Becoming female: Ovarian differentiation from an evolutionary perspective.

Differentiation of the bipotential gonadal primordium into ovaries and testes is a common process among vertebrate species. While vertebrate ovaries eventually share the same functions of producing oocytes and estrogens, ovarian differentiation relies on different morphogenetic, cellular, and molecular cues depending on species. The aim of this review is to highlight the conserved and divergent features of ovarian differentiation through an evolutionary perspective. From teleosts to mammals, each clade or species has a different story to tell. For this purpose, this review focuses on three specific aspects of ovarian differentiation: ovarian morphogenesis, the evolution of the role of estrogens on ovarian differentiation and the molecular pathways involved in granulosa cell determination and maintenance.

High androgen concentrations in follicular fluid of polycystic ovary syndrome women.

BACKGROUND: According to current definitions of Polycystic Ovary Syndrome (PCOS), hyperandrogenism is considered as a key element in the pathogenesis of this common endocrinopathy. However, until now, studies about ovarian androgen profile in women are very rare. Our aim was then to characterise the expression profile of the androgens in follicular fluid of 30 PCOS patients, and compare it to those of 47 Control women and 29 women with only polycystic ovary morphology on ultrasounds (ECHO group). METHODS: A retrospective, single-centre cohort study was performed. The intrafollicular concentrations of the key androgens were assessed and correlated with the intrafollicular levels of some adipokines of interest. Androgens were quantified by mass spectrophotometry combined with ultra-high-performance liquid chromatography, while adipokine concentrations were measured by ELISA assays. RESULTS: In PCOS patients, the intrafollicular concentrations of the androgens synthesised by ovarian theca cells, i.e., 17OH-pregnenolone, dehydroepiandrosterone, Δ4-androstenedione and testosterone, were significantly higher than those of the androgens of adrenal origin, and positively correlated with the main PCOS clinical and biological features, as well as with the adipokines mostly expressed in the follicular fluid of PCOS patients, i.e. resistin, omentin, chemerin and apelin. Conversely, Control women showed the highest levels of 17OH-progesterone, deoxycorticosterone and 11-deoxycortisol. Confirming these results, apelin levels were negatively associated with pregnenolone and deoxycorticosterone concentrations, while visfatin levels, which were higher in the Control group, negatively correlated with the Δ4-androstenedione and testosterone ones. CONCLUSIONS: PCOS is characterised by a selective increase in the intrafollicular levels of the androgens synthesised by theca cells, strengthening the hypothesis that ovarian hyperandrogenism plays a central role in its pathogenesis. Further, the significant correlation between the intrafollicular concentrations of the androgens and most of the adipokines of interest, including apelin, chemerin, resistin and omentin, confirms the existence of a close relationship between these two hormonal systems, which appear deeply involved in ovarian physiology and PCOS physiopathology.

Involvement of chemerin and CMKLR1 in the progesterone decrease by PCOS granulosa cells.

Polycystic ovarian syndrome (PCOS) is the main cause of infertility in women. It is frequently associated with reduced progesterone production by human luteinised granulosa cells (hlGCs). However, the molecular mechanisms involved in these steroidogenesis alterations in PCOS patients are unclear. In a dihydrotestosterone-induced PCOS mouse model, steroid production is maintained in the setting of chemokine-like receptor 1 (Cmklr1) knockout. Thus, chemerin and chemerin receptors in terms of expression and progesterone regulation could be different in control and PCOS hlGCs. We first confirmed that progesterone levels in both plasma (P < 0.0001) and follicular fluid (FF) (P < 0.0001) were significantly reduced in PCOS normal weight women compared to control women. These data were associated with a lower STAR mRNA expression in both in vivo (P < 0.0001) and in vitro (P < 0.0001) hlGCs from PCOS women. Secondly, chemerin FF levels (P < 0.0001) and RARRES2 (P < 0.05) and CMKLR1 (P < 0.0001) mRNA levels in GCs were higher in PCOS normal weight patients. Thirdly, treatment of hlGCs with a specific nanobody (the VHH CA4910) targeting the human receptor for CMKLR1 leading to its inactivation abolished chemerin-induced progesterone inhibition, suggesting the involvement of CMKLR1 in this process. Furthermore, the inhibition of progesterone secretion induced by chemerin was two-fold higher in PCOS hlGCs (P < 0.05). Moreover, the VHH CA4910 reinstated a normal progesterone secretion with lower concentrations in PCOS hlGCs, suggesting a different chemerin sensitivity between PCOS and control hlGCs. Thus, chemerin, through CMKLR1, could be involved in the steroidogenesis alterations in PCOS hlGCs.

