The acrosome reaction is a membrane fusion event that is prerequisite for sperm penetration through the zona pellucida. To elucidate the molecular mechanisms involved in membrane fusion, the expression and localization of Rab proteins, a subfamily of small GTPases that have been shown to play key roles in regulation of intracellular membrane traffic and exocytosis, were examined in rat testis and sperm. Reverse transcription polymerase chain reaction, immunoblot analysis, and immunofluorescence microscopy revealed that Rab3A protein, which is thought to be involved in regulation of exocytosis in neurons and endocrine cells, is associated with the sperm acrosome. The protein was undetectable in acrosome-free heads prepared by sucrose density gradient centrifugation. Immunogold electron microscopy performed on ultrathin cryosections provided further evidence that Rab3A protein is associated with the acrosomal membrane. Acrosome reaction assays revealed that synthetic peptide of the Rab3 effector domain inhibited acrosomal exocytosis triggered by calcium ionophore A23187 in a concentration-dependent fashion, suggesting that Rab3A acts as an inhibitory regulator in the acrosome reaction. In view of the putative role of Rab3A protein in membrane fusion systems, these results suggest that Rab3A could be involved in regulating the mammalian acrosome reaction by controlling the membrane fusion system in sperm.
A natural carotenoid crocin is contained in saffron and gardenia flowers (crocuses and gardenias) and is used as a food colorant. This study reports the potential inhibitory effects of crocin against inflammation-associated mouse colon carcinogenesis and chemically induced colitis in male ICR mice. In the first experiment, dietary crocin significantly inhibited the development of colonic adenocarcinomas induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) in mice by week 18. Crocin feeding also suppressed the proliferation and immunohistochemical expression of nuclear factor- (NF-) κB but increased the NF-E2-related factor 2 (Nrf2) expression, in adenocarcinoma cells. In the second experiment, dietary feeding with crocin for 4 weeks was able to inhibit DSS-induced colitis and decrease the mRNA expression of tumor necrosis factor α, interleukin- (IL-) 1β, IL-6, interferon γ, NF-κB, cyclooxygenase-2, and inducible nitric oxide synthase in the colorectal mucosa and increased the Nrf2 mRNA expression. Our results suggest that dietary crocin suppresses chemically induced colitis and colitis-related colon carcinogenesis in mice, at least partly by inhibiting inflammation and the mRNA expression of certain proinflammatory cytokines and inducible inflammatory enzymes. Therefore, crocin is a candidate for the prevention of colitis and inflammation-associated colon carcinogenesis.
To elucidate the molecular mechanisms involved with spermiogenesis in testis, we performed differential display screening to isolate genes that are developmentally up-regulated during rat testis development. One of the cDNAs isolated by differential display was highly expressed in testis. Both reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression level of the gene developmentally increased. By screening the rat testis cDNA library, we successfully isolated rat cDNA clones encoding the entire open-reading frame of 462 base pairs coding a small protein of 154 amino acids. Because in situ hybridization revealed that the gene was specifically expressed in haploid spermatids in the rat seminiferous tubules, it was designated as spergen-1 (spermatogenic cell-specific gene-1). The recently opened database of the full-length mouse cDNA collection contains a mouse gene that is homologous to rat spergen-1. Subcellular fractionation followed by immunoblot analysis revealed that spergen-1 protein was associated with mitochondria. The transfection experiments performed in COS-7 cells suggested that spergen-1 has a N-terminal mitochondria-targeting signal. We suggest that spergen-1 might be involved in spermiogenesis by transiently associating with spermatid mitochondria.
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