The FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is essential for the transport of reducing equivalents derived from glycolysis (or in sperm fructolysis) into the mitochondrial compartment for the synthesis of ATP. It has been shown that multiple (three) promoters of the mGPDH exist so that the expression is regulated in a tissue-restricted manner. Promoter A is expressed in brain, brown adipose tissue and pancreas, promoter B is ubiquitous and promoter C is proved to be a testis-specific one.
To evaluate if potential defects in the testis-specific promoter of the mGPDH gene could contribute to reduced metabolic activity in sperm followed by infertility, we investigated a pattern of gene expression and regulation in testis.
In the present study, nonradioactive in situ mRNA hybridization and immunohistochemistry were performed to determine the cellular localisation of mGPDH mRNA and protein in rat testes. First mGPHD transcripts are detected in stage 2 of round spermatids with intense specific staining till early-elongating spermatids (step 11). The specific binding between the mGPDH protein and polyclonal antibodies shown in immunochistochemitry confirmed delayed translation of the protein, with the first signals being detectable in the mature spermatozoa (steps 16 to 19). We next characterised the 5'end of mGPDH mRNA from rat testis using 5'-RACE (Rapid amplification of cDNA ends), which amplifies only full-length transcripts. The series of 5'-RACE clones obtained with the use of testis-specific primer were sequenced and analysed using computer software analysis. We also tried to investigate the promoter C activity by series of Transient Transfections in HepG2 (hepatoma cells), primary hepatocytes and TM3 (Leydig) cell lines.
Our results indicate that mGPDH mRNA expression within testis is distinctly germ cell-specific and stage-specific with the delayed translation of the protein that is explained with increasing metabolic activity in developing spermatozoa.
03 - 04 Dec 2001
Society for Endocrinology