Abstract
Disruption of murine Hook1 results in a disturbed spermatogenesis and consequently leads to male infertility in mice. Within these mice abnormal sperm development starts with a disorganization of the microtubular manchette in elongating spermatids that leads to an abnormal head shape as well as to distinctive structural changes in the flagella of the sperm. To elucidate Hook1 function in male germ cell differentiation a yeast two-hybrid screen was performed using a murine testicular library, which leads to the identification of several putative Hook1 interacting proteins. One of the isolated cDNA fragments encodes for the coiled-coil domain containing protein 181 (Ccdc181). The putative interaction of Ccdc181 with Hook1 was verified by FRET analysis and interacting regions were identified using yeast two-hybrid assays. Furthermore, Ccdc181 seems to interact directly with microtubules and localizes to the microtubular manchette of elongating spermatids, resembling the previously reported localization of Hook1. According to the observed immunostaining pattern the RNA expression of Ccdc181 is less prominent in pre-meiotic stages of sperm development but increases in the haploid phase of spermatogenesis and seems to be restricted to male germ cells. However, Ccdc181 expression is also observed to a lower extent in somatic tissues, particularly, in tissues containing ciliated epithelia. Additionally, Ccdc181 protein is found to localize to the sperm flagella and to the basal half of motile cilia, whereas Ccdc181 was not detected in primary non-motile cilia. Furthermore, we showed that Ccdc181 is a putative interacting partner of the different catalytic subunits of Pp1, raising the hypothesis that Ccdc181 plays a role in mediating ciliary motility.
Highlights
Spermatogenesis is characterized by successive periods of regulated cell proliferation, meiotic divisions and complex phases of differentiation of the newly formed haploid spermatids during spermiogenesis
Taken together our results suggest that coiled-coil domain containing protein 181 (Ccdc181) via its interaction with Pp1 might play an important role in the generation and regulation of flagellar as well as ciliary motility
We report the molecular characterization of a new member of the coiled-coil domain containing protein family encoded by the murine Ccdc181 gene
Summary
Spermatogenesis is characterized by successive periods of regulated cell proliferation, meiotic divisions and complex phases of differentiation of the newly formed haploid spermatids during spermiogenesis. The formation of the acrosome-acroplaxome-manchette complex is essential for proper spermatid development (Kierszenbaum and Tres, 2004). In spermatids of azh-mutant mice the microtubules of the manchette are often ectopically positioned, leading to abnormal sperm head shape and malformed sperm tails. This phenotype is caused by a deletion of two exons of the Hook gene, suggesting that Hook seems to be essential for the proper function of the manchette during sperm development (Meistrich et al, 1990; Mendoza-Lujambio et al, 2002; Mochida et al, 1999). To identify putative Hook cargoes in male germ cell development a yeast two hybrid screen
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