Adiponectin and visfatin expression profile in extra-embryonic annexes and role during embryo development in layer and broiler chickens.

Some evidence showed differences between layer and broiler embryo development. We recently showed that two adipokines, adiponectin and visfatin are expressed in the extra embryonic membranes and fluids. However, their role in the embryo development is unknown. Thus, our objectives were 1. to compare the expression of AdipoQ and its receptors AdipoR1 and AdipoR2 and visfatin in extra-embryonic annexes in broiler and layer breeders during the embryo development and 2. to investigate the role of two adipokines in embryo development in both broiler and layer breed after in ovo injection of blocking antibodies against chicken adiponectin or visfatin. We found that adiponectin, AdipoR1, AdipoR2 and visfatin were mainly more expressed in the allantoic that in amniotic membranes. In addition, these expressions increased according the stage of embryo development. We observed a higher expression in layer than in broiler of AdipoQ in allantoic membranes at ED14 and ED18, of AdipoR1 and AdipoR2 in both allantoic and amniotic membranes at ED7 and ED14 and of visfatin only in allantoic membrane from ED7 to ED18. AdipoQ and visfatin were absent in amniotic fluid at ED7 but present at ED14 or ED18 where higher concentrations were detected in layer than in broiler. Interestingly, we showed a strong positive correlation between Adipo and visfatin concentration in amniotic fluid and the body weight of embryo in both breeds. However, after in ovo injection of Adipo antibodies we did not observe any effect on the embryo mortality whereas injection of visfatin antibodies increased in a dose dependent manner the embryo mortality in both breeds. Taken together, Adipo and visfatin are higher expressed in layer than broiler in extra-embryonic membranes and amniotic fluid. Our data suggest also that visfatin could be a main regulator of embryo development.

Expression of chemerin and its receptors in extra-embryonic annexes and role of chemerin and its GPR1 receptor in embryo development in layer and broiler hens.

Intensive genetic selection of broiler breeders and layer hens resulted in differences in the mechanisms of growth and also in cell metabolism during embryogenesis. Previous research has shown that an adipokine named chemerin and one of these receptors, CMKLR1 were potentially involved in broiler embryo development. Here, our objectives were 1) to compare the expression of chemerin and its receptors CMKLR1, GPR1, and CCRL2 and chemerin concentration in extra-embryonic annexes (allantoic and amniotic membranes and fluids and plasma) in broiler and layer fertile eggs during the development (embryonic day (ED) 7, 14, and 18) by RT-qPCR and specific chicken ELISA and 2) to investigate the role of chemerin and one of its receptors GPR1 in embryo development after in ovo injections of neutralizing antibodies against chicken chemerin and GPR1. We found that chemerin expression in amniotic membranes was higher in layer than broiler eggs at ED7 and ED14 whereas the expression of the 3 receptors was higher in layer than broiler in the allantoic membranes at ED14 and ED18. Chemerin concentration was more important in layer than broiler at ED14 and ED18 in amniotic liquid and at all the studied stages in blood plasma. We also showed positive correlation between amniotic chemerin concentration and chemerin amniotic membrane expression, chemerin plasma concentration and embryo body weight in both breeds. Finally, in ovo injection of chicken chemerin and GPR1 neutralizing antibodies increased embryo mortality in both layer and broiler eggs. Taken together, even if chemerin concentration and chemerin system expression in embryonic membranes are mainly higher expressed in layer than in broiler, chemerin potentially through GPR1 could promote embryo development in both breeds.

Chemerin concentration in egg white in layer and broiler hens during the laying period for 2 successive generations.

