At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3ß-hydroxysteroid dehydrogenase (3ßHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3ßHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY ¼ funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A.

Maternal dietary supplementation with grape seed extract in reproductive hens increases fertility in females but decreases semen quality in males of the F1 generation.

Genetic selection in parental broiler breeders has increased their susceptibility to metabolic disorders and reproductive dysfunction. We have recently shown that maternal dietary grape seed extract (GSE) supplementation in hens improves fertility parameters, egg quality, oxidative stress in different tissues and the quality of F1 chicks. Here, we analysed the growth and fertility (both female and male) of the F1 generation animals and the quality of their offspring (F2 generation). Eggs issued from hens supplemented with GSE presented lower ROS production than control hens, suggesting a change in the embryonic environment. However, this did not affect the growth nor the body composition of male and female F1s from hatching to adulthood (37 weeks of age). At 37 weeks of age, the biochemistry analysis of the GSE-F1 muscle has revealed an increase in sensitivity to oxidative stress and a slight change in lipid composition. Both male and female F1-GSE groups presented a delay in puberty with a lower testis volume at 30 weeks of age and lower ovary development at 26 weeks of age. Adult GSE-F1 males did not present histological alterations of seminiferous tubules or semen production, but the semen quality was degraded due to higher oxidative stress and DNA-damaged spermatozoa compared with control F1 animals. In adult GSE-F1 females, despite the delay in puberty, the females laid more eggs of better quality (fewer broken eggs and a higher hatching rate). At hatching, the weight of the chicks from GSE-F1 females was reduced, and this effect was stronger in F2 male chicks (F2) compared with F2 control chicks (F2), because of the lower muscle volume. In conclusion, we can raise the hypothesis that maternal dietary GSE supplementation produces eggs with change in embryonic metabolism, which may affect in adulthood the fertility. The data obtained from the F1-GSE group pointed to a sex-specific modification with higher egg quality in females but semen sensitive to stress in males. Finally, male F2 chicks were leaner than control chicks. Thus, maternal dietary grape seed extract (GSE) supplementation in hens may impact on the fertility of the offspring in a sex-specific manner in subsequent generations.

Chemerin impairs food intake and body weight in chicken: Focus on hypothalamic neuropeptides gene expression and AMPK signaling pathway.

Unlike mammals, the role of adipokines and more particularly of chemerin in the regulation of food intake is totally unknown in avian species. Here we investigated the effect of chemerin on the food and water consumption and on the body weight in chicken. We studied the effects on the plasma glucose and insulin concentrations and the hypothalamic neuropeptides and AMPK signaling pathway. Female broiler chickens were intraperitoneally injected, daily for 13 days with either vehicle (saline; n = 25) or chemerin (8 µg/kg; n = 25 and 16 µg/kg; n = 25). Food and water intakes were recorded 24 h after each administration. Overnight fasted animals were sacrificed at day 13 (D13), 24 h after the last injection and hypothalamus and left cerebral hemispheres were collected. Chemerin and its receptors protein levels were determined by western-blot. Gene expression of neuropeptide Y (Npy), agouti-related peptide (Agrp), corticotrophin releasing hormone (Crh), pro-opiomelanocortin (Pomc), cocaine and amphetamine-regulated transcript (Cart) and Taste 1 Receptor Member 1 (Tas1r1) were evaluated by RT-qPCR. In chicken, we found that the protein amount of chemerin, CCRL2 and GPR1 was similar in left cerebral hemisphere and hypothalamus whereas CMKLR1 was higher in hypothalamus. Chemerin administration (8 and 16 µg/kg) decreased both food intake and body weight compared to vehicle without affecting water intake and the size or volume of different brain subdivisions as determined by magnetic resonance imaging. It also increased plasma insulin levels whereas glucose levels were decreased. These data were associated with an increase in Npy and Agrp expressions and a decrease in Crh, Tas1r1 mRNA expression within the hypothalamus. Furthermore, chemerin decreased hypothalamic CMKLR1 protein expression and AMPK activation. Taken together, these results support that chemerin could be a peripheral appetite-regulating signal through modulation of hypothalamic peptides expression in chicken.

The adiponectin agonist, AdipoRon, inhibits steroidogenesis and cell proliferation in human luteinized granulosa cells.

