Construction And Characterization Of Prokaryotic And Eukaryotic Vectors For Expression Of L153X Mutant Of Human Sperm-specific Lactate Dehydrogenase

Lactate dehydrogenase C4(LDH-C4)also known as sperm-specific or testis-specific lactate dehydrogenase is one of the lactate dehydrogenase isoenzymes identified by Goldberg and Blanco et al in 1960s.Expression of LDH-C4 reveals high tissue-specificity,which only presents in the reproductive system of mature male mammalian and birds.LDH-C4 catalyzes the reversible reaction between pyruvate and lactate and considered as a key enzyme in sperm energy production and relevant to spermatogenesis,metabolism and capactation.A T?G heterozygous mutation located on the 40th base of exon 5 of LDHC gene was identified in the fomer research about male infertility and LDHC gene mutation.The mutation resulted in an alteration of the TTA codon(encoding leucine)to TGA(a stop codon),and named as L153X mutation.Based on the early research,the mutant escapes nonsense-mediated mRN A degradation and generates a dominant negative L153X truncated peptide resulting in abnormal LDH-C4 activity.To clarify the underlying mechanism of male infertility caused by human LDHC heterozygous mutation,the prokaryotic expression vector pET-28a(+)-L153X was constructed to provide evidence for the hypothesis that the nonsense gene mutation of LDHC is responsible for the reduction or loss of human LDH-C4 activity in spermatozoa.The eukaryotic expression vectors pVAX1-LDHC and pVAX1-L153X were also constructed,and the subunit composition of individuals with homozygous wild type LDHC gene or LDHC gene with heterozygous or homozygous L153X mutant,was simulated by the TNT T7 rapid coupling in vitro transcription/translation system based on rabbit reticulocyte lysate.Detecting the LDH-C4 activity expressed in vitro,combining with the previous molecular biology predictions,the interactions between wild-type C subunits and mutant C subunits were analysed.The study provides a direct evidence for the hypothesis that LDHC gene mutation is responsible for male infertility from molecular and genetic perspective and also a basis for hypothesis attributing the dysfunction of LDH-C4 to the LDHC gene mutation.