Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

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.

Adipolin (C1QTNF12) is a new adipokine in female reproduction: expression and function in porcine granulosa cells.

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-IGF1Rß-to-IGFR1ß 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 Affects the Transcriptome of Porcine Luteal Cells during Early Pregnancy.

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.

Spexin role in human granulosa cells physiology and PCOS: expression and negative impact on steroidogenesis and proliferation†.

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.

Synergistic Action of MCL-1 Inhibitor with BCL-2/BCL-XL or MAPK Pathway Inhibitors Enhances Acute Myeloid Leukemia Cell Apoptosis and Differentiation.

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.

Concentration of Polycyclic Aromatic Hydrocarbons (PAHs) in Human Serum and Adipose Tissues and Stimulatory Effect of Naphthalene in Adipogenesis in 3T3-L1 Cells.

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.

Levels of spexin and its receptors GALR2 and GALR3 in the hypothalamus and ovary of letrozole-induced polycystic ovary syndrome in rats.

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.

Altered vitamin D3 metabolism in the ovary and periovarian adipose tissue of rats with letrozole-induced PCOS.

Vitamin D3 (VD3) 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 VD3 metabolism in the ovary during PCOS. Herein, we investigated differences in the expression of VD3 receptor (VDR) and key VD3 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, VD3 metabolite concentrations in plasma (25OHD) and tissues (ovary and POAT; 1,25(OH)2D3), 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)2D3 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 VD3 metabolism in the ovary and POAT of PCOS rats. The reduced 1,25(OH)2D3 concentration in those tissues suggests their contribution to VD3 deficiency observed in PCOS and might implicate in PCOS pathogenesis.

Expression and role of resistin on steroid secretion in the porcine corpus luteum.

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.

Anti-Apoptotic Effect of Apelin in Human Placenta: Studies on BeWo Cells and Villous Explants from Third-Trimester Human 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.

Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome.

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.

Role of vaspin in porcine ovary: effect on signaling pathways and steroid synthesis via GRP78 receptor and protein kinase A†.

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 pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells.

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.

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.

Adipokines expression profiles in both plasma and peri renal adipose tissue in Large White and Meishan sows: A possible involvement in the fattening and the onset of puberty.

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.

Novel Insights on the Corpus Luteum Function: Role of Vaspin on Porcine Luteal Cell Angiogenesis, Proliferation and Apoptosis by Activation of GRP78 Receptor and MAP3/1 Kinase Pathways.

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.

Phoenixin: More than Reproductive Peptide.

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.

Expression and Impact of Vaspin on In Vitro Oocyte Maturation through MAP3/1 and PRKAA1 Signalling Pathways.

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.

Flutamide Alters the Expression of Chemerin, Apelin, and Vaspin and Their Respective Receptors in the Testes of Adult Rats.

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.

Interstitial Leydig Cell Tumorigenesis-Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis.

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.