Identification and functional analysis of splice variants of the germ cellsoluble adenylyl cyclase

In mammalian germ cells, cAMP signaling is dependent on two forms of adenyl yl cyclase, the conventional membrane-bound ACIII and a soluble form of ade nylyl cyclase (SAC). Recent elucidation of the SAC sequence indicates that this enzyme is phylogenetically distinct from the membrane-bound AC, does n ot interact with G proteins, and its activity is regulated by bicarbonate i ons. Here we have investigated the properties and regulation of this enzyme during spermatogenesis. Two different transcripts encoding a full-length a nd truncated sAC were identified by reverse transcriptase-polymerase chain reaction and RNase protection analysis. The truncated sAC transcript lacks exon 11 with a premature termination of the open reading frame after the ca talytic domain. Reverse transcriptase-polymerase chain reaction with testis RNA from adult mouse and rat of different ages, as well as RNase protectio n, showed that both transcripts are absent at I I days of age, appear betwe en 20 and 30 days of age, and are retained in the adult test-is. The presen ce of corresponding proteins in testis, germ cells, and spermatozoa was dem onstrated by fast protein liquid chromatography and differential immunoprec ipitation with full-length sAC-specific antibodies. Bicarbonate ions activa ted both sAC forms and increased cAMP levels in germ cells isolated from 25 -and 50-day-old rats and adult rats in a concentration-dependent manner. Th ese findings provide evidence that full-length and truncated sAC are genera ted by alternate splicing. Both forms are active in spermatids, and the bic arbonate present in the seminiferous tubule may be a signal that regulates cAMP levels in these cells.