Pilot Study to Detect Genes Involved in DNA Damage and Cancer in Humans: Potential Biomarkers of Exposure to E-Cigarette Aerosols.

There is a paucity of data on how gene expression enables identification of individuals who are at risk of exposure to carcinogens from e-cigarette (e-cig) vaping; and how human vaping behaviors modify these exposures. This pilot study aimed to identify genes regulated from acute exposure to e-cig using RT-qPCR. Three subjects (2M and 1F) made three visits to the lab (nTOT = 9 visits); buccal and blood samples were collected before and immediately after scripted vaping 20 puffs (nTOT = 18 samples); vaping topography data were collected in each session. Subjects used their own e-cig containing 50:50 propylene glycol (PG):vegetable glycerine (VG) +3-6 mg/mL nicotine. The tumor suppressor TP53 was significantly upregulated in buccal samples. TP53 expression was puff volume and flow rate dependent in both tissues. In blood, the significant downregulation of N-methylpurine DNA glycosylase (MPG), a base excision repair gene, was consistent across all subjects. In addition to DNA repair pathway, cell cycle and cancer pathways were the most enriched pathways in buccal and blood samples, respectively. This pilot study demonstrates that vaping 20 puffs significantly alters expression of TP53 in human tissues; vaping behavior is an important modifier of this response. A larger study is needed to confirm these relationships.

Vaspin, a novel adipokine in woman granulosa cells physiology and PCOS pathogenesis?

Vaspin is a novel adipokine mainly expressed in visceral adipose tissue and closely related to obesity and insulin-resistance. Currently, data about its ovarian expression are limited to animal models and its role in human reproduction is largely unexplored. Our study's aims were then to characterise vaspin expression in the human ovary and to study in vitro its effects on granulosa cells physiology. Secondly, we assessed vaspin and its receptor GRP78 variations in granulosa cells and follicular fluid of a cohort of 112 infertile women undergoing an in vitro fertilisation procedure and allocated to three groups, each including normal-weight and obese subjects: 34 PCOS patients, 33 women with isolated polycystic ovary morphology (ECHO group) and 45 controls. Vaspin and GRP78 expression in the ovary was assessed by immunohistochemistry, RT-qPCR and Western blot. Granulosa cells and follicular fluid were analysed by RT-qPCR and ELISA, respectively. In vitro, granulosa cells metabolism was studied after stimulation with recombinant human vaspin, with and without a siRNA directed against GRP78. Vaspin was highly expressed in the human ovary and concentration-dependently enhanced granulosa cells steroidogenesis, proliferation and viability through GRP78 (P < 0.0001). Vaspin levels in both granulosa cells and follicular fluid were significantly higher in obese women (P < 0.0001) and in the normal-weight ECHO group (P < 0.001), which also had the highest expression rates of GRP78 (P < 0.05). Although further investigation is needed, vaspin appears as a novel modulator of human granulosa cells physiology and possibly plays a role in PCOS pathogenesis, notably protecting from insulin-resistance induced complications.

A grape seed extract maternal dietary supplementation improves egg quality and reduces ovarian steroidogenesis without affecting fertility parameters in reproductive hens.

