Spermatogenic cell-specific type 1 hexokinase is the predominant hexokinase in sperm

Hexokinase is the first enzyme in the glycolytic pathway and utilizes ATP to convert glucose to glucose-6-phosphate (G6P). We previously identified three variant transcripts of Hk1 that are expressed specifically in spermatogenic cells, have different 5' untranslated regions, and encode a protein (HK1S, spermatogenic cell-specific type 1 hexokinase) in which the porin-binding domain (PBD) of HK1 is replaced by a novel N-terminal spermatogenic cell-specific region (SSR). However, the level of expression of the individual variant transcripts or of the other members of the hexokinase gene family (Hk2, Hk3, and Gck) in spermatogenic cells remains uncertain. We show that Hk1, Hk2, and Hk3 transcripts levels are quite low in spermatocytes and spermatids and Gck transcripts are relatively abundant in spermatids, but that glucokinase (GCK) is not detected in spermatozoa. Using real time RT-PCR (qPCR) with primers specific for each of the three variant forms and RNA from whole testis and isolated germ cells, we found that transcripts for Hk1_v2 and Hk1_v3, but not for Hk1_v1, are relatively high in spermatids. Similar results were seen using spermatogenic cells isolated by laser-capture microdissection (LCM). Immunoblotting studies found that HK1S is abundant in sperm, and immunostaining confirmed that HK1S is located mainly in the principal piece of the sperm flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found. These results strongly suggest that HK1, HK2, HK3, and GCK are unlikely to have a role in glycolysis in sperm and that HK1S encoded by Hk1_v2 and Hk1_v3 serves this role.