Mice Lacking the USP 2 Deubiquitinating Enzyme Have Severe Male Subfertility Associated with Defects in Fertilization and Sperm Motility 1

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 / mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 þ/þ). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 / males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 þ/þ littermates. Spermatogenesis in Usp2 / mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 / mice, but some abnormal cells other than sperm were present in the lumen. Usp2 / epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 þ/þ spermatozoa, which largely maintained motility under this condition. Usp2 / and þ/þ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 / spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 þ/þ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization. fertilization, gamete biology, sperm motility and transport, ubiquitin, zona pellucida INTRODUCTION Spermatogenesis is a complex developmental program that includes many fundamental cellular processes, such as renewal of stem cells, proliferation of spermatogonia committed to spermatogenesis, meiosis, and remodeling of the haploid round spermatids to elongated spermatids [1–3]. Spermatids released from the testis undergo further maturation during transport through the epididymis [4]. Upon ejaculation, sperm acquire the capacity to fertilize eggs. All of these steps leading to the formation of mature sperm are precisely regulated, and this regulation depends significantly on the appropriate timing of expression of specific proteins that play critical roles in mediating these events [5–7]. The steady-state level of expression of specific proteins is influenced in a major way by the transcriptional control of genes encoding these proteins, as well as by the regulation of the translation of encoded mRNAs. However, it is also critically dependent on the rates of protein degradation. The ubiquitin-proteasome system is the major proteolytic system in the cell, responsible for the regulated degradation of many specific proteins [8]. This system plays important roles in the cell cycle, meiosis, and the terminal differentiation of various tissues. Ubiquitin is a small peptide that is covalently attached to proteins through a series of enzymatic reactions catalyzed by ubiquitin-activating enzyme, ubiquitin-conjugating enzymes, and ubiquitin protein ligases. Proteins can be monoubiquitinated (a single ubiquitin is attached at one or more lysines on the substrate) or polyubiquitinated, where a chain of multiple ubiquitin moieties is attached to the substrate. When polyubiquitin chains are attached to a protein substrate, particularly chains in which the C-terminal end of the distal ubiquitin is linked to lysine 48 in the proximal ubiquitin, the chain can mark the protein substrate for recognition and degradation by the 26S proteasome [9]. Alternatively, monoubiquitination and some non-lysine 48-linked forms of polyubiquitin chains can alter the function of the substrate protein or serve as docking sites for other proteins and thereby exert functions not mediated by proteasomal degradation [10]. Although the ubiquitin system is involved in many cellular processes, its functions during spermatogenesis remain poorly understood. In past studies, we have shown that ubiquitin conjugation is activated during spermatogenesis and that activation of conjugation is dependent on the UBC4/UBC5 family of ubiquitin-conjugating enzymes whose expression is markedly upregulated in spermatids [11]. Inactivation of the testisand spermatid-specific UBC4-testis gene results in mice with a mild delay in postnatal growth of the testis. The HR6B/ E214k isoform of UBC2 [12, 13] is expressed ubiquitously but at high levels in the testis [14]. Inactivation of this ubiquitinSupported by the Canadian Institutes of Health Research (grants MT12121 and MOP82734 to S.S.W.; IHO94381 to H.J.C.). S.S. W. was the recipient of a Chercheur National salary award from the Fonds de la Recherche en Santé du Québec. Correspondence: Simon S. Wing, Polypeptide Laboratory, Strathcona Anatomy and Dentistry Bldg, Room W315, 3640 University St., Montreal, Quebec, Canada H3A 2B2. FAX: 514 398 3923; e-mail: simon.wing@mcgill.ca Received: 14 September 2010. First decision: 3 October 2010. Accepted: 11 April 2011. 2011 by the Society for the Study of Reproduction, Inc. This is an Open Access article, freely available through Biology of Reproduction’s Authors’ Choice option. eISSN: 1529-7268 http://www.biolreprod. org ISSN: 0006-3363