In broiler hens, the genetic selection increased susceptibility to metabolic disorders and reproductive dysfunctions. In human ovarian cells, grape seed extracts (GSE) improved steroid production. Here, we investigated the effects of a GSE dietary supplementation on egg production and quality, fertility parameters, Reactive Oxygen Species (ROS) and steroid content in yolk egg associated to plasma adipokines in broiler hens. For this, we designed two in vivo experiments, the first one included three groups of hens: A (control), B and C (supplemented with GSE at 0.5% and 1% of the total diet composition, respectively, since week 4), and the second one used two groups of hens: A (control) and D (supplemented with GSE at 1% of the total diet composition since hatching). We assessed the egg production from 23th to 40th weeks and quality at 33th week. After artificial inseminations, the fertility parameters were calculated. In egg yolk, Reactive Oxygen Species (ROS) level and steroid production were evaluated by Ros-Glo H202 and ELISA assay, respectively. Expression of steroidogenic enzymes and adipokines and their receptors was determined by RT-qPCR in ovarian cells and plasma adipokines (RARRES2, ADIPOQ and NAMPT) were evaluated by specific ELISA assays. The fertility parameters and egg production were unaffected by GSE supplementation whatever the experiment (exp.). However, the rate of double-yolk eggs decreased for all GSE supplemented groups (exp. 1 P <0.01, exp.2, P<0.02). In exp.1, C group eggs were bigger and larger (P<0.0001) and the shell elasticity was higher for both B and C (P<0.0003) as compared to control. In the egg yolk, GSE supplementation in both exp. reduced ROS content and steroidogenesis consistent with a decrease in P450 aromatase and StAR mRNA expression and basal in vitro progesterone secretion in granulosa cells (P<0.001). Interestingly, in both exp. RARRES2 plasma levels were positively correlated while ADIPOQ and NAMPT plasma levels were negatively correlated, with steroids and ROS in yolk (P<0.0001). Taken together, maternal dietary GSE supplementation did not affect egg production and fertility parameters whereas it reduced ROS content and steroidogenesis in yolk egg. Furthermore, it ameliorated egg quality by decreasing the number of double-yolk eggs and by improving the size of normal eggs and the elasticity of the shell. Taken together, our data suggest the possibility of using dietary maternal GSE to improve egg quality.

In utero exposure to arsenite contributes to metabolic and reproductive dysfunction in male offspring of CD-1 mice.

In utero exposure to arsenite (iAs) is known to increase disease risks later in life. We investigated the effect of in utero exposure to iAs in the drinking water on metabolic and reproductive parameters in male mouse offspring at postnatal and adult stages. Pregnant CD-1 mice were exposed to iAs (as sodium arsenite) in the drinking water at 0 (control), 10 ppb (EPA standard for drinking water), and 42.5 ppm (tumor-inducing dose in mice) from embryonic day (E) 10-18. At birth, pups were fostered to unexposed females. Male offspring exposed to 10 ppb in utero exhibited increase in body weight at birth when compared to controls. Male offspring exposed to 42.5 ppm in utero showed a tendency for increased body weight and a smaller anogenital distance. The body weight in iAs-exposed pups continued to increase significantly compared to control at 3 weeks and 11 weeks of age. At 5 months of age, iAs-exposed males exhibited greater body fat content and glucose intolerance. Male offspring exposed to 10 ppb in utero had higher circulating levels of leptin compared to control. In addition, males exposed to 42.5 ppm in utero exhibited decreased total number of pups born compared to controls and lower average number of litters sired over a six-month period. These results indicate that in utero exposure to iAs at either human relevant concentration or tumor-inducing concentration is a potential cause of developmental origin of metabolic and reproductive dysfunction in adult male mice.

A grape seed extract maternal dietary supplementation in reproductive hens reduces oxidative stress associated to modulation of plasma and tissue adipokines expression and improves viability of offsprings.