The genetic selection progress in layers and broilers makes poultry production one of the fastest growing industries. Objectives of the breeding companies are the stability or the increase in the laying rate and the production of viable chicks. New biomarkers are necessary to improve reproductive and egg performances. Chemerin (Chem) produced by oviduct accumulates in egg white (EW). Here, we hypothesized that EW Chem concentration was dependent on the stage of laying and on the breed (layer vs. broiler). In addition, they could be associated to laying performance and fertility parameters. In breeding companies, we collected during 2 successive generations (G0 (mother) and G1 (daughter)) eggs from 100 layers and 100 broilers hens during 5 d at 3 stages: before, after laying peak and at the end of laying period. For each egg, the EW was sampled to measure Chem concentration by ELISA assay. In each generation at the end of laying period, magnums from oviduct, where the EG is formed, were collected in layers and broilers in order to investigate Chem differential expression by RT-qPCR between both breeds. Chem concentration in EW was dependent on the time of laying period and its profile was differently regulated in layers and broilers. Indeed, it increased at the end of laying in layers whereas it decreased after the laying peak in broilers. At the end of laying period, Chem concentration in EW was almost 2-fold higher in layers than in broilers and this was confirmed in both G0 and G1 generations at the Chem mRNA and protein levels in the magnum. For the 2 successive generations, Chem concentration in EW was negatively correlated with the laying rate and the fertility parameter in broiler hens whereas it was negatively correlated with the egg quality (weight of whole egg and weight of albumen) and positively with the fertility rate at some time of laying in layer hens. Taken together, the Chem concentration in EW could be a potential predictive tool for reproductive parameters in genetic selection.

Adipokines expression in reproductive tract, egg white and embryonic annexes in hen.

In mammals, molecules mainly secreted by white adipose tissue named adipokines are also synthetized locally in the reproductive tract and are able to influence reproductive functions. In avian species, previous studies indicated that the adipokine chemerin is highly abundant in the albumen, compared to the yolk and this was associated to high chemerin expression in the magnum. In addition, the authors observed that chemerin and its receptors are expressed by allantoic and amniotic membranes and chemerin is present in fluids during the embryo development. Here, we studied other adipokines, including adiponectin, visfatin, apelin, and adipolin in egg white and their known receptors in the active (egg-laying hen) and regressed (hen not laying) oviduct and embryonic annexes during embryo development. By using Western blot, RT-qPCR analysis and immunohistochemistry, we demonstrated the expression of different adipokines in the egg albumen (visfatin) and the reproductive tract (adiponectin, visfatin, apelin, adipolin, and their cognate receptors) according the position of egg in the oviduct. We showed that the expression of adipokines and adipokines receptors was strongly reduced in the regressed oviducts (arrested laying hen). Results indicated that visfatin and adiponectin appeared at ED11 to 14 and increased until ED18 in amniotic fluid whereas it was found from ED7 and was unchanged during embryo development in allantoic fluid. Taken together, adipokines and their receptors are expressed in the egg white, the reproductive tract and the embryonic annexes. Data obtained suggest important functions of theses metabolic hormones during the chicken embryo development. Thus, adipokines could be potential biomarkers to improve the embryo development.

The influence of selection in wild pheasant (Phasianus colchicus) breeding on reproduction and the involvement of the chemerin system.

Chemerin is a hormone produced mainly by adipose tissue and liver. We have recently shown that it is locally produced in the reproductive tract in hens, particularly at the magnum level, leading to its accumulation in the egg albumen. We have also determined that chemerin is necessary for egg fertilization, embryo development, and angiogenesis within the chorio-allantoic membrane in chicken species. We, therefore, hypothesize that chemerin, widely present in various gallinacean species, could be a marker of egg fertility in this animal order. To demonstrate this, we used a model close to the hen: the pheasant. By RT-qPCR, we have shown that chemerin and its three receptors CMKLR1, GPR1, and CCRL2 are expressed in the reproductive tract of females. In addition, chemerin is also produced predominantly in the magnum and accumulates in the egg albumen as determined by immunoblot. We then compared two lines of pheasants with different reproductive characteristics: the F11 and F22 breeds. F22 lays more eggs than F11, but have significantly lower fertility and hatchability rates. In addition, F22 exhibit a significantly lower amount of chemerin protein in their magnum (P < 0.01) and in the egg albumen (P < 0.0001) compared to F11. Finally, we observed a positive correlation between the chemerin amount in the albumen of F11 eggs and the hatching rate of the eggs (r = 0.5; P = 0.04) as well as a negative correlation between the chemerin quantity in the albumen of F22 eggs and the rate of unfertilized eggs (r = -0.37; P = 0.04). Finally, chemerin system (ligand and receptors) is also expressed within embryo annexes (chorioallantoic and amniotic membranes) during incubation. These data demonstrate an interspecies conservation of chemerin production in the magnum, its accumulation in the egg albumen and its possible use as a marker for determining the quality of eggs in term of fertility and embryo development.

