The Adipokines in Domestic Animal Reproduction: Expression and Role in the Regulation of Ovarian Function

Currently, it is clear that female reproduction is regulated by the hypothalamic–pituitary-ovary axis, which produces many hormones that control reproductive stages. It is therefore important to have knowledge of new regulators/hormones controlling reproduction in domestic animals. In female animals, energy metabolism and fertility are tightly connected, and reciprocally regulated. The adipose tissue is well known to be implicated in the secretion of several hormones, such as the adiponectin, resistin, chemerin, visfatin, vaspin and apelin, the so-called adipokines or “adipose tissue-derived hormones”. Many reports indicate that adipokines regulate the ovarian follicles’ development, the onset of puberty and/or ovulation. This chapter summarizes that several adipokines are expressed in the ovary and that they can regulate ovarian physiology such as the steroid hormone production, cell proliferation, apoptosis and oocyte maturation in different domestic animals like pigs, cows, goats, ewes, chickens and turkeys

Dynamic regulation of adipose tissue metabolism in the domestic broiler chicken – an alternative model for studies of human obesity

Background The domestic chicken is an attractive, but underutilized, animal model for studies of adipose tissue biology, metabolism and obesity: 1.) like humans, chickens rely on liver rather than adipose tissue for the majority of de novo lipogenesis; 2.) quantitative trait loci (QTLs) linked to fatness in chickens contain genes implicated in human susceptibility to obesity and diabetes; 3.) chickens are naturally hyperglycemic and insulin resistant; and 4.) a broad selection of genetic models exhibiting a range of fatness are available. To date, however, little is known about regulation of adipose metabolism in this model organism. Materials and methods Affymetrix arrays were used to profile gene expression in abdominal adipose tissue from broiler chickens fed ad libitum or fasted for five hours and from three distinct genetic lines with low (Fayoumi and Leghorn) or high (broiler) levels of adiposity. QPCR was used to validate microarray results for select genes. Western blotting was used to assay levels of signaling proteins. Tissue levels of beta-hydroxybutyrate were measured as an index of fatty acid oxidation using a colorimetric assay. Multiple testing was controlled using q-value. Mixed linear model and multivariate clustering analysis were implemented in SAS. The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/ webcite) was used for Gene Ontology (GO) and KEGG pathway enrichment analyses. Results A total of 1780 genes were differentially expressed in fasted vs. ad libitum fed (p\u3c0.05) tissue after correction for multiple testing. Gene Ontology and pathway analyses, combined with Western blot validation, indicated significant effects on a broad selection of pathways related to metabolism, stress signaling and adipogenesis. In particular, fasting upregulated rate-limiting genes in both the mitochondrial and peroxisomal pathways of beta-oxidation. Enhanced fatty acid oxidation in white adipose tissue was further suggested by a significant increase in tissue content of the ketone beta-hydroxybutyrate. Expression profiles suggested that, despite the relatively brief duration of feed withdrawal, fasting suppressed adipogenesis; expression of key genes in multiple steps of adipogenesis, including lineage commitment from mesenchymal stem cells, were significantly down-regulated in fasted vs. fed adipose tissue. Interestingly, fasting increased expression of several inflammatory adipokines and components of the toll-like receptor 4 signaling pathway. Microarray analysis of Fayoumi, Leghorn and broiler adipose tissue revealed that genetic leanness shared molecular signatures with the effects of fasting. In supervised clustering analysis, fasted broiler chickens clustered with lean Fayoumi and Leghorn lines rather than with the fed broiler group, suggesting that fasting manipulated expression profiles to resemble those of the lean phenotype. Conclusions Collectively, these data suggest that leanness in chickens is associated with increased fat utilization which, given the similarities between avian and human adipose tissue with regard to lipid metabolism, may have relevance for humans. The paradoxical increase in some inflammatory markers with an acute fast suggests that the dynamic relationship between inflammation and adipose metabolism may differ from what is observed in obesity. These results highlight chicken as a useful model in which to study the interrelationships between food intake, adipose development, metabolism, and cell stress

Intestinal kinetics from digestion of milk proteins in humans

Objective. We aim to assess in humans the intestinal digestion of two protein fractions from milk, i.e. a soluble (SP) and non-soluble (casein; CAS) proteins, that exhibit opposite chemical and digestive characteristics

