Eric D. Wieben

Cyclin-dependent kinase 5 is expressed in both sertoli cells and metaphase spermatocytes

OBJECTIVE To gain insight into the function of cyclin-dependent kinase 5 (Cdk5) in spermatogenesis. DESIGN The expression of the Cdk5 protein was determined with the use of immunohistochemical and immunoblot analysis. SETTING Academic research laboratory. ANIMAL(S) Adult mouse and archival human testicular tissue were used for the immunohistochemical analysis. Adult mice were used as the source of tissues for the immunoblot analysis. INTERVENTION(S) The immunohistochemical analysis was performed with an anti-Cdk5 antibody. The double immunohistochemical analysis was performed with anti-Cdk5 and alpha-tubulin antibodies. Immunoblotting was used to examine multiple mouse tissues for Cdk5 expression. MAIN OUTCOME MEASURE(S) Analysis of Cdk5 protein distribution. RESULT(S) Cdk5 was localized specifically within the cytoplasm of Sertoli cells and meiotic metaphase germ cells. The double immunohistochemistry analysis demonstrated the co-localization of Cdk5 and alpha-tubulin within the Sertoli cells. Western blot analysis revealed a high level of expression of Cdk5 in the testicular lysate. CONCLUSION(S) The cyclin-dependent kinases are known regulators of the cell cycle; however, Cdk5 expression previously has been described in terminally differentiated cells of the brain. The present evidence of an association between Cdk5 and microfilaments of Sertoli cells and meiotic metaphase germ cells suggests a role of Cdk5 in both seminiferous tubule function and meiosis.

The effects of androgen on the transcription of specific genes in guinea pig seminal vesicle epithelium

Steroid hormones have been shown to have highly differential effects on the expression of abundant cell-specific protein genes in a multitude of model tissues. In rat seminal vesicle, for example, DNA clones representing two major secretory protein genes have been used to show that both of the genes are differentially regulated by androgen. In this paper, we have examined the effects of androgen on the transcription of two major secretory protein genes in guinea pig seminal vesicle epithelium. Nuclear run-off experiments were used to show that castration of the adult resulted in a 3-fold decrease in total transcription activity. Surprisingly, the decrease in total transcriptional activity was not reflected in a differential decrease in the transcriptional activity of the two major secretory protein genes. When the effects of castration on the transcriptional activity of the major secretory protein genes were compared to the effects on other genes, it was found that the transcriptional activity of each gene examined was decreased by the same magnitude as the major secretory protein genes. Similarly, the transcriptional activity of every gene examined increased by the same magnitude as the major secretory protein genes during hormone repletion of the castrated adult. Thus, in contrast to the differential effects of steroids on the transcription of abundant cell-specific proteins in many other steroid-dependent tissues, the transcription of major secretory proteins in guinea pig seminal vesicle epithelium appears to be regulated in parallel with many other genes. The generalized effects of androgen on transcriptional activity could account for the generalized effects of androgen on seminal vesicle epithelial cell structure and function.

31 HUMAN METHYLENETETRAHYDROFOLATE REDUCTASE PHARMACOGENOMICS: GENE RESEQUENCING AND FUNCTIONAL GENOMIC STUDIES

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme in the folate metabolic pathway that contributes to DNA, protein and small molecule methylation. Previous studies have linked common genetic polymorphisms in the human MTHFR gene to hyperhomocysteinemia, a risk factor for cardiovascular disease. However, the full range of common genetic polymorphisms and haplotypes in the human MTHFR gene remains unclear. Furthermore, the underlying cellular mechanisms by which common nonsynonymous coding single nucleotide polymorphisms (cSNPs) might alter the function of this enzyme are not defined. The present study focused on the systematic identification and investigation of common polymorphisms in the MTHFR gene using a genotype-to-phenotype approach followed by studies of their molecular mechanisms for these functional effects. Specifically, we began by resequencing exons, splice junctions and portions of the 5′-flanking region of the human MTHFR gene using DNA samples from 60 African-American, 60 Caucasian-American, 60 Han-Chinese American, and 60 Mexican-American subjects obtained from the Coriell Cell Repository. A total of 66 polymorphisms, 63 SNPs and 3 insertion/deletion events, were observed. Ten nonsynonymous cSNPs were identified, including the known C677T (A222V) and A1298C (E429A) variants. We then performed functional genomic studies with all of the nonsynonymous cSNPs observed. Specifically, the wild-type (WT) and 10 variant allozymes were transiently expressed in COS-1 cells. After correction for transfection efficiency, activity for the variant allozymes ranged from 6.5% to 145% of WT activity. Levels of immunoreactive protein for the allozymes ranged from 15% to 118% of WT and were significantly correlated with levels of enzyme activity (Rp = .93, p = .0001), suggesting that a major mechanism by which nonsynoymous cSNPs influence the function of this gene is by alteration in the quantity of protein. These observations represent steps toward an understanding of molecular genetic mechanisms responsible for variation in MTHFR levels and/or properties that may contribute to the pathophysiology of human disease.