Chicken white egg chemerin as a tool for genetic selection for egg weight and hen fertility.

Embryo mortality rate, which can reach up to 40% in avian species, is a major issue for breeding. It is therefore important to identify new embryo development biomarkers for genetic selection to improve reproductive performances. We have recently shown that chemerin is expressed in the oviductal hen magnum, accumulates in egg white, is correlated with embryo survival and could thus be used as a molecular marker of embryo development. Eggs from seven hen breeds (n = 70) were collected during five successive days at the end of the laying period. After weighing eggs, yolk and albumen, an egg white sample from each egg was collected and a blood sample was taken from each hen. Chemerin concentrations in albumen and blood samples were measured by a specific home made ELISA assay. Hen's plasma and egg's albumen chemerin levels were found to be correlated with reproductive parameters such as fecundity, fertility, embryo mortality, hatchability and laying rates. The inter-hen chemerin level variability in albumen was higher than intra-hen except for one breed (R+). We observed significantly different levels of chemerin in egg white between breeds. However, chemerin concentrations in egg white were not significantly associated to variations of hen plasma chemerin levels. Interestingly, we observed negative correlations between albumen chemerin concentrations and egg weight (r = -0.43, p = 0.001), between albumen weight (r = -0.40, p = 0.002), and between yolk weight (r = -0.28, p = 0.03). We also showed negative correlations between egg white chemerin concentrations and fecundity (r = -0.32, p = 0.011) and fertility (r = -0.27, p = 0.04) whereas no significant correlation was observed with the laying rate. Taken together, these results suggest that egg white chemerin concentration might be a good biomarker for genetic selection for egg weight and fertility in hens, provided these data are confirmed on a larger scale.

Chemerin is secreted by the chicken oviduct, accumulates in egg albumen and could promote embryo development.

Understanding of the distribution of chemerin and its receptors, Chemokine-like Receptor 1 (CMKLR1), G Protein-coupled Receptor 1 (GPR1) and Chemokine (C-C motif) receptor-like 2 (CCRL2), in the egg and the embryonic annexes is currently lacking, and their role during embryogenesis remains unknown. By immunoblot using monoclonal anti-chicken antibodies and Enzyme Linked Immunosorbent Assays (ELISA), we found that chemerin is expressed 10 times higher in albumen eggs than in blood plasma, and it is also abundant in the perivitelline membrane but undetectable in yolk. Chicken chemerin can inhibit bacterial growth. By Reverse Transcription-quantitative Polymerisation Chain Reaction (RT-qPCR), western-blot, and immunofluorescence, we show that chemerin is locally produced by the oviduct magnum that participates in albumen formation. Using cultures of magnum explants, we demonstrate that progesterone (P4) and oestradiol (E2) treatment increases chemerin secretion into cultured media and expression in magnum. Chemerin and its three receptors are present in amniotic and Chorio Allantoic Membranes (CAM). Only CMKLR1 expression decreased from embryonic day (ED) 7 to ED11 and remained low until ED18. Chemerin concentrations strongly increased in amniotic fluid at D14 when egg albumen crossed the amniotic membrane. In ovo injections of neutralising chemerin and CMKLR1 antibodies (0.01, 0.1 and 1 µg) increased embryo mortality, which occurred mainly at ED12-13, in a dose-dependent manner. Chemerin treatment increased primary CAM viability. Finally, chemerin and CMKLR1 inhibition within the CAM led to a decrease in blood vessel development and associated angiogenic gene expression. Our results show an important function of the chemerin system during embryo development in chickens, suggesting the potential use of this adipokine as a predictive marker for egg fertility or hatchability.

