Developmental regulation of unique adenosine 3',5'-monophosphate-specific phosphodiesterase variants during rat...

Previous studies from our laboratory have shown that messenger RNAs (mRNAs) coding for a cAMP-specific phosphodiesterase (PDE4A) are present in mature rat and mouse germ cells. However, no information is available about the properties of the expressed proteins. To determine their structure and regulation, the PDE4A isoforms expressed in the rat testis were identified and compared to the variants expressed in the brain. Western blot analysis using an antiserum specific for PDE4A demonstrated the presence in testis extracts of two distinct proteins with apparent masses of 98.8 and 86 kDa. The electrophoretic mobilities of these proteins differ from those of proteins detected in the brain extracts (113 and 76 kDa). Reverse transcriptase-PCR of the different splicing mRNA variants expressed in testis confirmed the presence of at least one novel PDE4A mRNA that is distinct from the PDE4A splicing variants identified in the brain and other tissues. Expression of the complementary DNA encoding this variant in a heterologous system resulted in an increase in PDE activity and the appearance of an immunoreactive protein with a mass of 98.8 kDa. No 86-kDa protein could be generated with this transfection. Upon fractionation of testis extracts by HPLC diethylaminoethyl-chromatography, a peak of cAMP-PDE activity coeluted with the two immunoreactive species. During testicular development, the 98.8-kDa protein is present in trace amounts at 10 days, and its level increases with the age of the animals, reaching a plateau at 40 days. The 86-kDa protein appears at 20 days of age and reaches its maximum at 40 days. Studies on the cellular site of expression demonstrated that the two polypeptides are most abundant in round spermatids and are expressed in trace amounts in pachytene spermatocytes, whereas they could not be detected in Sertoli or interstitial cells. The 98.8-kDa, but not the 86-kDa, protein was also expressed in epididymal spermatozoa. These data demonstrate the expression of novel cAMP-specific PDEs coded by the PDE4A gene. The expression of these isoforms is maximal in round spermatids and is maintained in mature spermatozoa. The genesis of the lower mol wt species remains to be determined.