Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells.
To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.
Xie
Fang
F
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.
Conti
Marco
M
D000262:Adenylyl Cyclases / Q000235:genetics / Q000502:physiology*; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis*; D003593:Cytoplasm / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D020869:Gene Expression Profiling; D005854:Germ Cells / Q000166:cytology; D007150:Immunohistochemistry; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D013091:Spermatogenesis / Q000502:physiology*
D000906:Antibodies; D000242:Cyclic AMP; D000262:Adenylyl Cyclases