescence role in disturbed functions of canine testis. Moreover, the application of studied here senescence regulating molecules for detection and prevention against pathologies of the male gonad should be furtherly considered., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

In female, energy metabolism influences reproductive function by modulating the Hypothalamic Pituitary Ovarian axis including the hypothalamic GnRH neuronal network, the pituitary gonadotropin secretion and the ovarian follicle growth and steroidogenesis. Several hormones and neuropeptides or metabolites are important signals between energy balance and reproduction. These energy sensors mediate their action on reproductive cells through specific kinases or signaling pathways. This review focuses on the role of three main enzymes-specifically, mTOR, AMPK, and SIRT1 at the hypothalamic pituitary and ovarian axis in normal female fertility and then we discuss their possible involvement in some women reproductive disorders known to be associated with metabolic complications, such as polycystic ovary syndrome (PCOS) and premature ovarian failure (POF)., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vitamin D 3 (VD 3 ) plays an important role in the ovary and its deficiency is associated with ovarian pathologies, including polycystic ovary syndrome (PCOS). However, there is no data related to VD 3 metabolism in the ovary during PCOS. Herein, we investigated differences in the expression of VD 3 receptor (VDR) and key VD 3 metabolic enzymes, 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1), in the ovary and periovarian adipose tissue (POAT) of control (proestrus and diestrus) and PCOS induced by letrozole rats. Vdr, Cyp27b1 and Cyp24a1 mRNA expression was determined, their protein abundance was examined and immunolocalized. Furthermore, VD 3 metabolite concentrations in plasma (25OHD) and tissues (ovary and POAT; 1,25(OH) 2 D 3 ), and plasma calcium level were determined. 25OHD concentration decreased markedly in letrozole-treated rats in comparison with controls, whereas calcium concentration did not vary among the examined groups. The amount of 1,25(OH) 2 D 3 decreased in both ovary and POAT of PCOS rats. In the ovary, we found decreased Cyp27b1 and increased Vdr mRNA expression in letrozole-treated and diestrus control group. Corresponding protein abundances were down-regulated and up-regulated, respectively but only following letrozole treatment. In POAT, only Cyp27b1 transcript level and CYP27B1 protein abundance were decreased in letrozole-treated rats. VDR was immunolocalized in healthy and cystic follicles, while CYP27B1 and CYP24A1 were found exclusively in healthy ones. Concluding, our results provide the first evidence of disrupted VD 3 metabolism in the ovary and POAT of PCOS rats. The reduced 1,25(OH) 2 D 3 concentration in those tissues suggests their contribution to VD 3 deficiency observed in PCOS and might implicate in PCOS pathogenesis.

Oocyte maturation is a critical stage in embryo production and female reproduction. The aims of this study were to determine: (i) the mRNA and protein expression of vaspin and its receptor 78-kDa glucose-regulated (GRP78) in porcine cumulus-oocyte complexes (COCs) by real-time PCR and Western blot analysis, respectively, and their localisation by immunofluorescence; and (ii) the effects of vaspin on in vitro oocyte maturation (IVM) and the involvement of mitogen ERK1/2 (MAP3/1)- and AMPKα (PRKAA1)-activated kinases in the studied processes. Porcine COCs were matured in vitro for 22 h or 44 h with vaspin at a dose of 1 ng/mL and nuclear maturation assessed by Hoechst 33342 or DAPI staining and the measurement of progesterone (P4) level in the maturation medium. We showed that vaspin and GRP78 protein expression increased in oocytes and cumulus cells after IVM. Moreover, vaspin enhanced significantly porcine oocyte IVM and P4 concentration, as well as MAP3/1 phosphorylation, while decreasing PRKAA1. Using pharmacological inhibitors of MAP3/1 (PD98059) and PRKAA1 (Compound C), we observed that the effect of vaspin was reversed to the control level by all studied parameters. In conclusion, vaspin, by improving in vitro oocyte maturation via MAP3/1 and PRKAA1 kinase pathways, can be a new factor to improve in vitro fertilisation protocols.

