ブタ凍結乾燥精子の特性およびその精子からの産子作製法に関する研究

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Successful offspring production from freeze-dried (FD) sperm has been reported in laboratory animals but not in pigs. The integrity of the DNA in the FD sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in FD sperm, and on the in vitro and in vivo development of porcine oocytes. Boar spermatozoa were sonicated and suspended in buffer supplemented with 1) 50mM EGTA, 2) 50mM EDTA, 3) 10mM EDTA, or 4) no chelating agent. A fertilization medium (Pig-FM) was used as a control. Spermatozoa were put into a glass ampoule, precooled at - 40℃ for 6h, and lyophilized for 6h. The ampoules were stored at4℃ and the contents rehydrated with distilled water. The rehydrated spermatozoa were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed. However, the rates in all experimental groups did not increase, even at 180 min. These rates were all significantly lower (P<0.05) than that of the control group. Sperm heads from all experimental and control groups were incubated for 0-60, 60-120, or 120-180 min and then injected into in vitro-matured porcine oocytes. The rate of blastocyst formation in the control group was significantly lower (P<0.05) than those in the 50-mM EGTA and 10-mM EDTA groups incubated for 120-180 min. Finally, we transferred oocytes injected sperm from 50-mM EGTA or control groups incubated for 0-60 min into recipients. One of the two recipients of the 50-mM EGTA oocytes became pregnant but aborted on day 29 after transfer. The two recipients of the control oocytes became pregnant, and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in FD boar sperm is one of the causes of decreased in vitro development of injected oocytes to the blastocyst stage. Supplementation with 50mM EGTA in a freeze-drying buffer improves this ability. Furthermore, porcine oocytes injected with FD sperm heads were able to grow to 39-day fetuses.
麻布大学の論文

ブタ凍結乾燥精子の特性およびその精子からの産子作製法に関する研究

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