In reproductive hens, a feed restriction is an usual practice to improve metabolic and reproductive disorders. However, it acts a stressor on the animal. In mammals, grape seed extracts (GSE) reduces oxidative stress. However, their effect on endocrine and tissue response need to be deepened in reproductive hens. Here, we evaluated the effects of time and level of GSE dietary supplementation on growth performance, viability, oxidative stress and metabolic parameters in plasma and metabolic tissues in reproductive hens and their offsprings. We designed an in vivo trial using 4 groups of feed restricted hens: A (control), B and C (supplemented with 0.5% and 1% of the total diet composition in GSE since week 4, respectively) and D (supplemented with 1% of GSE since the hatch). In hens from hatch to week 40, GSE supplementation did not affect food intake and fattening whatever the time and dose of supplementation. Body weight was significantly reduced in D group as compared to control. In all hen groups, GSE supplementation decreased plasma oxidative stress index associated to a decrease in the mRNA expression of the NOX4 and 5 oxidant genes in liver and muscle and an increase in SOD mRNA expression. This was also associated to decreased plasma chemerin and increased plasma adiponectin and visfatin levels. Interestingly, maternal GSE supplementation increased the live body weight and viability of chicks at hatching and 10 days of age. This was associated to a decrease in plasma and liver oxidative stress parameters. Taken together, GSE maternal dietary supplementation reduces plasma and tissue oxidative stress associated to modulation of adipokines without affecting fattening in reproductive hens. A 1% GSE maternal dietary supplementation increased offspring viability and reduced oxidative stress suggesting a beneficial transgenerational effect and a potential use to improve the quality of the progeny in reproductive hens.

Unraveling the Adipose Tissue Proteome of Transition Cows through Severe Negative Energy Balance.

Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points (one month before calving, one and sixteen weeks after calving) that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins (annexin A2, actin-related protein 10, glyceraldehyde-3-phosphate dehydrogenase, and fatty acid-binding protein) involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum.

Impact of the severity of negative energy balance on gene expression in the subcutaneous adipose tissue of periparturient primiparous Holstein dairy cows: Identification of potential novel metabolic signals for the reproductive system.

The severity of negative energy balance (NEB) in high-producing dairy cows has a high incidence among health diseases. The cow's energy status during early lactation critically affects metabolic and reproductive parameters. The first objective of this study was to investigate by RNA-seq analysis and RT-qPCR the gene expression profile in white adipose tissue and by gene ontology and upstream regulation tools the relationships with energy metabolism and reproduction in two groups of primiparous dairy cows with extreme NEB statuses (NEB < -9 Mcal/day vs. NEB > -9 Mcal/day) around parturition. The second objective was to determine the potential involvement of a new adipokine identified as a candidate for the regulation of ovarian function in our RNA-seq analysis by using bovine primary granulosa culture, thymidine incorporation to determine cell proliferation and ELISA assays to measure progesterone secretion. The RNA-seq analysis revealed that 514 genes were over-expressed and 695 were under-expressed in the adipose tissue of cows with severe NEB (SNEB) and cows with moderate NEB (MNEB) during the -4 and 16 wkpp period. In addition, 491 genes were over-expressed and 705 genes were under-expressed in the adipose tissue of SNEB cows compared to MNEB cows. Among these differently expressed genes (DEGs), 298 were related to metabolic functions and 264 to reproductive traits. A set of 19 DEGs were validated by RT-qPCR, including CCL21 (C-C motif chemokine ligand 21). Moreover, CCL21, a gene known to be secreted by adipose tissue, was chosen for further analysis in plasma and ovaries. The use of next-generation sequencing technologies allowed us to characterise the transcriptome of white adipose tissue from primiparous cows with different levels of NEB during lactation. This study highlighted the alteration of the expression of genes related to lipid metabolism, including CCL21, which is released in the bloodstream and associated with the in vitro regulation of ovarian functions.

Ovarian Expression of Adipokines in Polycystic Ovary Syndrome: A Role for Chemerin, Omentin, and Apelin in Follicular Growth Arrest and Ovulatory Dysfunction?

