Identification and functional analysis of splice variants of the germ cell soluble adenylyl cyclase.
In mammalian germ cells, cAMP signaling is dependent on two forms of adenylyl cyclase, the conventional membrane-bound ACIII and a soluble form of adenylyl cyclase (sAC). Recent elucidation of the sAC sequence indicates that this enzyme is phylogenetically distinct from the membrane-bound AC, does not interact with G proteins, and its activity is regulated by bicarbonate ions. Here we have investigated the properties and regulation of this enzyme during spermatogenesis. Two different transcripts encoding a full-length and 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 catalytic domain. Reverse transcriptase-polymerase chain reaction with testis RNA from adult mouse and rat of different ages, as well as RNase protection, showed that both transcripts are absent at 11 days of age, appear between 20 and 30 days of age, and are retained in the adult testis. The presence of corresponding proteins in testis, germ cells, and spermatozoa was demonstrated by fast protein liquid chromatography and differential immunoprecipitation with full-length sAC-specific antibodies. Bicarbonate ions activated 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. These findings provide evidence that full-length and truncated sAC are generated by alternate splicing. Both forms are active in spermatids, and the bicarbonate present in the seminiferous tubule may be a signal that regulates cAMP levels in these cells.
Jaiswal
B S
BS
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317, USA.
Conti
M
M
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005854:Germ Cells / Q000201:enzymology*; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility; D013737:Testis / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases