Spermatogenesis and the regulation of Ca(2+)-calmodulin-dependent protein kinase IV localization are not dependent on calspermin.

Calcium plays a central role in numerous biological processes, including cell proliferation, protein secretion, and muscle contraction. Many of these cellular effects are mediated by the ubiquitous intracellular calcium receptor calmodulin, which when bound to calcium can activate a variety of enzymes, including protein kinases, phosphatases, and phosphodiesterases (6). Because calmodulin is present in all tissues, cell-type-specific functions are determined by the complement of its downstream targets. Calmodulin is especially abundant in the testis, which led to the identification of the testis-specific binding protein, calspermin. Calspermin was initially purified from rat and pig testes as a potent inhibitor of the calmodulin-dependent cyclic nucleotide phosphodiesterase (11). Purified calspermin binds calmodulin in the presence of Ca2+ (10, 11) and contains a calmodulin-binding domain close to the N terminus (12). Calspermin and Ca2+-calmodulin-dependent protein kinase IV (CaMKIV) are both products of the Camk4 gene (7, 9) and are derived by alternative transcriptional initiation (7). The calspermin promoter and its testis-specific first exon are located within the 10th CaMKIV intron, and the two proteins share the final two exons of the Camk4 gene (15). In vitro transcription assays with the rat calspermin promoter demonstrated that a fragment spanning from −80 to +361 (with +1 as the transcriptional initiation site) yielded maximal activity (16). Within this region, two cyclic AMP response element (CRE) motifs bind the testis-specific transcriptional activator CREMτ (16), which regulates transcription of several male germ cell-specific genes (5). Mice deficient in CREMτ fail to express calspermin even if heterozygous, suggesting that calspermin is exquisitely sensitive to levels of CREMτ (8). In transfection assays, CREMτ must be phosphorylated for full activity, and in vitro both protein kinase A (PKA) and CaMKIV can phosphorylate CREMτ. Cotransfection of CREMτ with either PKA or CaMKIV can stimulate transcriptional activity from the calspermin promoter in NIH 3T3 cells (16). That the calspermin promoter functions in vivo has been demonstrated by transgenic mice bearing the β-galactosidase gene driven by the calspermin promoter, which exhibit male germ cell-specific X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining (15). Furthermore, β-galactosidase activity can be activated in mouse embryonic fibroblasts from these mice by transfection of CREMτ with PKA or CaMKIV (15). These results led to the prediction that CaMKIV might regulate expression of calspermin and other CRE-dependent testis-specific genes by phosphorylation and activation of CREMτ (14). However, this model has since been refuted by two lines of evidence. (i) CREMτ is not phosphorylated in vivo in germ cells. Rather, CREMτ appears to interact with the transcriptional regulator ACT to stimulate transcription in postmeiotic spermatids (4). (ii) Expression of CRE-containing genes, including calspermin, is unaltered in CaMKIV-deficient mice (18). Instead, the absence of CaMKIV results in the failure of spermatogenesis and reflects a requirement for CaMKIV in the replacement of transition protein by protamine during spermiogenesis (18). CaMKIV and calspermin are both expressed in spermatids (17) and, although CaMKIV is not required for the proper expression and localization of calspermin, the relationship between these two proteins is not understood. Indeed, the function of calspermin in the testis remains unknown. One plausible role that it may serve is to regulate the abundance of calmodulin levels within the testis. Calmodulin is essential during cell replication, but it may be inhibitory during differentiation in the last stages of sperm production (3). CREMτ−/− mice lack calspermin and exhibit defective spermatogenesis, but they also fail to express several other CRE-containing genes, including those encoding the protamines and transition proteins, which are also required for sperm production (1, 8). To clarify the role of calspermin in the testis, we have specifically deleted calspermin gene expression in mice. In doing so, it was critical to leave the kinase intact since CaMKIV has been shown to have important effects in several tissues, including the testis. We report here that calspermin is not required for fertility and that neither the expression of CaMKIV nor its targeting to the nuclear matrix are dependent on calspermin.