Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs.
We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP alphaT). No differences were observed in COMP alphaT values among GSH-treated and control groups. COMP alphaT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.
Cheng
Wen-Min
WM
Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences and Technology, China Agricultural University, Beijing.
An
Lei
L
Wu
Zhong-Hong
ZH
Zhu
Yu-Bo
YB
Liu
Jing-Hao
JH
Gao
Hong-Mei
HM
Li
Xi-He
XH
Zheng
Shi-Jun
SJ
Chen
Dong-Bao
DB
Tian
Jian-Hui
JH
D000818:Animals; D002843:Chromatin / Q000187:drug effects*; D004229:Dithiothreitol / Q000494:pharmacology*; D047108:Embryonic Development; D005260:Female; D005978:Glutathione / Q000494:pharmacology*; D008297:Male; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000187:drug effects*; D013552:Swine
D002843:Chromatin; D005978:Glutathione; D004229:Dithiothreitol