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

Expression and in vitro effect of phoenixin-14 on the porcine ovarian granulosa cells

International audiencePhoenixin-14 (PNX-14) regulates energy metabolism via the G protein-coupled receptor 173 (GPR173); elevated plasma levels have been described in patients with polycystic ovary syndrome. The aims were to investigate the ovarian expression of PNX-14/GPR173 and the in vitro effect of PNX-14 on granulosa cells (Gc) function. Transcript and protein levels of PNX-14/GRP173 were analysed by real-time PCR, western blot and immuno-histochemistry in the porcine ovarian follicles at days 2-3, 10-12 and 16-18 of the oestrous. For in vitro ex-periments, Gc were isolated from follicles at days 10-12 of the oestrous (4-6 mm) and PNX-14 at doses 1-1000 nM was added for 24-72 h to determine Gc proliferation. Cell cycle progression, E2 secretion, expression of proliferating cells nuclear antigen, cyclins, mitogen-activated kinase (MAP3/1; ERK1/2), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) were studied. The involvement of these kinases in PNX-14 action on Gc proliferation was analysed using pharmacological inhibitors. Levels of GPR173 were increased in the ovarian follicles with oestrous progression, while only PNX-14 protein was the highest at days 10-12 of the oestrous. Immuno-signal of PNX-14 was detected in Gc and theca cells and oocyte, while GPR173 was mostly in theca. Interestingly, PNX-14 stimulated Gc proliferation, E2 secretion, cell cycle progression and cyclins expression and had a modulatory effect on MAP3/1, AKT and STAT3 activation. Our study suggests that PNX-14 could be an important factor for porcine reproduction by influencing ovarian follicle growth through direct action on Gc function

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

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

Haploinsufficiency of Dmxl2, Encoding a Synaptic Protein, Causes Infertility Associated with a Loss of GnRH Neurons in Mouse

International audienceCharacterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3a, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3a was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3a controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3a as a key controller of neuronal and endocrine homeostatic processes

Automated Extraction of API Usage Patterns from Source Code for Vulnerability Identification

In the past decades, numerous methods have been proposed to partially automate the discovery of those well understood classes of software security vulnerabilities, which lend themselves easily to generalization. Despite these efforts, vulnerability identification remains an inherently creative process, in which an auditor must carefully examine the intentions of the developer and the code chosen to express them, and it is in the subtle differences between intentions and implementations that vulnerabilities are commonly found. Modularization, the definition of auxiliary functions and the presence of conventions imposed by external interfaces, naturally lead to the formulation of these intentions by means of application-specific, often sparsely documented programming patterns, which in consequence give rise to application-specific sets of vulnerabilities. In this work, the use of unsupervised learning algorithms as developed in the field of machine learning is proposed to exploit the statistical properties of the target code-base and arrive at a vectorial representation of source-code based on dominant programming patterns suitable to assist in manual source-code auditing. Furthermore, by measuring distances within th

Impact of metformin on reproductive tissues: an overview from gametogenesis to gestation.

peer reviewedMetformin is an oral anti-hyperglycemic drug that acts as an insulin sensitizer in the treatment of diabetes mellitus type 2. It has also been widely used in the treatment of polycystic ovary syndrome (PCOS) and gestational diabetes. This drug has been shown to activate a protein kinase called 5' AMP-activated protein kinase or AMPK. AMPK is present in many tissues making metformin's effect multi factorial. However as metformin crosses the placenta, its use during pregnancy raises concerns regarding potential adverse effects on the mother and fetus. The majority of reports suggest no significant adverse effects or teratogenicity. However, disconcerting reports of male mouse offspring that were exposed to metformin in utero that present with a reduction in testis size, seminiferous tubule size and in Sertoli cell number suggest that we do not understand the full suite of effects of metformin. In addition, recent molecular evidence is suggesting an epigenetic effect of metformin which could explain some of the long-term effects reported. Nevertheless, the data are still insufficient to completely confirm or disprove negative effects of metformin. The aims of this review are to provide a summary of the safety of metformin in various aspects of sexual reproduction, the use of metformin by gestating mothers, and its possible side-effects on offspring from women who are administered metformin during pregnancy