Adipokines in metabolic and reproductive functions in birds: An overview of current knowns and unknowns.

Adipose tissue is now recognized as an active endocrine organ, which synthesizes and secretes numerous peptides factors called adipokines. In mammals, they exert pleiotropic effects affecting energy metabolism but also fertility. In mammals, secretion of adipokines is altered in adipose tissue dysfunctions and may participate to obesity-associated disorders. Thus, adipokines are promising candidates both for novel pharmacological treatment strategies and as diagnostic tools. As compared to mammals, birds exhibit several unique physiological features, which make them an interesting model for comparative studies on endocrine control of metabolism and adiposity and reproductive functions. Some adipokines such as leptin and visfatin may have different roles in avian species as compared to mammals. In addition, some of them found in mammals such as CCL2 (chemokine ligand 2), resistin, omentin and FGF21 (Fibroblast Growth factor 21) have not yet been mapped to the chicken genome model and among its annotated gene models. This brief review aims to summarize data (structure, metabolic and reproductive roles and molecular mechanisms involved) related to main avian adipokines (leptin, adiponectin, visfatin, and chemerin) and we will briefly discuss the adipokines that are still lacking in avian species.

Chemerin Impairs In Vitro Testosterone Production, Sperm Motility, and Fertility in Chicken: Possible Involvement of Its Receptor CMKLR1.

The chemokine chemerin is a novel adipokine involved in the regulation of energy metabolism but also female reproductive functions in mammals. Its effects on male fertility are less studied. Here, we investigated the involvement of chemerin in chicken male reproduction. Indeed, the improvement of the sperm of roosters is a challenge for the breeders since the sperm quantity and quality have largely decreased for several years. By using specific chicken antibodies, here we show that chemerin and its main receptor CMKLR1 (chemokine-like receptor 1) are expressed within the chicken testis with the lowest expression in adults as compared to the embryo or postnatal stages. Chemerin and CMKLR1 are present in all testicular cells, including Leydig, Sertoli, and germinal cells. Using in vitro testis explants, we observed that recombinant chicken chemerin through CMKLR1 inhibits hCG (human chorionic gonadotropin) stimulated testosterone production and this was associated to lower 3ßHSD (3beta-hydroxysteroid dehydrogenase) and StAR (steroidogenic acute regulatory protein) expression and MAPK ERK2 (Mitogen-Activated Protein Kinase Extracellular signal-regulated kinase 2) phosphorylation. Furthermore, we demonstrate that chemerin in seminal plasma is lower than in blood plasma, but it is negatively correlated with the percentage of motility and the spermatozoa concentration in vivo in roosters. In vitro, we show that recombinant chicken chemerin reduces sperm mass and individual motility in roosters, and this effect is abolished when sperm is pre-incubated with an anti-CMKLR1 antibody. Moreover, we demonstrate that fresh chicken sperm treated with chemerin and used for artificial insemination (AI) in hen presented a lower efficiency in terms of eggs fertility for the four first days after AI. Taken together, seminal chemerin levels are negatively associated with the rooster fertility, and chemerin produced locally by the testis or male tract could negatively affect in vivo sperm quality and testosterone production through CMKLR1.

Adipokines Expression and Effects in Oocyte Maturation, Fertilization and Early Embryo Development: Lessons from Mammals and Birds.

Some evidence shows that body mass index in humans and extreme weights in animal models, including avian species, are associated with low in vitro fertilization, bad oocyte quality, and embryo development failures. Adipokines are hormones mainly produced and released by white adipose tissue. They play a key role in the regulation of energy metabolism. However, they are also involved in many other physiological processes including reproductive functions. Indeed, leptin and adiponectin, the most studied adipokines, but also novel adipokines including visfatin and chemerin, are expressed within the reproductive tract and modulate female fertility. Much of the literature has focused on the physiological and pathological roles of these adipokines in ovary, placenta, and uterine functions. The purpose of this review is to summarize the current knowledge regarding the involvement of leptin, adiponectin, visfatin, and chemerin in the oocyte maturation, fertilization, and embryo development in both mammals and birds.