During obesity, excess body weight is not only associated with an increased risk of type 2-diabetes, but also several other pathological processes, such as infertility. Adipose tissue is the largest endocrine organ of the body that produces adipokines, including adiponectin. Adiponectin has been reported to control fertility through the hypothalamic-pituitary-gonadal axis, and folliculogenesis in the ovaries. In this study, we focused on a recent adiponectin-like synthetic agonist called AdipoRon, and its action in human luteinized granulosa cells. We demonstrated that AdipoRon activated the adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor alpha (PPAR) signalling pathways in human luteinized granulosa cells. A 25 µM AdipoRon stimulation reduced granulosa cell proliferation by inducing cell cycle arrest in G1, associated with PTEN and p53 pathway activation. In addition, AdipoRon perturbed cell metabolism by decreasing mitochondrial activity and ATP production. In human luteinized granulosa cells, AdipoRon increased phosphodiesterase activity, leading to a drop in cyclic adenosine monophosphate (cAMP) production, aromatase expression and oestrogens secretion. In conclusion, AdipoRon impacted folliculogenesis by altering human luteinized granulosa cell function, via steroid production and cell proliferation. This agonist may have applications for improving ovarian function in metabolic disorders or granulosa cancers.

Microbiota Changes Due to Grape Seed Extract Diet Improved Intestinal Homeostasis and Decreased Fatness in Parental Broiler Hens.

In poultry, the selection of broilers for growth performance has induced a deterioration in the health of the parental hens associated with poor reproductive efficiency. To improve these parameters, we administered to laying parental broiler hens a regular diet supplemented or not (Control) with a moderate (1%) or a high level (2%) of grape seed extract (GSE). The 1% GSE diet was administered from a young age (from 4 to 40 weeks of age) and the high level of 2% GSE was administered only during a 2-week period (from 38 to 40 weeks of age) in the laying period. The analysis of 40-week-old hens showed that 2% GSE displayed a reduction in the fat tissue and an improvement in fertility with heavier and more resistant eggs. Seven monomer phenolic metabolites of GSE were significantly measured in the plasma of the 2% GSE hens. GSE supplementation increased the relative abundance of the following bacteria populations: Bifidobacteriaceae, Lactobacilliaceae and Lachnospiraceae. In conclusion, a supplementation period of only 2 weeks with 2% GSE is sufficient to improve the metabolic and laying parameters of breeder hens through a modification in the microbiota.

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.

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.

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.

Metformin Improves Quality of Post-Thaw Canine Semen.

Sperm cryopreservation is an assisted reproductive technique routinely used in canine species for genetic conservation. However, during cryopreservation, the DNA damages are still elevated, limiting the fertilization rate. The present study was conducted to evaluate whether supplementation of canine semen extender with a molecule limiting the metabolic activities can improve the quality of frozen-thawed canine spermatozoa. We used metformin, known to limit the mitochondrial respiratory and limit the oxidative stress. Before and during the freezing procedure, metformin (50µM and 500µM) has been added to the extender. After thawing, sperm exposed to metformin conserved the same viability without alteration in the membrane integrity or acrosome reaction. Interestingly, 50µM metformin improved the sperm motility in comparison to the control, subsequently increasing mitochondrial activity and NAD+ content. In addition, the oxidative stress level was reduced in sperm treated with metformin improving the sperm quality as measured by a different molecular marker. In conclusion, we have shown that metformin is able to improve the quality of frozen-thawed dog semen when it is used during the cryopreservative procedure.

Assessment of the body development kinetic of broiler breeders by non-invasive imaging tools.

In order to determine the body composition of parental broilers during growth from hatching to adulthood (32 wk of age), we evaluated the kinetics of fattening, growth rate, reproduction parameters, and body composition of the animals by using non-invasive tools such as medical imaging (ultrasound and CT scan) and blood sample analysis. The use of CT scanner allowed us to monitor the development of the body composition (fatness, bone, muscle, ovary, and testis growth) of these same animals. These analyses were accompanied by biochemical blood analyses such as steroids, metabolites, and some adipokines concentration. Difference in the body composition between males and females appeared at 16 wk of age. From 20 wk of age, shortly before the onset of lay, the females had 1.6-fold more adipose tissues than males (P < 0.001) and 8-fold more elevated plasma triglycerides levels. In addition, females, from 16 wk of age, presented a weakened bone quality in comparison to males (P < 0.001). The ratio of the tibia volume/tibia length was 33.2% lower in female compared to male chicken at 32 wk of age (P < 0.001). However, the pectoral muscle had the same volume in both sexes. The production of steroids by gonad started at 16 wk of age for both sexes, and the testis and ovary development could be measured by imaging tools at 24 wk. The follicle development was correlated to the ovarian fat tissue (r = 0.80) and fatness. In conclusion, the use of CT scanner and ultrasound system has allowed investigate the body composition of live animals and actual parental breeds with to the aim of using them for genetic selection.

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.

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.