Knockdown of regulatory associated protein of TOR (raptor) in hypothalamus-stimulated folliculogenesis and induced ovarian cysts.

CONTEXT: Mammalian target of rapamycin complex 1 (mTORC1) is an essential sensor that regulates fundamental biological processes like cell growth, proliferation and energy metabolism. The treatment of disease by sirolimus, a mTORC1 inhibitor, causes adverse effects, such as female fertility disorders. AIMS: The objective of the study was to decipher the reproductive consequences of a downregulation of mTORC1 in the hypothalamus. METHODS: The reduced expression of mTORC1 was induced after intracerebroventricular injection of lentivirus expressing a short hairpin RNA (shRNA) against regulatory associated protein of TOR (raptor) in adult female mice (ShRaptor mice). KEY RESULTS: The ShRaptor mice were fertile and exhibited a 15% increase in the litter size compared with control mice. The histological analysis showed an increase in antral, preovulatory follicles and ovarian cysts. In the hypothalamus, the GnRH mRNA and FSH levels in ShRaptor mice were significantly elevated. CONCLUSIONS: These results support the hypothesis that mTORC1 in the central nervous system participates in the regulation of female fertility and ovarian function by influencing the GnRH neuronal activity. IMPLICATIONS: These results suggest that a lower mTORC1 activity directly the central nervous system leads to a deregulation in the oestrous cycle and an induction of ovarian cyst development.

Chicken cGAS Senses Fowlpox Virus Infection and Regulates Macrophage Effector Functions.

The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids. In multiple species, the pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) senses viral DNA as an essential component of the innate response. cGAS initiates a range of signaling outputs that are dependent on generation of the second messenger cGAMP that binds to the adaptor protein stimulator of interferon genes (STING). Here we show that in chicken macrophages, the cGAS/STING pathway is essential not only for the production of type-I interferons in response to intracellular DNA stimulation, but also for regulation of macrophage effector functions including the expression of MHC-II and co-stimulatory molecules. In the context of fowlpox, an avian DNA virus infection, the cGAS/STING pathway was found to be responsible for type-I interferon production and MHC-II transcription. The sensing of fowlpox virus DNA is therefore essential for mounting an anti-viral response in chicken cells and for regulation of a specific set of macrophage effector functions.

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens.

It is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC. RESEARCH HIGHLIGHTS Use of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host-pathogen interactions. pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons. pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

Potential involvement of several signaling pathways in initiation of the chicken acrosome reaction.

Avian sperm biology has demonstrated specific features in preparation for fertilization. For example, capacitationlike processes and motility hyperactivation do not exist in the form described in mammals. The present study investigated the potential involvement of several signaling pathways, including protein kinase A (PKA), phosphatidylinositol 3 kinase (PIK3), mitogen-activated protein kinase 3/1 (MAPK3/1), and MAPK14 in the chicken acrosome reaction (AR). The presence in chicken spermatozoa of key proteins involved in these signaling pathways (i.e., cAMP-responsive element-binding protein [CREB], AKT, MAPK1, and MAPK14 and their respective phosphorylated forms) was detected using immunoblotting and localized by immunocytochemistry, mainly in the heads. The potential involvement of these pathways in the AR induced by inner perivitelline layer (IPVL) and Ca(2+) was then examined using specific inhibitors and phosphorylation status measurements. The effects of the specific inhibitors on motility were also measured. Phosphorylations of AKT, CREB, and MAPK1, but not MAPK14, were increased at the time of AR. Phosphorylation of AKT was increased in the presence of IPVL alone, whereas both IPVL and Ca(2+) were needed to increase CREB and MAPK1 phosphorylations. Inhibition of the three corresponding pathways blocked the increase in phosphorylation and significantly decreased AR. Inhibitions of the PKA and MAPK1 pathways also significantly decreased motility, whereas MAPK14 and PIK3 inhibition had no effect on motility. Our results suggest that the AR could be mediated by activation of the PKA, PIK3, and MAPK1 pathways through a sequential action involving, successively, PIK3 and then PKA and MAPK1 activations.