Involvement of Novel Adipokines, Chemerin, Visfatin, Resistin and Apelin in Reproductive Functions in Normal and Pathological Conditions in Humans and Animal Models.

It is well known that adipokines are endocrine factors that are mainly secreted by white adipose tissue. Their central role in energy metabolism is currently accepted. More recently, their involvement in fertility regulation and the development of some reproductive disorders has been suggested. Data concerning the role of leptin and adiponectin, the two most studied adipokines, in the control of the reproductive axis are consistent. In recent years, interest has grown about some novel adipokines, chemerin, visfatin, resistin and apelin, which have been found to be strongly associated with obesity and insulin-resistance. Here, we will review their expression and role in male and female reproduction in humans and animal models. According to accumulating evidence, they could regulate the secretion of GnRH (Gonadotropin-Releasing Hormone), gonadotropins and steroids. Furthermore, their expression and that of their receptors (if known), has been demonstrated in the human and animal hypothalamo-pituitary-gonadal axis. Like leptin and adiponectin, these novel adipokines could thus represent metabolic sensors that are able to regulate reproductive functions according to energy balance changes. Therefore, after investigating their role in normal fertility, we will also discuss their possible involvement in some reproductive troubles known to be associated with features of metabolic syndrome, such as polycystic ovary syndrome, gestational diabetes mellitus, preeclampsia and intra-uterine growth retardation in women, and sperm abnormalities and testicular pathologies in men.

Apelin (APLN) and Apelin Receptor (APLNR) in Human Ovary: Expression, Signaling, and Regulation of Steroidogenesis in Primary Human Luteinized Granulosa Cells.

Apelin (APLN) is a recently discovered adipokine involved in the regulation of various metabolic functions. Its receptor, APLNR, is expressed in reproductive tissues, however, its role in human ovarian cells is unknown. In this study, we identified APLN and APLNR in human ovarian follicles and analyzed their expression in granulosa cells and follicular fluid obtained from obese and nonobese patients, with or without polycystic ovary syndrome (PCOS). We also investigated the effect of APLN on steroidogenesis in cultured human luteinized granulosa cells (hGCs) from nonobese patients without PCOS. Using RT-PCR and immunoblotting, we found that APLN and APLNR were expressed in hGCs and cumulus and theca cells. We confirmed these data immunohistochemically and observed that APLNR and APLN are present in human oocytes at different stages of follicular development. In patients with PCOS, we observed that follicular fluid APLN concentration and granulosa cell APLN and APLNR mRNA expression was higher than that observed in control patients. In cultured hGCs from nonobese patients without PCOS, insulin-like growth factor 1 (IGF1) increased APLNR expression, and recombinant human APLN (APLN-13 and APLN-17) increased both basal and IGF1-induced steroid secretion. These effects on steroid production were reversed when cultured in the presence of ML221, an APLNR antagonist, which was associated with an increased 3beta-hydrosteroid dehydrogenase (HSD3B) protein concentration. We showed that these effects were dependent on the activation of the AKT and MAPK3/1 pathways using pharmacological inhibitors. Our results show that APLN and APLNR are present in human ovarian cells and APLN increases IGF1-induced steroidogenesis in granulosa cells through an increase in HSD3B protein expression and activation of the MAPK3/1 and Akt pathways. Therefore, APLN and APLNR may play a role in human follicular development and the pathogenesis of PCOS.

VISFATIN (NAMPT) Improves In Vitro IGF1-Induced Steroidogenesis and IGF1 Receptor Signaling Through SIRT1 in Bovine Granulosa Cells.

