Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function.

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

Genetic ablation of pannexin1 counteracts liver fibrosis in a chemical, but not in a surgical mouse model.

Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis.

TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice.

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice.

OBJECTIVE: Liver fibrosis results from the perpetuation of the normal wound healing response to several types of injury. Despite the wealth of knowledge regarding the involvement of intracellular and extracellular signaling pathways in liver fibrogenesis, information about the role of intercellular communication mediated by gap junctions is scarce. METHODS: In this study, liver fibrosis was chemically induced by carbon tetrachloride in mice lacking connexin32, the major liver gap junction constituent. The manifestation of liver fibrosis was evaluated based on a series of read-outs, including collagen morphometric and mRNA analysis, oxidative stress, apoptotic, proliferative and inflammatory markers. RESULTS: More pronounced liver damage and enhanced collagen deposition were observed in connexin32 knockout mice compared to wild-type animals in experimentally triggered induced liver fibrosis. No differences between both groups were noticed in apoptotic signaling nor in inflammation markers. However, connexin32 deficient mice displayed decreased catalase activity and increased malondialdehyde levels. CONCLUSION: These findings could suggest that connexin32-based signaling mediates tissue resistance against liver damage by the modulation of the antioxidant capacity. In turn, this could point to a role for connexin32 signaling as a therapeutic target in the treatment of liver fibrosis.

S-nitroso-N-acetylcysteine attenuates liver fibrosis in experimental nonalcoholic steatohepatitis.

S-Nitroso-N-acetylcysteine (SNAC) is a water soluble primary S-nitrosothiol capable of transferring and releasing nitric oxide and inducing several biochemical activities, including modulation of hepatic stellate cell activation. In this study, we evaluated the antifibrotic activity of SNAC in an animal model of nonalcoholic steatohepatitis (NASH) induced in Sprague-Dawley rats fed with a choline-deficient, high trans fat diet and exposed to diethylnitrosamine for 8 weeks. The rats were divided into three groups: SNAC, which received oral SNAC solution daily; NASH, which received the vehicle; and control, which received standard diet and vehicle. Genes related to fibrosis (matrix metalloproteinases [MMP]-13, -9, and -2), transforming growth factor ß-1 [TGFß-1], collagen-1α, and tissue inhibitors of metalloproteinase [TIMP-1 and -2] and oxidative stress (heat-shock proteins [HSP]-60 and -90) were evaluated. SNAC led to a 34.4% reduction in the collagen occupied area associated with upregulation of MMP-13 and -9 and downregulation of HSP-60, TIMP-2, TGFß-1, and collagen-1α. These results indicate that oral SNAC administration may represent a potential antifibrotic treatment for NASH.

Microsomal triglyceride transfer protein and nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease is currently one of the most common forms of liver disease, covering cases from simple steatosis without inflammation, to cases of steatohepatitis and fibrosis, and may lead to liver cirrhosis and hepatocellular carcinoma. The pathophysiology of nonalcoholic fatty liver disease is based on multiple events; changes in the secretion of lipoproteins can lead to steatosis. Liver lipid secretion is mediated by apoB100 and microsomal triglyceride transfer protein (MTP). The pharmacological suppression of MTP is suggested as a possible treatment for hyperlipidemia, although the upregulation of this protein can be a treatment for nonalcoholic steatohepatitis.