In pig, backfat deposition is strongly related to the growth and reproductive performance. However, the molecular regulatory mechanisms of adipose tissue are not clearly understood. Adipose tissue is now recognized as an important endocrine organ that secretes a variety of factors including adipokines. However, the regulation of expression pattern of these adipokines in both plasma and visceral white adipose tissue (WAT) in lean and fat pig is unclear. In the present study, we used two representative porcine breeds (Large White, LW; Meishan, MS) with contrasting backfat thickness and sexual maturity age. Using specific ELISA assays, we determined the plasma profile of eight adipokines, leptin, adiponectin, visfatin, apelin, chemerin, resistin, omentin and vaspin in LW and MS sows. By RT-qPCR and western-blot we also investigated the mRNA and protein levels of these adipokines and their cognate receptors (LEPR, ADIPOR1, ADIPOR2, CMKLR1, CCRL2, GPR1, APLNR, TLR4, ROR1, CAP1 and HSPA5) in the peri renal WAT, respectively. At both plasma and peri renal WAT level, we found that the amounts of leptin, chemerin, resistin and vaspin were higher whereas those of adiponectin and omentin were lower in MS than LW sows. Plasma and adipose tissue visfatin and apelin levels were not different between the two breeds. Moreover, we noted that the variations of peri renal WAT adipokines observed between MS and LW were similar at the protein and mRNA level except for chemerin and apelin suggesting post-transcriptional modifications for these two adipokines. Finally, among the eight adipokines studied, we showed that only the plasma concentrations of leptin and chemerin were positively and those of adiponectin, negatively associated with the thickness of fat and opposite correlation was found for the onset of puberty in both LW and MS animals. Taken together, these results support a potential involvement of adipokines in WAT regulation and its link with the onset of the puberty in sows., (Copyright © 2020 Elsevier Inc. All rights reserved.)

One of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

Vaspin, visceral adipose tissue-derived serine protease inhibitor, plays important roles in inflammation, obesity, and glucose metabolism. Our recent research has shown the expression and role of vaspin in the function of ovarian follicles. However, whether vaspin regulates steroidogenesis and luteolysis in the corpus luteum (CL) is still unknown. The aim of this study was first to determine the expression of vaspin and its receptor GRP78 in porcine CL at the early, middle, and late stages of the luteal phase. Next, we investigated the hormonal regulation of vaspin levels in luteal cells in response to luteinizing hormone (LH), progesterone (P4), and prostaglandin PGE2 and PGF2α. Finally, we determined vaspin's direct impact on luteal cells steroidogenesis, luteolysis and kinases phosphorylation. Our results are the first to show higher vaspin/GRP78 expression in middle and late vs early stages; immunohistochemistry showed cytoplasmic vaspin/GRP78 localization in small and large luteal cells. In vitro, we found that LH, P4, PGE2, and PGF2α significantly decreased vaspin levels. Furthermore, vaspin stimulated steroidogenesis by the activation of the GRP78 receptor and protein kinase A (PKA). Also, vaspin increased the ratio of luteotropic PGE2 to luteolytic PGF2α secretion via GRP78 and mitogen-activated kinase (MAP3/1). Moreover, vaspin, in a dose-dependent manner, decreased GRP78 expression, while it, in a time-dependent manner, increased kinases PKA and MAPK3/1 phosphorylation. Taken together, we found that vaspin/GRP78 expression depends on the luteal phase stage and vaspin affects luteal cells endocrinology, indicating that vaspin is a new regulator of luteal cells steroidogenesis and CL formation.

Phoenixin (PNX) neuropeptide is a cleaved product of the Smim20 protein. Its most common isoforms are the 14- and 20-amino acid peptides. The biological functions of PNX are mediated via the activation of the GPR173 receptor. PNX plays an important role in the central nervous system (CNS) and in the female reproductive system where it potentiates LH secretion and controls the estrus cycle. Moreover, it stimulates oocyte maturation and increases the number of ovulated oocytes. Nevertheless, PNX not only regulates the reproduction system but also exerts anxiolytic, anti-inflammatory, and cell-protective effects. Furthermore, it is involved in behavior, food intake, sensory perception, memory, and energy metabolism. Outside the CNS, PNX exerts its effects on the heart, ovaries, adipose tissue, and pancreatic islets. This review presents all the currently available studies demonstrating the pleiotropic effects of PNX.

Formation and limited lifespan of corpus luteum (CL) are important for proper ovarian periodicity and fertility. Failed vascularization, imbalance between proliferation and apoptosis leads to luteal phase deficiency and infertility. The aim of this study was to examine the effect of vaspin on angiogenesis, apoptosis and proliferation as well as the involvement of 78-kDa glucose-regulated protein receptor (GRP78) and mitogen-activated kinase (MAP3/1) in these processes. Porcine luteal cells were incubated with vaspin (0.1-10 ng/mL) for 24 h to 72 h and then mRNA and protein expression of angiogenesis: vascular endothelial growth factor (VEGFA), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), VEGFA receptors (VEGFR1, VEGFR2), apoptosis: caspase 3, bcl-2-like protein 4 (BAX), B-cell lymphoma (BCL2), and proliferation: proliferating cells nuclear antigen (PCNA), cyclin A factors as well as secretion of VEGFA, FGF2, ANGT1 were measured by real-time polymerase chain reaction (PCR), immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Moreover, apoptosis was assessed by caspase activity using the Caspase-Glo 3/7 assay, while proliferation was by alamarBlue. We found that vaspin enhanced luteal cell angiogenesis, proliferation, and significantly decreased apoptosis. Additionally, using GRP78 siRNA and the pharmacological inhibitor of MAP3/1 (PD98059), we observed that the effect of vaspin was reversed to the control level in all investigated processes. Taken together, our results suggest that vaspin is a new regulator of female fertility by direct regulation of CL formation and maintenance of luteal cell function.

Childhood maltreatment, which can take the form of physical or psychological abuse, is experienced by more than a quarter of all children. Early life stress has substantial and long-term consequences, including an increased risk of drug abuse and psychiatric disorders in adolescence and adulthood, and this risk is higher in women than in men. The neuronal mechanisms underlying the influence of early life adversities on brain functioning remain poorly understood; therefore, in the current study, we used maternal separation (MS), a rodent model of early-life neglect, to verify its influence on the properties of neurons in the ventral tegmental area (VTA), a brain area critically involved in reward and motivation processing. Using whole-cell patch-clamp recordings in brain slices from adolescent female Sprague-Dawley rats, we found an MS-induced increase in the excitability of putative dopaminergic (DAergic) neurons selectively in the medial part of the VTA. We also showed an enhancement of excitatory synaptic transmission in VTA putative DAergic neurons. MS-induced alterations in electrophysiology were accompanied by an increase in the diameter of dendritic spine heads on lateral VTA DAergic neurons, although the overall dendritic spine density remained unchanged. Finally, we reported MS-related increases in basal plasma ACTH and corticosterone levels. These results show the long-term consequences of early life stress and indicate the possible neuronal mechanisms of behavioral disturbances in individuals who experience early life neglect., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 The Authors.)

Phoenixin (PNX) is a newly discovered peptide produced by proteolytic cleavage of a small integral membrane protein 20 (Smim20), which acts as an important regulator of energy homeostasis and reproduction. Since dysfunction of reproduction is characteristic in polycystic ovarian syndrome (PCOS), the role of PNX in pathogenesis of PCOS needs further investigation. The objective of this study was to determine expression of Smim20, PNX-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue (PAT) of letrozole induced PCOS rats. Phosphorylation of extracellular signal-regulated kinase (ERK1/2), protein kinases A (PKA) and B (Akt) were also estimated. We observed that PCOS rats had high weight gain and a number of ovarian cyst, high levels of testosterone, luteinizing hormone and PNX-14, while low estradiol. Smim20 mRNA expression was higher in the ovary and PAT, while PNX-14 peptide production was higher only in the ovary of PCOS rat. Moreover, in PCOS rats Gpr173 level was lower in PAT but at the protein level increased only in the ovary. Depending on the tissues, kinases phosphorylation were significantly differ in PCOS rats. Our results showed higher levels of PNX-14 in PCOS rats and indicated some novel findings regarding the mechanisms of PCOS pathophysiology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Using JEG-3 and BeWo cells, we examined the effect of "real life" mixtures of polycyclic aromatic hydrocarbons (PAHs), at doses reported in maternal blood (Mix I) and in placental tissue (Mix II), on human chorionic gonadotropin (hCG), placental lactogen (hPL) and placental growth factor (hPLGF) secretion, protein expression and immunolocalization. Additionally, the action of PAH mixtures on basal and hormone-stimulated matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) protein expression was evaluated. Under basal conditions, the PAH mixtures increased hCG and decreased hPLGF levels in both cell lines, while hPL expression was stimulated in JEG-3 and inhibited in BeWo. There was no effect on the MMP-2/MMP-9 ratio or VEGF expression. In hormone-stimulated cells, PAH mixtures changed the MMP-2/MMP-9 ratio in JEG-3 cells in favor of MMP-9, while in BeWo MMP-2 was favored. The effect on VEGF expression was cell specific and dependent on the mixture. In hCG-treated cells, only Mix II inhibited VEGF expression in JEG-3 cells. Neither PAH mixtures affected this protein in BeWo cells. In hPL-treated cells, Mix I had a stimulatory effect in JEG-3 cells, while Mix II exerted an inhibitory effect in BeWo cells. In hPLGF-treated cells, Mix II decreased in JEG-3 cells, but in BeWo cells, both mixtures increased VEGF expression. Considering that the evaluated protein hormones play crucial roles in angiogenesis and neovascularization in the placenta, "real life" PAH mixtures by disrupting protein hormones levels, the MMP-2/MMP-9 ratio and VEGF expression can lead to insufficiency and many pregnancy-related disorders., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

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., (© 2020 Wiley Periodicals LLC.)

Adipokines influence energy metabolism and have effects on male reproduction, including spermatogenesis and/or Sertoli cell maturation; however, the relationship between these active proteins and androgens in testicular cells is limited. Here, we studied the impact of short-term exposure to flutamide (an anti-androgen that blocks androgen receptors) on the expression of chemerin, apelin, vaspin and their receptors (CCRL2, CMKLR1, GPR1, APLNR, GRP78, respectively) in adult rat testes. Moreover, the levels of expression of lipid metabolism-modulating proteins (PLIN1, perilipin1; TSPO, translocator protein) and intercellular adherens junction proteins (nectin-2 and afadin) were determined in testicular cells. Plasma levels of adipokines, testosterone and cholesterol were also evaluated. Gene expression techniques used included the quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The androgen-mediated effects observed post-flutamide treatment were found at the gonadal level as chemerin, apelin, and vaspin gene expression alterations at mRNA and protein levels were detected, whereas the cellular targets for these adipokines were recognised by localisation of respective receptors in testicular cells. Plasma concentrations of all adipokines were unchanged, whereas plasma cholesterol content and testosterone level increased after flutamide exposure. Differential distribution of adipokine receptors indicates potential para- or autocrine action of the adipokines within the rat testes. Additionally, changes in the expression of PLIN1 and TSPO, involved in the initial step of testosterone synthesis in Leydig cells, suggest that testicular cells represent a target of flutamide action. Increase in the gene expression of PLIN1 and TSPO and higher total plasma cholesterol content indicates enhanced availability of cholesterol in Leydig cells as a result of androgen-mediated effects of flutamide. Alterations in adherens junction protein expression in the testis confirm the flutamide efficacy in disruption of androgen signalling and presumably lead to impaired para- and autocrine communication, important for proper functioning of adipokines.

Vaspin, visceral-adipose-tissue-derived serine protease inhibitor, is involved in the development of obesity, insulin resistance, inflammation, and energy metabolism. Our previous study showed vaspin expression and its regulation in the ovary; however, the role of this adipokine in ovarian cells has never been studied. Here, we studied the in vitro effect of vaspin on various kinase-signaling pathways: mitogen-activated kinase (MAP3/1), serine/threonine kinase (AKT), signal transducer and activator of transcription 3 (STAT3) protein kinase AMP (PRKAA1), protein kinase A (PKA), and on expression of nuclear factor kappa B (NFKB2) as well as on steroid synthesis by porcine ovarian cells. By using western blot, we found that vaspin (1 ng/ml), in a time-dependent manner, increased phosphorylation of MAP3/1, AKT, STAT3, PRKAA1, and PKA, while it decreased the expression of NFKB2. We observed that vaspin, in a dose-dependent manner, increased the basal steroid hormone secretion (progesterone and estradiol), mRNA and protein expression of steroid enzymes using real-time PCR and western blot, respectively, and the mRNA of gonadotropins (FSHR, LHCGR) and steroids (PGR, ESR2) receptors. The stimulatory effect of vaspin on basal steroidogenesis was reversed when ovarian cells were cultured in the presence of a PKA pharmacological inhibitor (KT5720) and when GRP78 receptor was knocked down (siRNA). However, in the presence of insulin-like growth factor type 1 and gonadotropins, vaspin reduced steroidogenesis. Thus, vaspin, by activation of various signaling pathways and stimulation of basal steroid production via GRP78 receptor and PKA, could be a new regulator of porcine ovarian function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis-controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors ( n = 20) were obtained from patients (31-45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients ( n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

Placentation requires the production of numerous growth factors, hormones and transcription factors. Many of them, like the adipose tissue‑derived leptin or adiponectin, have been identified in the placenta and their role has been established in the proliferation and subsequent development of the placenta. Apelin is another adipokine known for proliferative effects in different cell types. PCR, immunoblotting and immunocytochemistry were used to study mRNA and protein expression of apelin and its receptor (APJ) in syncytiotrophoblast (BeWo) and cytotrophoblast (JEG‑3) cells as well in immunohistochemistry in human normal placenta slides. The effect of apelin on cell proliferation study was investigated by alamarBlue® and Cell Counting Kit‑8 assays, the cell cycle by the flow cytometry method and the protein expression of cyclins and phosphorylation level of extracellular signal‑regulated kinases (ERK)1/2, phosphatidylinositol 3'‑kinase/protein kinase B (Akt), signal transducer and activator of transcription 3 (Stat3) and 5'‑monophosphate‑activated protein kinase (AMPKα) were studied by western blotting. Apelin was increased in JEG‑3 compared with in BeWo cells, while APJ was the same in both placenta cell lines. Immunocytochemical analyses revealed high cytoplasmic and/or membrane apelin localisation in JEG‑3, while BeWo cells exhibited markedly weaker apelin signal in the cytoplasm. Apelin increased cell proliferation as well as the percentage of cells in the G2/M phase of the cell cycle, cyclin proteins and the expression of all kinases mentioned above. In conclusion, apelin by promotion of trophoblast cell proliferation by APJ and ERK1/2, Stat3 and AMPKα signalling could be a new important adipokine in the regulation of early placental development.

Vaspin, a visceral adipose tissue-derived serine protease inhibitor, is expressed in the porcine ovary; it induces the activation of various kinases and steroidogenesis. The aim of this study was to examine the effect of vaspin on granulosa (Gc) proliferation, cell cycle regulation, and apoptosis. Porcine Gc was incubated with vaspin (0.01-10 ng/mL) for 24 to 72 h, proliferation was measured using alamarBlue assay, cell cycle progression was assessed using flow cytometry, and cyclin (D, E, and A) protein expression was measured using immunoblotting. Apoptosis was assessed by measuring caspase activity using Caspase-glo 3/7 assay. Furthermore, histone-associated DNA fragments levels were measured using a cell-death detection ELISA; BAX (bcl-2-like protein 4), BCL2 (B-cell lymphoma 2), caspases (-3, -8, and -9), p53 mRNA, and protein expression were assessed using real time PCR and immunoblotting. We found that vaspin significantly enhanced Gc proliferation and cell cycle progression into the S and G2/M phases and decreased apoptosis. We observed that siRNA silencing of the glucose-regulated protein (GRP78) receptor and pharmacological inhibitors of mitogen-activated kinase (MAP3/1/ERK1/2), Janus kinase (STAT3) and protein kinase B (AKT) blocked the ability of vaspin cell proliferation and enhanced caspase-3/7 activities. These results suggest that vaspin via mitogenic effect on porcine Gc acts as a new regulator of ovarian growth, development, or folliculogenesis.

Earwigs (Dermaptera) use different strategies to increase their reproductive success. Most species lay eggs; however, viviparity of the matrotrophic type has been reported in two groups: Hemimeridae and Arixeniidae. In Arixeniidae, offspring develop in two separate places: inside an ovary (the intraovarian phase) and within a uterus (the intrauterine phase). Both morphological and physiological aspects of viviparity in Arixeniidae have begun to be unraveled only recently. Here, we characterize how the first instar larvae of Arixenia esau, developing inside the mother's reproductive system, manage respiration and gas exchange. Using modern light and electron microscopy techniques as well as immunological approach, we provide a detailed account of the maternal and larval tissue interactions during the intrauterine development. We demonstrate that respiration in the Arixenia first instar larvae relies on the extensive tracheal system of the mother as well as a respiratory pigment (hemocyanin) present within the body cavity of the larvae. Our results indicate that the larval fat body tissue is the likely place of the hemocyanin synthesis. Our study shows that characteristic cone-shaped lobes of the outgrowths located on the larval abdomen are a part of a placenta-like organ and mediate the gas exchange between the maternal and larval organisms. Based on the obtained results, we propose that Arixenia esau evolved a unique biphasic system supporting respiration of the first instar larvae during their development inside the mother's reproductive tract.