VISFATIN is a novel adipokine, also known as a nicotinamide phosphorybosyltransferase (NAMPT), that is able to modulate different processes, including lipid and glucose metabolism, oxidative stress, inflammation, and insulin resistance. Recent data suggest that it also plays a role in reproductive function in rats, humans, and chickens. Here we identified VISFATIN in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and proliferation and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found VISFATIN in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, we showed that IGF1 (10(-8) M) and VISFATIN (10 and 100 ng/ml) but not FSH (10(-8) M) increased mRNA expression levels of NAMPT after 48 h of stimulation. Moreover, we observed that human recombinant VISFATIN (hVisf, 10 ng/ml, 48 h) increased the release of progesterone and estradiol secretion, and this was associated with an increase in the protein level of STAR, the HSD3B activity, and the phosphorylation levels of IGF1R and MAPK ERK1/2 in the presence or absence of IGF1 (10(-8) M). All these effects were abolished when NAMPT was knocked down and when the sirtuin pharmacological inhibitors CHIC-35 (60 nM) and EX-527 (0.5 µM) were preincubated in bovine granulosa cells. Thus, in cultured bovine granulosa cells, VISFATIN improves basal and IGF1-induced steroidogenesis and IGF1 receptor signaling through SIRT1.

Adipokines in human reproduction.

Adipose tissue communicates with other central and peripheral organs by the synthesis and release of substances called adipokines. The most studied adipokine is leptin but others have been recently identified including resistin, adiponectin, chemerin, omentin and visfatin. These adipokines have a critical role in the development of obesity-related complications and inflammatory conditions. However, they are also involved in other functions in the organism including reproductive functions. Indeed, many groups have demonstrated that adipokine receptors, such as adiponectin and chemerin, but also adipokines themselves (adiponectin, chemerin, resistin, visfatin and omentin) are expressed in human peripheral reproductive tissues and that these adipokines are likely to exert direct effects on these tissues. After a brief description of these new adipokines, an overview of their actions in different human reproductive organs (hypothalamus, pituitary, ovary, testis, uterus and placenta) will be presented. Finally, comments will be made on the eventual alterations of these adipokines in reproductive disorders, with special attention to polycystic ovary syndrome, a disease characterized by dysfunction of gonadal axis and systemic nerve endocrine metabolic network with a prevalence of up to 10% in women of reproductive age.

Expression of adiponectin, chemerin and visfatin in plasma and different tissues during a laying season in turkeys.

BACKGROUND: In mammals, adipose tissue is able to secrete various hormones called adipokines including adiponectin (ADP), chemerin (Chem) and visfatin (Visf) which are involved in controlling energy metabolism as well as reproductive functions. Visf receptor is still unknown whereas ADP and Chem mainly act through AdipoR1, AdipoR2 and CMKLR1 and GPR1 receptors, respectively. No studies have yet demonstrated the presence of these three adipokines in peripheral tissues, ovarian cells or turkey plasma. Here, we investigated the expression (mRNA and protein) of ADP, Chem, Visf and their receptors in peripheral tissues and ovarian cells (granulosa and theca cells) from hierarchical follicles. Furthermore, we determined the plasma profile of ADP, Visf and Chem at different physiological stages: start, peak and end of the laying period in Meleagris gallopavo turkeys. This data was correlated with the metabolic data (plasma glucose, triglycerides, cholesterol and phospholipids). METHODS: Tissue and ovarian cells mRNA and protein expression levels were determined by RT-qPCR and immunoblot, respectively. Plasma adipokines were measured by chicken ELISA and immunoblotting. RESULTS: In turkeys, Chem is mainly expressed in the liver while ADP and Visf are mainly expressed in the abdominal adipose tissue and pectoral muscles,respectively. As in mammals, AdipoR1 and AdipoR2 expression levels (mRNA and protein) are highly present in muscle and liver, respectively, whereas the mRNA expression of CMKLR1 and GPR1 is ubiquitous. In ovarian cells, ADP, Visf, Chem and their receptors are more highly expressed in theca cells than in granulosa cells excepted for AdipoR1. Furthermore, we found that plasma levels of ADP, Chem and Visf were reduced at the end of the laying period compared to the start of this period. At the plasma levels, the levels of these adipokines are strongly negatively correlated with glucose and only plasma Chem is negatively correlated with cholesterol, triglycerides and phospholipids. CONCLUSIONS: In turkeys, ADP, Visf and Chem and their receptors are expressed in peripheral tissues and ovarian cells. Plasma concentration of ADP, Visf and Chem decrease at the end of laying period and only plasma Chem is negatively correlated with levels of cholesterol, triglycerides and phospholipids levels during the entire laying period.

