MOLECULAR REPRODUCTION AND DEVELOPMENT 74:341–359 (2007) Structural Abnormalities in Spermatids Together With Reduced Sperm Counts and Motility Underlie the Reproductive Defect in HIP1 / Mice KARINE KHATCHADOURIAN, 1 CHARLES E. SMITH, 2 MARTINA METZLER, 3 MARY GREGORY, 4 MICHAEL R. HAYDEN, 1 DANIEL G. CYR, 1,4 AND LOUIS HERMO 1 * 1 Department of Anatomy and Cell Biology, McGill University, Montreal, Canada 2 Departement de Stomatologie, Universite´de Montre´al, Montreal, Canada 3 Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada 4 INRS-Institut Armand Frappier, Universite´du Que´bec,Pointe-Claire, Canada ABSTRACT Huntingtin interacting protein 1 (HIP1) is an endocytic adaptor protein with clathrin assembly activity that binds to cytoplasmic proteins, such as F-actin, tubulin, and huntingtin (htt). To gain insight into diverse functions of HIP1, we characterized the male reproductive defect of HIP1 / mice from 7 to 30 weeks of age. High levels of HIP1 protein were expressed in the testis of wild-type mice as seen by Western blots and as a reaction over Sertoli cells and elongating spermatids as visualized by immunocyto- chemistry. Accordingly, major structural abnormalities were evident in HIP1 / mice with vacuolation of seminiferous tubules caused by an apparent loss of postmeiotic spermatids and a significant reduction in mean profile area. Remaining spermatids revealed deformations of their heads, flagella, and/or acro- somes. In some Sertoli cells, ectoplasmic specializa- tions (ES) were absent or altered in appearance accounting for the presence of spherical germ cells in the epididymal lumen. Quantitative analyses of sperm counts from the cauda epididymidis demonstrated a significant decrease in HIP1 / mice compared to wild-type littermates. In addition, computer-assisted sperm analyses indicated that velocities, amplitude of lateral head displacements (ALH), and numbers and percentages of sperm in the motile, rapid, and progressive categories were all significantly reduced in HIP1 / mice, while the numbers and percentages of sperm in the static category were greatly increased. Taken together, these various abnormalities corrobo- rate reduced fertility levels in HIP1 / mice and suggest a role for HIP1 in stabilizing actin and microtubules, which are important cytoskeletal ele- ments enabling normal spermatid and Sertoli cell morphology and function. Mol. Reprod. Dev. 74: 341–359, 2007. ß 2006 Wiley-Liss, Inc. Key Words: germ cells; fertility; electron micro- scopy; actin; microtubules INTRODUCTION Huntingtin interacting protein 1 (HIP1) was first identified through its interaction with huntingtin (htt), a polyglutamine-containing protein associated with neurodegeneration in Huntington Disease (Kalchman et al., 1997; Wanker et al., 1997). HIP1 is a 116-kDa endocytic adaptor protein and a component of clathrin- coated vesicles at the plasma membrane (Metzler et al., 2001; Mishra et al., 2001; Waelter et al., 2001). HIP1 contains consensus sites for binding to clathrin and AP2 and has clathrin assembly activity that is regulated through its direct binding to clathrin light chain (Legendre-Guillemin et al., 2004, 2005; Chen and Brodsky, 2005). Moreover, HIP1 contains an AP180 N-terminal homology (ANTH) domain, which is similar in structure to the Epsin N-terminal homology (ENTH) domain (Ford et al., 2002). Both domains bind to phosphatidylinositol-containing membranes and are present in several endocytic proteins playing important roles in the formation of clathrin-coated pits and clathrin-coated vesicles (Ford et al., 2001; Itoh et al., 2001; Legendre-Guillemin et al., 2004). It has been shown that the ENTH/ANTH domains, including that of HIP1, bind to tubulin heterodimers and to assembled microtubules (Hussain et al., 2003). In addition to its interaction with tubulin/microtubules, HIP1 interacts directly with actin via an I/LWEQ module (talin homology domain) located at its C-terminus (Senetar ß 2006 WILEY-LISS, INC. Grant sponsor: CIHR; Grant sponsor: NSERC; Grant sponsor: National Institutes of Health; Grant number: DE013237; Grant sponsor: Canadian Health Research; Grant number: MOP-9133; Grant sponsor: Merck-Frosst Canada; Grant sponsor: Canadian Genetics Disease Network. *Correspondence to: Dr. Louis Hermo, Department of Anatomy & Cell Biology, McGill University, 3640 University Street, Montreal, QC, Canada H3A 2B2. E-mail: louis.hermo@mcgill.ca Received 20 February 2006; Accepted 8 May 2006 Published online 11 September 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20564