Adipokines are a potential link between reproduction and energy metabolism and could partly explain some infertilities related to some pathophysiology, such as polycystic ovary syndrome (PCOS). However, adipokines were predominantly assessed in blood samples, while very little is known concerning their variations in follicular fluid (FF) and ovarian granulosa cells (GCs) of PCOS women. Thus, the objectives of our study were to investigate adiponectin, chemerin, resistin, visfatin, omentin, and apelin ovarian expression in PCOS women in comparison with controls and women with only a polycystic ovary morphology. In total, 78 women undergoing an in vitro fertilization procedure were divided into three groups: 23 PCOS women, 28 women presenting only ≥12 follicles per ovary (ECHO group), and 27 control women. Each group almost equally included normal weight and obese women. Follicular fluid (FF) concentration and granulosa cells (GCs) mRNA expression of adipokines and their receptors were assessed by ELISA and RT-qPCR, respectively. Omentin levels in FF and GC were higher in PCOS than in ECHO and control women, while apelin expression was increased in both PCOS and ECHO groups. FF chemerin concentration was predominant in normal-weight PCOS women compared to BMI (Body Mass Index)-matched ECHO and control women, while GC mRNA levels were higher in the obese PCOS group than in the ECHO one. Compared to PCOS, ECHO women had increased FF adiponectin concentrations and lower plasma AMH levels. The FF concentration of all adipokines was higher in obese subjects except for adiponectin, predominant in normal-weight women. In conclusion, women with PCOS expressed higher GC chemerin and omentin, whereas the ECHO group presented higher levels of FF adiponectin and apelin and lower plasma AMH and LH concentrations. Chemerin, omentin, and apelin expression was differently regulated in women with PCOS, suggesting their possible role in follicular growth arrest and ovulatory dysfunction characterizing PCOS pathogenesis.

Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells.

Reactive oxygen species (ROS) which lead to oxidative stress affect ovarian function. Grape seed extract (GSE) could be proposed as an effective antioxidant, particularly due to its proanthocyanidin content. In this study, we investigated a dose effect (0, 0.01, 0.1, 1, 10, 50, and 100 µg/mL) of GSE and proanthocyanidin B2 (GSPB2) on the ROS content, cell proliferation, cell viability, and steroidogenesis in both primary luteinized granulosa cells (hGC) and the tumor granulosa cell line (KGN). The levels of ROS were measured using ROS-Glo assay. Cell proliferation and viability were evaluated by [3H]-thymidine incorporation and Cell Counting Kit-8 (CCK8) assay, respectively. Steroid secretion was evaluated by radioimmunoassay. We also analyzed the cell cycle component protein level and signaling pathways by immunoblot and the NOX4 mRNA expression by RTqPCR. From 0.1 to 1 µg/mL, GSE and GSBP2 reduced the ROS cell content and the NOX4 mRNA levels, whereas, GSE and GSBP2 increased the ROS cell content from 50 to 100 µM in both hGC and KGN. GSE and GSPB2 treatments at 50 and 100 µg/mL induced a delay in G1 to S phase cell cycle progression as determined by fluorescence-activated cell sorting. Consequently, they reduced cell growth, cyclin D2 amount, and Akt phosphorylation, and they increased protein levels of p21 and p27 cyclin-dependent kinase inhibitors. These data were also associated with an increase in cell death that could be due to a reduction in Bcl-2-associated death promoter (BAD) phosphorylation and an increase in the cleaved-caspase-3 level. All these negative effects were not observed at lower concentrations of GSE and GSPB2 (0.01 to 10 µg/mL). Interestingly, we found that GSE and GSPB2 treatments (0.1 to 100 µg/mL) improved progesterone and estradiol secretion and this was associated with a higher level of the cholesterol carriers, StAR (steroidogenic acute regulatory protein), CREB (Cyclic adenosine monophosphate Response Element-binding protein), and MAPK ERK1/2 (Mitogen-Activated Protein Kinases Extracellular signal-Regulated Kinases 1/2) phosphorylation in both hGC and KGN cells. Taken together, GSE and GSPB2 (0.1-10 µg/mL) in vitro treatments decrease oxidative stress and increase steroidogenesis without affecting cell proliferation and viability in human granulosa cells.

