No abstract

Cdk5 is ubiquitously expressed in all tissues, but its activators, p35 and p39, are principally found in brain, and Cdk5 activity has mostly been associated with brain development, particularly neuronal differentiation and migration. Here we show that the p35 transcript and protein are also present in the testis, and an active Cdk5⅐p35 complex exists in this tissue as well. Cdk5 and p35 are prominently observed in elongating spermatid tails, particularly over the tail outer dense fibers (ODF). The appearance of Cdk5⅐p35 proceeds from the proximal to the distal end of elongating spermatids, coinciding with the proximal to distal assembly of ODF along the length of the tail axoneme. Incidentally, increased Cdk5⅐p35 activity is observed in isolated elongating spermatids and at a time when elongating spermatids appear in the developing testis, suggesting a role for Cdk5⅐p35 in spermiogenesis. The presence of Cdk5 and p35 in ODF isolated from rat sperm tails implies a strong association among these proteins. In vitro ODF phosphorylation by Cdk5⅐p35 and decreased in vivo sperm tail ODF phosphorylation in p35-deficient mice indicate that Cdk5⅐p35 is an integral component of ODF and that ODF is a functional Cdk5⅐p35 target in the testis. Our results demonstrate for the first time that Cdk5⅐p35 may participate in the regulation of sperm tail development via a mechanism involving ODF phosphorylation. Apparently, as in brain development, Cdk5⅐p35 plays a part in testis development.

The efficacies of N-acetylcysteine (NAC), phenylethyl isothiocyanate (PEITC), and 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ) at preventing the neurotoxicity and testicular toxicity of acrylamide (ACR) were investigated in rats. To this end, Sprague-Dawley males were given 0.02% ACR in drinking water, with or without 1% NAC, 0.5% PEITC or 0.1% HTHQ in the diet for four weeks. A group of untreated controls was also included in the study. All ACR-treated animals exhibited progressive neurotoxicity as judged by gait scores, and among the chemicals co-administered, only HTHQ caused any suppression by the end of the experiment, and this was slight. The severity of the neurotoxicity, as judged by axonal degeneration in the spinal gracile fasciculus and sciatic nerve (distal portion) and aberrant dot-like synaptophysin immunoreactivity, reflecting nerve terminal degeneration in the cerebellar molecular layer, was not clearly reduced by co-administration of HTHQ, NAC or PEITC either. ACR-induced sciatic nerve axon atrophy was marginally and non-significantly reduced by HTHQ. In contrast, in terms of ACR-induced testicular toxicity, exfoliation of spermatids into seminiferous lumen was clearly reduced by co-administered PEITC and was marginally reduced by co-administered HTHQ. These antioxidative agents may therefore reduce/prevent ACR-induced toxicity, at least in the testes.

No abstract

S U M M A R YWe have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients Ͼ 0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, Ն 200 cells were necessary for accurate measurement in 10-m-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.