Detection Of Human Lactate Dehydrogenase C4 Gene Mutation And Its Role In Pathogenesis Of Male Infertility

Lactate dehydrogenase-C4(LDH-C4) is a specific isoenzyme of the LDH family, which presents only in mature mammalian testis and spermatozoa, and plays a pivotal role in spermatic energy metabolism. It is also implicated in sperm motility, capacitation and sperm-egg fusion. Abnormal LDH-C4 function or activity is associated with infertility.In this study, we modified 2-hydroxyvaleric acid based method, a histochemical staining technique for detecting LDH-C4 activity in human spermatozoa, in which 2-hydroxyvaleric acid was used as LDH-C4 specific substrate, and a normal reference range for enzyme activity was constructed. In terms of the plotted ROC curve, this method yielded a sensitivity of 89.5% and specificity of 91.2%. Another novel 2-hydroxybutyrate based method for measuring LDH-C4 activity was developed as well. With the staining method, we detected a group of patients with unexplained infertile status, aiming at finding Ldhc gene mutation, which would be further identified by T cloning technique. As described in the results, a Tâ†'G heterozygous mutation located on the 40 th base of exon 5 was identified. The mutation resulted in an alteration of the TTG codon(encoding leucine) to TAG(a stop codon), and therefore named as L153 X mutation.In order to know the mechanism underlying the process in which L153 X mutation led to male sterility, we constructed the wild prokaryotic and eukaryotic recombinant vectors of Ldhc through molecular cloning, and obtained L153 X recombinant vectors via the artificial mutagenesis technique. The effects of homo- or heterozygous mutation on LDH-C4 activity as well as the interaction between wild and mutant C subunit, were simulated or evaluated by induced fusion-protein expression in bacteria, and He La cell transfection. The results revealed that homo- or heterozygous mutation of L153 X was a culprit for the loss or reduction of LDH-C4 activity in spermatozoa. Simultaneously, the data also provide evidence that the wild C subunit might intact with the mutant C subunit. The study employed computational algorithms to depict the three-dimensional computer model of L153 X mutant and made predictions upon its possible structure and function. The results showed that L153 X mutant led to a loss of all the amino acid residues positioned from 153 to 332, as well as many enzyme active and substrate binding sites, suggesting that the truncated polypeptides were unable to further polymerize due to the missing large parts of protein functional sitesThe study further evaluated the effects of L153 X mutation on spermatic energy metabolism, motility and function by utilizing a series of analyses as energy metabolism assay, CASA and capacitation test. We finally found that L153 X mutated spermatozoa yielded abnormal energy metabolism and impaired capacitation.The current study may provide the direct evidence for the hypothesis that Ldhc gene mutation is responsible for the causes of infertility, and also provide a new etiological explanation for male sterility from a molecular and genetic perspective. Moreover, the novel 2-hydroxybutyrate based method for measuring sperm LDH-C4 activity has the potential of wide application in the laboratory.