Mechanisms of Adiponectin Action in Fertility: An Overview from Gametogenesis to Gestation in Humans and Animal Models in Normal and Pathological Conditions.

Adiponectin is the most abundant plasma adipokine. It mainly derives from white adipose tissue and plays a key role in the control of energy metabolism thanks to its insulin-sensitising, anti-inflammatory, and antiatherogenic properties. In vitro and in vivo evidence shows that adiponectin could also be one of the hormones controlling the interaction between energy balance and fertility in several species, including humans. Indeed, its two receptors-AdipoR1 and AdipoR2-are expressed in hypothalamic⁻pituitary⁻gonadal axis and their activation regulates Kiss, GnRH and gonadotropin expression and/or secretion. In male gonads, adiponectin modulates several functions of both somatic and germ cells, such as steroidogenesis, proliferation, apoptosis, and oxidative stress. In females, it controls steroidogenesis of ovarian granulosa and theca cells, oocyte maturation, and embryo development. Adiponectin receptors were also found in placental and endometrial cells, suggesting that this adipokine might play a crucial role in embryo implantation, trophoblast invasion and foetal growth. The aim of this review is to characterise adiponectin expression and its mechanism of action in male and female reproductive tract. Further, since features of metabolic syndrome are associated with some reproductive diseases, such as polycystic ovary syndrome, gestational diabetes mellitus, preeclampsia, endometriosis, foetal growth restriction and ovarian and endometrial cancers, evidence regarding the emerging role of adiponectin in these disorders is also discussed.

Possible involvement of the RARRES2/CMKLR1-system in metabolic and reproductive parameters in Holstein dairy cows.

BACKGROUND: In dairy cows, the energy cost of milk yield results in a negative energy balance (EB) and body fat mobilization that impairs reproductive efficiency. Emerging evidence suggests that the novel adipokines, Retinoic acid receptor responder protein 2 (RARRES2), and its main receptor, Chemokine-like receptor 1 (CMKLR1) are involved in the regulation of metabolic and ovarian functions. So, we investigated in a first experiment the plasma RARRES2, and RARRES2 and CMKLR1 mRNA expression levels in subcutaneous adipose tissue (SAT) and granulosa cells (GC) at different times of body fat mobilization in dairy cows (4, 8, 20 and 44 weeks postpartum, wk. pp. for SAT and 8, 20 and 44 wk. pp. for GC). Then, in a second experiment we examined the effect of high (HE) and low energy (LE) diets on the RARRES2 system and its links with metabolic and reproductive parameters. METHODS: The first experiment included 9 animals fed with HE diet from 4 to 44 wk. pp. and the second one included animals fed either a HE diet (n = 8) or a LE diet (n = 8) from - 4 to 16 wk. peripartum. In both experiments, various metabolic and reproductive parameters were determined and associated with plasma RARRES2 as measured by bovine ELISA. RARRES2 and CMKLR1 mRNA expression levels were analyzed by RT-qPCR in SAT after biopsy and GC after aspiration of follicles. RESULTS: Plasma RARRES2 levels were higher at 4 wk. pp. as compared to 20 and 44 wk. pp. and they were positively correlated with body fat mobilization and milk yield. RARRES2 and CMKLR1 mRNA expression levels increased from 4 to 8 wk. pp. (fat mobilization, EB < 0) and remained unchanged at 20 and 44 wk. pp. (fat reconstitution, EB > 0) as compared to 4 wk. pp. in SAT. RARRES2 and CMKLR1 mRNA levels decreased from 8 to 44 wk. pp. in GC from small follicles. In the second experiment, plasma RARRES2 increased from - 4 to 8 wk. peripartum similarly in both LE and HE cows. In addition, the area under of plasma RARRES2 curve was highly negatively associated with the number of small follicles obtained in HE animals during the cycle before the first artificial insemination. In SAT of HE cows, RARRES2 mRNA expression decreased at 1 wk. pp. compared to - 4 and 16 wk. peripartum whereas opposite expression patterns were obtained for CMKLR1. Similar results were observed for CMKLR1 mRNA expression in LE cows while there was no variation in RARRES2 mRNA expression. Moreover, RARRES2 mRNA was higher expressed in LE than in HE cows at 1 wk. pp. CONCLUSIONS: The lactation-induced fat and energy mobilization influenced plasma RARRES2 profile and mRNA expression pattern of RARRES2 and CMKLR1 similarly in both SAT and GC. In addition, the energy content of the diet did not affect plasma RARRES2 but it altered RARRES2 mRNA expression in SAT and the area under the curve of plasma RARRES2 that was negatively associated to the number of small follicles in HE animals. Thus, RARRES2 could be a metabolic or ovarian signal involved in the interactions between metabolic and reproductive functions in dairy cows.

