Selection of Male Donors in Local Chicken Breeds to Implement the Italian Semen Cryobank: Variability in Semen Quality, Freezability and Fertility.

Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5-16%) and between birds within a breed (BP = 3-7%; BS = 7-31%; PP = 6-22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen's fertilizing ability are required.

Molecular imaging provides novel insights on estrogen receptor activity in mouse brain.

Estrogen receptors have long been known to be expressed in several brain areas in addition to those directly involved in the control of reproductive functions. Investigations in humans and in animal models suggest a strong influence of estrogens on limbic and motor functions, yet the complexity and heterogeneity of neural tissue have limited our approaches to the full understanding of estrogen activity in the central nervous system. The aim of this study was to examine the transcriptional activity of estrogen receptors in the brain of male and female mice. Exploiting the ERE-Luc reporter mouse, we set up a novel, bioluminescence-based technique to study brain estrogen receptor transcriptional activity. Here we show, for the first time, that estrogen receptors are similarly active in male and female brains and that the estrous cycle affects estrogen receptor activity in regions of the central nervous system not known to be associated with reproductive functions. Because of its reproducibility and sensitivity, this novel bioluminescence application stands as a candidate as an innovative methodology for the study and development of drugs targeting brain estrogen receptors.

Gender differences in endothelial function and inflammatory markers along the occurrence of pathological events in stroke-prone rats.

Spontaneously hypertensive stroke-prone rats (SHRSP) feature an established model for human cerebrovascular disease. SHRSP, kept on a high-salt permissive diet (JPD), develop hypertension, renal and brain damage. In this report we compared the behavior of female and male SHRSP regarding the main aspects of their pathological condition. Brain abnormalities, detected by magnetic resonance imaging, developed spontaneously in males after 42+/-3 days, in females after 114+/-14 days from the start of JPD. Survival was >3-fold longer for females than for males. The development of brain damage was preceded, in both genders, by an inflammatory condition characterized by the accumulation in serum and urine of acute-phase proteins. The increase in thiostatin level was significantly lower and delayed in female in comparison to male SHRSP. During JPD female and male SHRSP developed massive proteinuria, its worsening being significantly slower in females. The alterations of vasculature-bound barriers in kidney and brain were connected with endothelial dysfunction and relative deficiency in nitric oxide (NO). In thoracic aortic rings, basal release of NO was significantly higher in female than in male SHRSP, both if receiving and if not receiving JPD. The gender differences in SHRSP thus appear to be connected to a more efficient control in females of inflammation and of endothelial dysfunction.

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor.

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

The endogenous estrogen status regulates microglia reactivity in animal models of neuroinflammation.

It has been previously demonstrated that 17beta-estradiol (E(2)) inhibits the response of microglia, the resident brain macrophages, to acute injuries in specific brain regions. We here show that the effect of E(2) in acute brain inflammation is widespread and that the hormone reduces the expression of inflammatory mediators, such as monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and TNF-alpha, induced by lipopolysaccharide, demonstrating that microglia are a direct target of estrogen action in brain. Using the APP23 mice, an animal model of Alzheimer's disease reproducing chronic neuroinflammation, we demonstrate that ovary ablation increases microglia activation at beta-amyloid (Abeta) deposits and facilitates the progression of these cells toward a highly reactive state. Long-term administration of E(2) reverts the effects of ovariectomy and decreases microglia reactivity compared with control animals. In this animal model, these events do not correlate with a reduced number of Abeta deposits. Finally, we show that E(2) inhibits Abeta-induced expression of scavenger receptor-A in macrophage cells, providing a mechanism for the effect of E(2) on Abeta signaling observed in the APP23 mice. Altogether, our observations reveal a substantial involvement of endogenous estrogen in neuroinflammatory processes and provide novel mechanisms for hormone action in the brain.

Regulation of the lipopolysaccharide signal transduction pathway by 17beta-estradiol in macrophage cells.

We have previously shown that 17beta-estradiol (E2) prevents the activation of brain macrophages, i.e. microglia cells, both in vitro and in vivo. Hormone exerts this inhibitory effect by inhibiting pro-inflammatory gene expression. In this study we further investigated on the molecular mechanism of E2 action in the RAW 264.7 macrophage cell line. We show here that these cells express the alpha-isoform of the estrogen receptor (ERalpha) and not ERbeta. Similarly to its activity in brain macrophages, E2 is able to inhibit the activation program induced by lipopolysaccharide (LPS) in RAW 264.7 cells, as shown by the inhibitory effect of hormone on the morphological conversion and matrix metalloproteinase-9 (MMP-9) expression induced by the endotoxin. In addition, we demonstrate that hormone treatment is not associated with a reduction in the steady-state expression of Toll-like receptor-4 (TLR-4) and CD14, two components of the LPS receptor complex. Our results further confirm the anti-inflammatory role of ERalpha in macrophages and propose that the mechanism of hormone action on macrophage reactivity involves signaling molecules which are down-stream effectors of the LPS membrane receptors.

Estrogens in the nervous system: mechanisms and nonreproductive functions.

The past decade has witnessed a growing interest in estrogens and their activity in the central nervous system, which was originally believed to be restricted to the control of reproduction. It is now well accepted that estrogens modulate the activity of all types of neural cells through a multiplicity of mechanisms. Estrogens, by binding to two cognate receptors ERalpha and ERbeta, may interact with selected promoters to initiate the synthesis of target proteins. Alternatively, the hormone receptor complex may interfere with intracellular signaling at both cytoplasmic and nuclear levels. The generation of cellular and animal models, combined with clinical and epidemiological studies, has allowed us to appreciate the neurotrophic and neuroprotective effects of estrogens. These findings are of major interest because estradiol might become an important therapeutic agent to maintain neural functions during aging and in selected neural diseases.

Estrogen receptor-alpha mediates the brain antiinflammatory activity of estradiol.

Beyond the key role in reproductive and cognitive functions, estrogens have been shown to protect against neurodegeneration associated with acute and chronic injuries of the adult brain. Current hypotheses reconcile this activity with a direct effect of 17beta-estradiol (E2) on neurons. Here we demonstrate that brain macrophages are also involved in E2 action on the brain. Systemic administration of hormone prevents, in a time- and dose-dependent manner, the activation of microglia and the recruitment of peripheral monocytes induced by intraventricular injection of lipopolysaccharide. This effect occurs by limiting the expression of neuroinflammatory mediators, such as the matrix metalloproteinase 9 and lysosomal enzymes and complement C3 receptor, as well as by preventing morphological changes occurring in microglia during the inflammatory response. By injecting lipopolysaccharide in estrogen receptor (ER)-null mouse brains, we demonstrate that hormone action is mediated by activation of ERalpha but not of ERbeta. The specific role of ERalpha is further confirmed by comparing the effects of ERs on the matrix metalloproteinase 9 promoter activity in transient transfection assays. Finally, we report that genetic ablation of ERalpha is associated with a spontaneous reactive phenotype of microglia in specific brain regions of adult ERalpha-null mice. Altogether, these results reveal a previously undescribed function for E2 in brain and provide a mechanism for its beneficial activity on neuroinflammatory pathologies. They also underline the key role of ERalpha in brain macrophage reactivity and hint toward the usefulness of ERalpha-specific drugs in hormone replacement therapy of inflammatory diseases.