Analysis of Hook1 interacting proteins in spermiogenesis

The murine Hook1 gene has an essential role in the differentiation process of haploid male germ cells. In homozygous azh-mice (abnormal spermatozoon head shape) Hook1 function is disrupted due to the deletion of exons 10 and 11 leading to a truncated Hook1 polypeptide. Wild type Hook1 protein consists of three putative domains: an N-terminal microtubule binding domain, a central homodimerization domain and a C-terminal organelle binding domain. In azh-males the mutated Hook1 bears only the N-terminal domain while the two other domains are deleted. The loss of Hook1 functions results in an abnormal head shape of all spermatozoa, most probably due to a disturbed structure of the microtubular manchette. Moreover, a fragile connection between the spermatid head and the flagella is often observed leading to a detachment of head and tail. Immunohistochemical localization studies of the Hook1 wild type protein to the manchette and to the spermatid head - tail implantation region suggest different putative functions of Hook1 in male germ cell differentiation. In addition, interaction of Hook1 with other male germ cell proteins can be assumed. Therefore, we performed a yeast-two-hybrid screen using a murine testicular library and identified different polypeptides as putative Hook1 interacting proteins. In the intended project six of these polypeptides and the corresponding genes will be analysed. In a first step the interaction of the six different polypeptides with Hook1 will be proven by immunoprecipitation assays as well as putative co-localization in male germ cells will be analysed. If the interaction between Hook1 and the six polypeptides has been confirmed the function of the six genes in male germ cell differentiation will be analysed in more detail. The inactivation of the genes by homologous recombination will demonstrate the role of the six genes for male germ cell differentiation. One other interesting question is to clarify whether the expression or the localisation of one of the six polypeptides is altered if Hook1 function is missing. Analyses of the six genes in azh- mice will contribute to clarification of this question. Taken together, the molecular characterization of Hook1 and its putative interacting proteins will increase our knowledge concerning the processes during spermiogenesis and will add information concerning the role of the manchette in this differentiation process. Due to the high similarity of murine and human Hook1 proteins it can be assumed that both have a comparable function in spermatogenesis. Therefore, it can be expected that the intended project will also add substantial information to the understanding of human haploid male germ cell differentiation and will give clues to the reasons of male infertility.

Male infertility is an increasing problem of our society. Our knowledge concerning the molecular processes during male germ cell development is still limited. The murine Hook1 gene plays an important role in spermiogenesis, because loss of Hook1 function results in an abnormal head morphology, looping in the midpiece of the sperm flagellum and detached sperm heads from tails. However, the exact function of Hook1 is still unclear. Our previous results indicate that Hook1 is associated with the microtubular manchette of the spermatid and might be involved in transport processes along this organelle. This hypothesis concerning Hook1 function implies an interaction with other polypeptides within male germ cells. Using the yeast-two-hybrid system 26 different putative Hook1 interacting proteins had been identified. In our project we have proved the interaction of six proteins using different technologies. We were able to localize the interacting domains in Hook1 as well as in the six proteins. Ift81 and Ccdc181 two of the interacting proteins were analysed in male germ cell development. Both proteins were detected in different stages of male germ cell development. Interestingly, Ift81 antibody staining was observed along the whole sperm flagella, whereas Ccdc181 was localized to the basal part of motile cilia and sperm flagella. These results suggest a ciliary function of both proteins. Moreover, Ccdc181 is not only interacting with Hook1 but also with a subunit of the protein phosphatase1 within the male germ cell. This finding indicates that Ccdc181 is involved in the regulation of sperm motility via protein phosphatise1. However, further experiments are necessary to prove this assumption. Therefore, we have cloned constructs to inactivate the different genes tissue specific.