The Reseach On Alternative Splicing Of Testis-specific Lactate Dehydrogenase C Gene In Testes Of Mammals

Lactate dehydrogenaseC (LDH-C) catalyzes the interconversion of pyruvate andL-lactate，which plays crucial roles in spermatogenesis and capcitation by involving incellular energy metabolism. RT-PCR was employed to clone the open reading frame ofldhc from testes of mammals including pig, dog, rabbit, cat, rat and mouse as well aspigeon，and two to six kinds of variants of ldhc were found in the seven species as aresult of deletion of one or more exons or insertion of partial sequence of an intron in themature mRNA. The deleted exons occur mostly in exon5,4,6and3. The insertion ofpartial sequence of introns, which resulted in abnormal stop codon in inserted intronsequence, was observed only in dog and rat; the deletion of exons resulted in readingframe shift and formation of stop codon in some variants.No splice variants wereobserved for either ldha or ldhb gene in testes and skeletal muscles of adult yak in ourpresent experiment.Native polyacrylamide gel electrophoresis and Western blot analysisrevealed no LDH-C4protein activity derived from expressed ldhc variants.Ldhc genes from the testes of yak and cattle-yak were cloned，and the analysis ofsequencing results revealed there emerged a total of eight splice variants of ldhc as aresult of deletion of one or more exons in the mature mRNA，and more ldhc transcriptswere spliced in the testes of sexually immature yak calf or sterile male hybrid cattle-yakthan those of adult yak. The deletion of exons alike resulted in reading frame shift andformation of stop codon in some variants. Quantitative real-time RT-PCR analysis usingldhc variant-specific primers showed the mRNA level of full length ldhc decreaseddramatically in the testes of yak calf and cattle-yak compared to that of adult yak. Therelative expressions of the ldhc variants assayed differed significantly among adult yak,yak calf and cattle-yak.Our results suggest that the widespread and unique existence of alternative splicingof ldhc gene in testes of mammals. In terms of the differences of mRNA levels andrelative percentages of ldhc splice variants in normal and immature/abnormal testes of yaks and cattle-yaks,we guessed the alternative splicing might play a role in theregulation of ldhc expression in testes of yak and cattle-yak.