Successful Microinsemination of Rabbit Oocytes with Round Spermatids.

Abstract

Although the first report for rabbit offspring by round spermatid injection (ROSI) has already been published in 1994, the production of rabbit ROSI offspring is considered difficult due to the high incidence of chromosomal aberrations. The ROSI oocytes need to be activated artificially because sperm-borne oocyte-activating factor, SOAF, is present in elongating spermatids and spermatozoa but not round spermatids, and metaphase-promoting factor, MPF, in the oocytes causes premature chromosome condensation in injected spermatid nuclei. The present study was designed to investigate the effect of oocyte activation regimens on developmental ability of rabbit ROSI oocytes. Round spermatid cells were isolated from seminiferous tubules of Japanese White rabbits (Kbs:JW) with proven fertility and resuspended in GL-PBS (PBS supplemented with 5.6 mM glucose, 5.4 mM sodium lactate and 0.01% PVP). Oocytes were recovered from oviductal ampullae of Kbs:JW females superovulated with 3 AU FSH and 75 IU hCG, and were freed from cumulus cell layers. An individual round spermatid was introduced into the denuded oocyte using a piezo-driven micromanipulator. For activation regimens, oocytes were subjected to 5-min treatment with 5-10 μM ionomycin in RPMI1640/DMEM (RD) medium 50 min before ROSI (Group-1; n=108) or 10 min after ROSI (Group-2; n=87), 50 min before and 10 min after ROSI (Group-3; n=82), or 50 min before and 10 min after ROSI followed by incubation for 1 h in the RD medium and subsequent 1 h in the RD medium supplemented with 5 mg/ml cycloheximide and 2 mM 6-dimethylaminopurine (Group-4; n=131). All activation treatments were performed at 38.5°C. Oocytes microinjected with ejaculated spermatozoa (intracytoplasmic sperm injection; ICSI) were served as controls (Group-5; n=163). The ROSI/ICSI oocytes were cultured in TCM199+20% fetal bovine serum for 22-24 h at 38.5°C in 5% CO2 in air. Oocytes cleaving to the 2-8 cell stage were selected for oviductal transfer to recipient rabbits. The activation regimens influenced cleavage rate of microinseminated oocytes (39, 29, 51, 68 and 60% in the Group-1, -2, -3, -4 and -5, respectively). The cleavage rate of ROSI oocytes in Group-4 (68%) was not significantly different from that of ICSI oocytes (60%), but higher than those of ROSI oocytes in the Group-1 (39%) and Group-2 (29%). However, proportions of advanced 4-8 cell-stage embryos among the cleaved ROSI embryos (45, 36, 43 and 52% in the Group-1, -2, -3 and -4, respectively) were significantly lower than that of ICSI embryos (67% in the Group-5). Caesarean section of recipient rabbits 4 weeks after the embryo transfer showed the presence of implantation signs in the Group-3 (2%) and Group-4 (9%), and full-term development only in the Group-4 (7%). The control ICSI (Group-5) resulted in implantation signs at 4% and full-term development at 8%. All the full-term offspring derived from the ROSI (n=6) / ICSI (n=8) appeared to be normal. In conclusion, rabbit ROSI oocytes were capable of developing into full-term when the oocytes were activated with a combined treatment of ionomycin and cycloheximide + 6-dimethylaminopurine.