Chicken Is a Useful Model to Investigate the Role of Adipokines in Metabolic and Reproductive Diseases.

Reproduction is a complex and essential physiological process required by all species to produce a new generation. This process involves strict hormonal regulation, depending on a connection between the hypothalamus-pituitary-gonadal axis and peripheral organs. Metabolic homeostasis influences the reproductive functions, and its alteration leads to disturbances in the reproductive functions of humans as well as animals. For a long time, adipose tissue has been recognised as an endocrine organ but its ability to secrete and release hormones called adipokines is now emerging. Adipokines have been found to play a major role in the regulation of metabolic and reproductive processes at both central and peripheral levels. Leptin was initially the first adipokine that has been described to be the most involved in the metabolism/reproduction interrelation in mammals. In avian species, the role of leptin is still under debate. Recently, three novel adipokines have been discovered: adiponectin (ADIPOQ, ACRP30), visfatin (NAMPT, PBEF), and chemerin (RARRES2, TIG2). However, their mode of action between mammalian and nonmammalian species is different due to the different reproductive and metabolic systems. Herein, we will provide an overview of the structure and function related to metabolic and reproductive mechanisms of the latter three adipokines with emphasis on avian species.

Adipokines expression profile in liver, adipose tissue and muscle during chicken embryo development.

In broiler chickens, the intense genetic selection for rapid growth has resulted in an increase in growth rate and fat deposition. Adipose tissue is now recognized as an important endocrine organ that secretes a variety of factors including adipokines. However, the expression pattern of these adipokines is unclear in chicken embryo development. In the present study, we determined the expression profile of three novel adipokines, NAMPT, RARRES2 and ADIPOQ, and their cognate receptors in metabolic tissues (liver, muscles and adipose tissue) of chicken embryo/chicks from 15 days of incubation (E15) to hatching (D0). From E15 to hatching, embryos gradually gained weight and started to develop subcutaneous adipose tissue at E15. We conducted western blot and RT-qPCR tests and found that ADIPOQ expression increased over time and was positively correlated with adipose tissue weight. In addition, NAMPT expression increased only in muscles. By using a new homemade chicken RARRES2 specific antibody we showed that RARRES2 protein levels increased specifically at hatching in adipose tissue, liver and pectoralis major and this was associated with an increase in the weight of embryo. Taken together, these results support a potential involvement of adipokines in metabolic regulation during chicken embryo development.

Effect of different levels of feed restriction and fish oil fatty acid supplementation on fat deposition by using different techniques, plasma levels and mRNA expression of several adipokines in broiler breeder hens.

