Alterations in mitochondrial function and spermatozoal motility in goat spermatozoa following incubation with a human lysozyme plasmid.
Spermatozoa mediated gene transfer has become a promising technology to generate transgenic animals with disease resistance. However, exogenous DNA invasion may cause changes in spermatozoon natural defense system which result in spermatozoon dysfunction.The objective of this study was to investigate the changes of mitochondrial function and motility in goat spermatozoa after pre-incubation and incubation with and without the human lysozyme plasmid pFLAG-hLY. The results demonstrated that human lysozyme plasmid pFLAG-hLY could bind to the surface of the spermatozoon membrane at 186,000 copies/spermatozoa and incorporate to spermatozoon nucleus at 78 copies/spermatozoa after incubation. However, the treated spermatozoon samples showed a significant lower motility (29.7+/-2.2% vs. pre-incubation control 48.0+/-1.4% and incubation control 54.5+/-1.5%, P < 0.05), and percentage of rapid progressive motile spermatozoa(16.4+/-2.4% vs. pre-incubation control 31.4+/-0.6% and incubation control 37.0+/-0.5%,P < 0.01). Meanwhile, the incubation with plasmids caused significant reduction of mitochondrial membrane potential (31.44+/-2.17% vs. pre-incubation control 51.79+/-2.08% and incubation control 58.81+/-1.76%, P < 0.05). In addition, dichlorofluorescin relative fluorescence intensity (32.81+/-2.41%) and malondialdehyde levels (2.18+/-0.21 nM/108 spermatozoa), which represents mitochondrial function, showed a significant increase after incubation (P < 0.05). The cytochrome c release from the mitochondrial inner membrane space, and enzymatic activities of caspase-3 (0.086+/-0.024) and caspase-9 (0.083+/-0.019)(P < 0.05) also increased, in which resulted in spermatozoon dysfunction. In conclusion, this study confirmed that goat spermatozoa could capture human lysozyme plasmid pFLAG-hLY,but the incubation with the plasmids resulted in a decrease of spermatozoa motility and partial rupture of mitochondrial membrane, and further prompted the expression of cytochrome c, and generation of oxidative stress in vitro and finally led to spermatozoon dysfunction.
Ma
Huiming
H
College of Veterinary Medicine, Key Laboratory of Animal Reproductive Physiology & Embryo Technology of Ministry of Agriculture, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi Province, PR China.
Quan
Fusheng
F
Chen
Dongmei
D
Zhang
Bowei
B
Zhang
Yong
Y
D000818:Animals; D018014:Gene Transfer Techniques; D006041:Goats / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D009113:Muramidase / Q000235:genetics* / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010957:Plasmids / Q000494:pharmacology*; D013081:Sperm Motility / Q000187:drug effects* / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology
D008315:Malondialdehyde; D009113:Muramidase