Type 4 cyclic adenosine monophosphate-specific phosphodiesterases are expressed in discrete subcellular compartments during rat spermiogenesis

The type 4 cAMP-specific phosphodiesterases (PDE4) are a family of closely related enzymes with similar catalytic domains and divergent amino- and car boxyl-terminus domains. Multiple PDE proteins with heterogeneous amino term ini are derived fi om each gene. To understand the significance of this het erogeneity, the expression and localization of variants derived from PDE4A and PDE4D genes was investigated during spermatogenesis in the rat. RNase p rotection analysis with mRNA for testes at different ages of development sh owed that two transcripts (PDE4D I and PDE4D2) are expressed at day 10 and 15 of age and become undetectable thereafter. An additional PDE4D transcrip t appears at day 30 and increased during testid maturation. This latter tra nscript codes for a long Variant of the PDE4D gene and is expressed in germ cells as demonstrated by RNase protection with RNA from isolated pachytene spermatocytes and round spermatids. The presence of a corresponding PDE4D protein with a molecular mass of 98 kDa was established by immunoprecipitat ion and Western blot analysis with antibodies specific for PDE4D and by imm unoaffinity chromatography purification of the 98 kDa variant from isolated germ cells. PDE4A transcripts were also expressed in pachytene spermatocyt es and round spermatids. Two polypeptides encoded by these PDE4A transcript s were expressed in pachytene spermatocytes, reached a maximum in round spe rmatids, and declined thereafter. Immunofluorescence analysis demonstrated a localization of the PDE4D protein in the manchette and in a periacrosomal region of the developing spermatid, a localization confirmed by immunogold electron microscopy. Conversely, the PDE4A was mostly soluble in the cytop lasm of round spermatids. These data demonstrate that PDE4D and PDE4A varia nts are expressed at different stages and localized in distinct subcellular structures of developing spermatids. Different properties of the mRNAs der ived fi om the two genes and localization signals are responsible for the t emporal and spatial expression of the different PDE4 isoenzymes.