[Chemerin: a pro-inflammatory adipokine involved in the reproduction function?]. / La chémérine - Une adipocytokine pro-inflammatoire impliquée dans la fonction de reproduction ?

Chemerin is a pro-inflammatory adipokine secreted and expressed predominantly by adipocytes. Chemerin is initially involved in the regulation of the immune system, the adipogenesis and the energy metabolism. However, several recent studies show that this adipokine and its receptors are present in the gonads. In vitro, chemerin reduces steroidogenesis in ovarian and testicular cells in rodents and humans. Chemerin and its receptors are also present in the placenta. Chemerin plays an important role in the regulation of fetal and maternal metabolism, fetal growth and metabolic homeostasis during pregnancy. This review describes the role of chemerin in metabolism and reproduction.

Expression and effect of NAMPT (visfatin) on progesterone secretion in hen granulosa cells.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

Expression and regulation of INTELECTIN1 in human granulosa-lutein cells: role in IGF-1-induced steroidogenesis through NAMPT.

INTELECTIN (ITLN) is an adipokine involved in the regulation of insulin sensitivity and inflammatory and immunity responses. Serum ITLN levels are lower in obese, diabetic, and polycystic ovary syndrome (PCOS) women than in control subjects. ITLN has never been studied in ovarian cells. Here, we identified ITLN1 in human ovarian follicles and investigated the molecular mechanisms involved in the regulation of its expression in response to the insulin sensitizers metformin and rosiglitazone, in human granulosa-lutein cells (hGLCs) and in a human ovarian granulosa-like tumor cell line (KGN). We also studied the effects of human recombinant ITLN1 (hRom1) on steroid production and on the activation of various signaling pathways. Using RT-PCR, immunoblotting, and immunohistochemistry, we found that INTL1 is present in human follicular cells. Using ELISA, we showed that INTL levels are similar in plasma and follicular fluid (FF) in control patients, whereas they are higher in FF than in plasma in PCOS patients. In KGN cells and hGLCs, insulin (10(-8) M), insulin-like growth factor-1 (IGF-1; 10(-8) M), and metformin (10(-2) M or 10(-3) M) increased INTL1 expression (mRNA and protein) after 12 and 24 h of stimulation. For metformin, this effect was mediated by adenosine monophosphate-activated kinase (PRKA). Furthermore, hRom1 increased nicotinamide phosphoribosyltransferase (NAMPT) expression in KGN and hGLCs. We also showed that hRom1 increased IGF-1-induced progesterone and estradiol secretion and this was associated with an increase in the STAR and CYP19A1 protein levels and an increase in IGF-1R signaling. Furthermore, all these data were abolished when NAMPT was knocked down in KGN cells, suggesting that INTL1 improves IGF-1-induced steroidogenesis through induction of NAMPT in hGLCs.

Adipokines and the female reproductive tract.

It is well known that adipose tissue can influence puberty, sexual maturation, and fertility in different species. Adipose tissue secretes molecules called adipokines which most likely have an endocrine effect on reproductive function. It has been revealed over the last few years that adipokines are functionally implicated at all levels of the reproductive axis including the gonad and hypothalamic-pituitary axis. Many studies have shown the presence and the role of the adipokines and their receptors in the female reproductive tract of different species. These adipokines regulate ovarian steroidogenesis, oocyte maturation, and embryo development. They are also present in the uterus and placenta where they could create a favorable environment for embryonic implantation and play a key role in maternal-fetal metabolism communication and gestation. Reproductive functions are strongly dependent on energy balance, and thereby metabolic abnormalities can lead to the development of some pathophysiologies such as polycystic ovary syndrome (PCOS). Adipokines could be a link between reproduction and energy metabolism and could partly explain some infertility related to obesity or PCOS.

CHEMERIN (RARRES2) decreases in vitro granulosa cell steroidogenesis and blocks oocyte meiotic progression in bovine species.

CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation.