Differential display to identify and isolate novel genes expressed during spermatogenesis

Spermatogenesis is a complex differentiation process in which diverse stage-specific proteins are co-ordinately expressed. Previously, subtractive hybridization and differential hybridization have been used in the identification of differentially expressed mRNAs, Although these techniques have been successfully used they require large amounts of RNA and are time consuming. To overcome these problems we have made use of the recently described mRNA differential display technique. The technique is an effective method which can identify and separate cDNAs that are differentially expressed between various cell-types. By comparing RNA from testes of mature (>60 days old) and prepubertal (15-16 days old) mice we have identified nine differential cDNA bands expressed in mature testes. The differential display cDNA band DDC8 was used to screen a testis cDNA library and the full length cDNA was isolated and sequenced. DDC8 cDNA is 1965 bp with an open reading frame of 533 amino acids which codes far a predicted hydrophilic protein with a calculated molecular weight of 62.04 kDa. RNase protection assays indicate DDC8 to be expressed during the postmeiotic stages of spermatogenesis and database searches using both nucleotide and amino acid sequences show DDC8 to have similarities to structural, cytoskeletal and associated proteins.