BACKGROUND: Reproductive hens are subjected to a restricted diet to limit the decline in fertility associated with change in body mass. However, endocrine and tissue responses to diet restriction need to be documented. OBJECTIVE: We evaluated the effect of different levels of feed restriction, with or without fish oil supplementation, on metabolic parameters and adipokine levels in plasma and metabolic tissues of reproductive hens. METHODS: We designed an in vivo protocol involving 4 groups of hens; RNS: restricted (Rt) unsupplemented, ANS: ad libitum (Ad, receiving an amount of feed 1.7 times greater than animals on the restricted diet) unsupplemented, RS: Rt supplemented, and AS: Ad supplemented. The fish oil supplement was used at 1% of the total diet composition. RESULTS: Hens fed with the Rt diet had a significantly (P < 0.0001) lower growth than Ad hens, while the fish oil supplementation had no effect on these parameters. Furthermore, the bioelectrical impedance analysis (BIA) and the fat ultrasonographic examinations produced similar results to the other methods that required animals to be killed (carcass analysis and weight of adipose tissue). In addition, the Rt diet significantly (P < 0.05) decreased plasma levels of triglycerides, phospholipids, glucose and ADIPOQ, and fish oil supplementation decreased plasma levels of RARRES2. We also showed a positive correlation between insulin values and ADIPOQ or NAMPT or RARRES2 values, and a negative correlation of fat percentage to RARRES2 values. Moreover, the effects of the Rt diet and fish oil supplementation on the mRNA expression depended on the factors tested and the hen age. CONCLUSIONS: Rt diet and fish oil supplementation are able to modulate metabolic parameters and the expression of adipokines and their receptors in metabolic tissue.

Food restriction but not fish oil increases fertility in hens: role of RARRES2?

Overfed hens selected for their rapid growth become fatter and develop reproductive disorders. Herein, we aimed to demonstrate that food restriction leading to a weight reduction and/or a supplementation with fish oil may be effective in preventing reproductive disorders through the regulation of adipokine expression in broiler hens. This study included four groups of food restricted (Rt) or ad libitum hens (Ad, feeding at a rate 1.7 times greater than Rt hens) supplemented or unsupplemented with fish oil (1%). The Rt diet significantly increased plasma chemerin (RARRES2) levels during the laying period, delayed sexual maturity by one week and improved egg quality and fertility. These effects were associated with higher progesterone production in response to IGF1 (or LH) in cultured granulosa cells and in vivo egg yolk, as compared with Ad hens. Fish oil supplementation had similar effects to the Rt diet on progesterone (P < 0.05), but without any effect on fertility. Using RT-PCR, we found that RARRES2 levels were lower in theca cells of Rt hens and NAMPT levels were increased by the fish oil supplementation. A significant positive correlation between RARRES2 expression in granulosa cells and the weight of F1 preovulatory follicle was observed, as well as a negative correlation of plasma RARRES2 levels with hatchability. Thus, food restriction but not fish oil supplementation improved fertility, and this was associated with variations in RARRES2 plasma and ovarian expression in hens.

Adiponectin and resistin: potential metabolic signals affecting hypothalamo-pituitary gonadal axis in females and males of different species.

Adipokines, including adiponectin and resistin, are cytokines produced mainly by the adipose tissue. They play a significant role in metabolic functions that regulate the insulin sensitivity and inflammation. Alterations in adiponectin and resistin plasma levels, or their expression in metabolic and gonadal tissues, are observed in some metabolic pathologies, such as obesity. Several studies have shown that these two hormones and the receptors for adiponectin, AdipoR1 and AdipoR2 are present in various reproductive tissues in both sexes of different species. Thus, these adipokines could be metabolic signals that partially explain infertility related to obesity, such as polycystic ovary syndrome (PCOS). Species and gender differences in plasma levels, tissue or cell distribution and hormonal regulation have been reported for resistin and adiponectin. Furthermore, until now, it has been unclear whether adiponectin and resistin act directly or indirectly on the hypothalamo-pituitary-gonadal axis. The objective of this review was to summarise the latest findings and particularly the species and gender differences of adiponectin and resistin on female and male reproduction known to date, based on the hypothalamo-pituitary-gonadal axis.