JOURNAL OF CELLULAR PHYSIOLOGY 209:755–766 (2006) Acrosome-Specific Gene AEP1: Identification, Characterization and Roles in Spermatogenesis JOHN M. LUK, 1 * NIKKI P.Y. LEE, 1 CATHY K. SHUM, 1 BRIAN Y. LAM, 1 ANNIE F.M. SIU, 2 CHI-MING CHE, 2 PO-CHOR TAM, 1 ANNIE N.Y. CHEUNG, 3 Z.M. YANG, 5 YI-NAN LIN, 6 MARTIN M. MATZUK, 6 KAI-FAI LEE, 4 AND WILLIAM S.B. YEUNG 4 * 1 Department of Surgery, The University of Hong Kong, Pokfulam, Hong Kong 2 Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Pokfulam, Hong Kong 3 Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong 4 Department of Obstetrics and Gynecology, The University of Hong Kong, Pokfulam, Hong Kong 5 College of Life Sciences, Northeast Agricultural University, Harbin, China 6 Department of Pathology, Molecular and Cellular Biology, and Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas Spermatogenesis is a tightly regulated process leading to the development of spermatozoa. To elucidate the molecular spermatogenic mechanisms, we identified an acrosome-specific gene AEP1 in spermatids, which is located in rat chromosome 17p14 with a transcript size of 3,091 bp encoding a signal peptide, zinc finger-like motif, coiled-coil region, several predicted glycosylation and phosphorylation sites. Northern blot and RT-PCR analyses revealed the restricted expression of AEP1 to the testis only. In postnatal rat testes, AEP1 mRNA became detectable from postnatal 25 dpp (round spermatids) and onwards. By using in situ hybridization (ISH) and flow cytometry-fluorescent ISH, only the haploid spermatids yielded the positive AEP1 signal. Immunohistochemistry showed that AEP1 was expressed in the acrosomal cap of late-staged germ cells in rat testis, and co-localized with the acrosomal marker, peanut agglutinin. The spatial expression of AEP1 immunoreactivity in testis was conserved among diverse mammalian species (rat, pig, monkey, human). To further study its roles in spermatogenesis, we showed AEP1 and b-actin was associated together in complex by co-immunoprecipitation in adult germ cells and by immunofluorescence assay in isolated spermatozoon. In human testes diagnosed with hypospermatogenesis, lower expression of AEP1 was observed, whereas there was no detectable signal in undescended testes. In short, AEP1 is an evolutionary-conserved acrosome-specific gene and likely functions in acrosome-cap formation. J. Cell. Physiol. 209: 755 – 766, 2006. ß 2006 Wiley-Liss, Inc. Spermatogenesis, the generation of spermatozoa, involves mitosis and meiosis of germ cells, such that diploid spermatogonia are transformed into haploid spermatozoa (Johnson et al., 1970; Byers et al., 1993; Cheng and Mruk, 2002). An enzyme-containing cap, the acrosome, is formed in the anterior part of the sperm head during spermiogenesis. Its enzymes are released in acrosome reaction to enable the penetration of sperma- tozoa through the zona pellucida surrounding the oocyte and are crucial for the fertilization process. During spermatogenesis, germ cells move across the seminiferous epithelium from the basal compartment to the adluminal compartment. This movement of the germ cells occurs in a cyclic manner with the continuous generation of spermatogonia and subsequent release of spermatozoa into the seminiferous lumen (Mruk and Cheng, 2004). The spermatogenic cycle is divided into stages according to the types of spermatogenic cells present in the seminiferous tubules. For instance, 14 spermatogenic stages (I – XIV) are found in rats (Leblond and Clermont, 1952). As spermatogenesis does not start synchronously, various spermatogenic stages can be found in different seminiferous tubules within a testis at any time of the cycle. Furthermore, spermatogenesis is a rapid process involving nuclear and cytoplasmic re- organizations, and many proteins are transiently or temporally expressed in a particular stage of the spermatogenic cycle (Cheng and Mruk, 2002). Thousands of genes are expressed specifically in the testis (Schultz et al., 2003) and hundreds of genes have already been shown to play roles in male infertility (Matzuk and Lamb, 2002). To identify some of these cell specific and testis specific genes, we have employed the vitamin A-deficient (VAD) ß 2006 WILEY-LISS, INC. This article includes Supplementary Material available from the authors upon request or via the Internet at http://www. interscience.wiley.com/jpages/0021-9541/suppmat. Abbreviations: AEP1, acrosome expressed protein 1; dpp, day postpartum; ISH, in situ hybridization; IHC, immunohistochem- istry; MS, mass spectrometry; VAD, vitamin A-deficient. John M. Luk and Nikki P.Y. Lee are contributed equally. Contract grant sponsor: Research Grants Council of Hong Kong; Contract grant numbers: HKU 7272/01M, HKU 7537/05M. Brian Y. Lam’s present address is Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK. *Correspondence to: John M. Luk and William S.B. Yeung, Department of Surgery, University of Hong Kong, Jockey Club Clinical Research Centre, 21 Sassoon Road, Pokfulam, Hong Kong. E-mail: jmluk@hkucc.hku.hk; wsbyeung@hkucc.hku.hk Received 9 May 2006; Accepted 8 June 2006 DOI: 10.1002/jcp.20746