An earlier study using RT-PCR reported that is expressed in pachytene spermatocytes, round spermatids, and condensing spermatids in the mouse (Visconti et al
An earlier study using RT-PCR reported that is expressed in pachytene spermatocytes, round spermatids, and condensing spermatids in the mouse (Visconti et al., 1996), but the reasons for these variations are not obvious. 5 untranslated areas, but the open reading frames are alike except for a 69 nucleotide place in that we refer to as the place (SBI) (Mori et al., 1993). A novel feature common to all three variants is the encoding of a 24 amino-acid sequence in the N-terminus that we refer as the spermatogenic cell-specific region (SSR) (Mori et al., 1993). The N-terminal 20 amino Loxoprofen acids of the ubiquitously Loxoprofen indicated form of HK1 define the porin binding website (PBD) (Arora et al., 1990; Griffin et al., 1991) that binds HK1 to porin (also known as voltage-dependent anion channels; VDACs) within the outer CD2 mitochondrial membrane; presumably providing HK1 preferential access to ATP produced by oxidative phosphorylation, (see Adams et al., 1991, Ceser and Wilson, 1998). Open in a separate windows Fig. 1 The constructions of the cDNAs of hexokinase gene-family users. The coding regions of the variants and are related in length, while that for is definitely approximately half that of the additional hexokinase family members. The 5untranslated regions of differ in their lengths and sequences. Pair of facing arrows shows the positions of the sequence-specific primers used in this study. Previous reports indicated that a monoclonal antibody to rat mind HK1 bound to the proximal and middle portion of the mouse sperm flagellum (Visconti et al., 1996), while two antisera to the SSR region localized HK1S to the principal piece region in the mouse sperm flagellum (Mori et al., 1998; Travis et al., 1998). One SSR antiserum also bound to the surface of the head and the midpiece region of the flagellum (Travis et al., 1998). In this study, we used real time RT-PCR (qPCR) to examine mRNA from testes of juvenile mice during the relatively synchronous first wave of spermatogenesis (days 10C30) to compare the steady-state transcript levels of the users of the hexokinase gene family (variants Loxoprofen are first indicated and to compare their levels during this period. In addition, the relative steady-state levels for the variant transcripts and for the additional hexokinase gene-family users in individual spermatogenic cell types were determined by qPCR with RNA from isolated mouse pachytene spermatocytes, round spermatids, and elongating spermatids, and with RNA from spermatogonia, pachytene spermatocytes, early spermatids and late spermatids collected by laser-capture microdissection (LCM). Western blotting and immunohistochemistry were used to determine when HK1 and GCK are indicated in testis and if they are present in sperm. Most of the ATP required for mouse sperm motility is definitely produced by glycolysis (Miki et al., 2004). The present study confirms and stretches previous suggestions that variant transcripts encode the hexokinase isozyme that participates in glycolysis Loxoprofen in mouse spermatozoa. MATERIALS AND METHODS All reagents were purchased from Sigma-Aldrich (Saint Louis, MO) unless indicated normally. The CD-1 mice utilized for isolation of RNA, immunohistochemistry and germ cell isolation were from Charles River Laboratories (Raleigh, NC). the C57BL6/J mice utilized for laser capture studies were from Japan SMC (Hamamatsu, Japan). The care and attention and use of animals were carried out relating to U.S. Public Health Service (USPHS) recommendations and the studies were approved in advance from the Institutional Animal Care and Use Committee of NIEHS or the University or college of North Carolina, or were performed in accordance with Chiba University animal experimentation recommendations. Isolated spermatogenic cells Spermatogenic cells were isolated as previously explained (OBrien, 1993). Purities of pachytene spermatocytes and round spermatids (methods 1C8) exceeded 90%. Elongating spermatids isolated by this method contained 30C40% nucleated spermatids (methods 9C16) and cytoplasts derived primarily from these same cells. Two self-employed preparations for each of the germ cell types were used. Quantitative Real-Time RT-PCR (qPCR) Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA) from testes of mice 10 to 30 days of age, mind of adults, and purified populations of spermatogenic cells. The cDNA themes for qPCR were synthesized from RNA samples using reverse-transcriptase (Applied Biosystems, Foster City, CA). Gene specific primer pairs for transcript variants, and for transcripts of ribosomal protein L7 (and variants amplified by each primer pair are demonstrated in.