MOLECULAR DIVERSITY OF TEX101, A GERM CELLSPECIFIC GLYCOPROTEIN IN THE PROCESS OF SPERMATOGENESIS ANALYZED BY MONOCLONAL ANTIBODIES

Abstract

TEX101, a glycosylphosphatidyl inositol (GPI)-anchored glycoprotein we identified, is characterized as a unique germ cell-specific marker protein that shows sexually dimorphic expression during mouse gonad development. Our previous study using a specific monoclonal antibody (mAb) to TEX101 (designated TES101), showed that TEX101 is expressed constitutively on the plasma membrane of prespermatogonia within seminiferous cords before initiation of spermatogenesis. Then, it appears on meiotic cells including spermatocytes, spermatids, and testicular sperm after puberty. Recently, we identified three proteins, cellubrevin (a member of the family of soluble N-ethylmaleimide-sensitive factor attachment protein receptors), annexin 2 (a calcium- and phospholipidbinding protein), and ly6k (a member of urokinase type plasminogen activator receptor/Ly-6 family) as the candidate of TEX101-associated proteins using liquid chromatography/tandem mass spectrometry. Although morphological/biochemical characteristics of TEX101 are gradually known, the biological function(s) of TEX101 has not yet been clarified. To elucidate the function of TEX101 more precisely, we established new mAbs against TEX101 by immunizing with the molecule displayed by the baculovirus expression system suitable for cell membrane proteins with their complex structures like GPI-anchored proteins. Among mAbs established, we characterized two of new anti-TEX101 mAbs, designated #6002 and #6035. Like TES101 mAb, Western blot analysis revealed that these mAbs detected a band at an apparent molecular mass of 38 kDa under non-reducing conditions in Triton X-100-soluble fraction of testicular extract from sexually mature mice as expected. Unlike TES101 mAb, these new mAbs possessed an advantage to be also reactive with TEX101 under reducing conditions. The reactivity under reducing conditions was observed after deglycosylation of TEX101 with Nglycanase, confirmed that antigen determinants of these mAbs are not N-linked carbohydrate-related moieties. In addition, both #6002 and #6035 are immunohistchemically reactive in the mice testicular specimen of Bouin's fixation, whereas the reactivity of TES101 mAb to the section was not observed. In comparison to previous immunomorphological studies using TES101 mAb, we investigated the reactivity of #6002 and #6035 in the frozen testicular sections from sexually mature mice fixed with paraformaldehyde. Somehow different from TES101 mAb staining pattern, #6002 is reactive with only plasma membrane of spermatocytes, whereas the reactivity was observed on spermatocytes and weakly on round spermatids when #6035 was used as a primary antibody. Neither #6002 nor #6035 reacted to tails of testicular sperm, a strongly positive area stained with TES101 mAb. These results strongly suggest that the characteristics of TEX101 such as molecular association or steric structure may vary in the process of spermatogenesis, and the mAbs newly established should be valuable tools for further analysis of TEX101 including its function(s). (Supported by Grants-in-Aid for "High-Tech Research Center" Project for Private Universities: matching fund subsidy and General Scientific Research No. 18591813 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.) (poster)