TESTICULAR SPERMATOGENESIS, MORPHOGENESIS, AND GENE EXPRESSION
7:30 AM-10:00 AM
(483) ELUCIDATION OF THE POTENTIAL MECHANISM FOR THE FORMATION OF 32-KILODALTON SPERMINOGEN FROM PROACROSIN IN BOAR SPERMATOZOA.
Yi, Lee1, Pyoung, Il Sun1, 1 Dept of Biological Science, Suwon, KR
ABSTRACT- The potential mechanism(s) to produce 32 kDa boar sperminogen has been investigated by RT-PCR, Northern blot and Western blot analyses. The novelty of sperminogen, originally discovered from the acid extracts of human sperm as a novel protease, has been the subject of controversy. Composed of three proteolytic protein bands, the molecular masses of sperminogen were estimated to be in the 32 to 36 kDa range. Previously, the lowest molecular mass sperminogen, 32 kDa sperminogen, was characterized as part of the proacrosin/acrosin system from peptide sequence analysis. However, the mechanism(s) for the formation of 32 kDa sperminogen--e.g., by differential splicing of the precursor to proacrosin mRNA or posttranslational modification of preproacrosin--has not been elucidated. When RT-PCR was performed using the primer deduced from the known amino acid sequence of 32 kDa sperminogen, a single band was amplified at around 1 Kbp, the same length which is expected when amplified from proacrosin mRNA. If the 32 kDa sperminogen is the product of differential splicing of proacrosin mRNA, then more than one band will be detected when screened with the partial DNA sequences of the proacrosin gene. Therefore, we have attempted northern blot analysis of boar testicular mRNA with 4 different DNA probes, each of which corresponds to the exon of the boar proacrosin genomic sequence. Northern blot analysis demonstrated that all 4 probes detected only one species of mRNA signifying that 32 kDa sperminogen is not produced by differential splicing of the precursor to proacrosin mRNA. We have also tested the possibility that 32 kDa sperminogen might be produced by the random breakdown of proacrosin during the preparation of the acid extracts of boar spermatozoa. Proacrosin (53-55 kDa) was purified from the acid extracts of boar spermatozoa by SDS-PAGE and electroelution and allowed to autoactivate in vitro. When this sample was analyzed by Western blot analysis, the monospecific polyclonal 32 kDa sperminogen antibody did not detect any protein band which corresponds to 32 kDa sperminogen. This strongly implies that 32 kDa sperminogen is not produced as a breakdown by-product during the preparation steps; rather, it is produced by specific posttranslational modification mechanism(s) in the male germ cells during the formation of spermatozoa.
KEY WORDS: spermatozoa, sperminogen, proacrosin