A Novel Mouse Enolase 1-like Gene is Expressed Specifically in Spermatogenic Cells and Encodes a Protein Present in the Principal Piece Region of the Sperm Flagellum.

Abstract

The main source of ATP for sperm motility in the mouse is glycolysis and several spermatogenic cell-specific glycolytic enzymes are confined to the principal piece region of the flagellum. During a proteomics study using MALDI/MS to analyze non-ionic detergent-soluble sperm proteins, peptide sequences similar to those present in the glycolytic enzyme enolase 1 (alpha enolase) were identified. Other investigators reported that an enolase sperm-specific isozyme (ENOS) is present in the flagellum of spermatozoa of several mammalian species that is unlike other enolase family members (ENO1, ENO2 and ENO3) in electrophoretic mobility and enzymatic properties. However, the peptide and nucleotide sequences of this novel enolase have not been reported. Database mining identified multiple Eno1-like sequences in the mouse genome, but EST expression profiles and deduced protein sequence analyses strongly suggested that a gene located on chromosome 19 is the Enos gene. The use of PCR and 5 RACE determined that there are two Enos transcript variants expressed in mouse testis (Enos_v1 and Enos_v2), each with a 5 sequence different from those present in transcripts of Eno1 or of other enolase family members. Northern analysis using a probe from the 3′ region of the Enos transcript recognized mRNA only from testis. Northern analysis of RNA from testes of 10- to 35-day-old juvenile mice first detected Enos transcripts on day 14, corresponding to when pachytene spermatocytes appear in the first wave of spermatogenesis. In immunoblotting assays, a pan-enolase antibody reacted with a protein in 1 % Triton X-100, 1 % NP-40 and 0.1 % SDS lysates of testes and sperm, while enolase 2 or 3 were not detected in lysates of testis and sperm with antibodies specific to these enzymes. Immunofluorescence microscopy with this antibody recognized a protein localized to the principal piece region of sperm flagellum, strongly suggesting that the protein recognized by the pan-enolase antibody is ENOS. Extraction of sperm with NP-40 and then 6M urea and immunoblotting assays determined that ENOS is partially extracted by NP-40 and the remainder is extracted by 6M urea. These results suggest that ENOS is not an integral protein of the fibrous sheath like GAPDHS, AKAP4 and GSTM5, but is loosely associated like HK1S and PFKM. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.