Activation regimens for full‐term development of rabbit oocytes injected with round spermatids

Abstract

The present study was designed to investigate the effect of activation regimens on full-term development of rabbit oocytes after round spermatid injection (ROSI). In the first series, rabbit oocytes were treated with 5 microM ionomycin before ROSI, after ROSI, or before and after ROSI. In addition, non-treated oocytes were subjected to intracytoplasmic sperm injection (ICSI) using ejaculated spermatozoa. Cleavage rate of ROSI oocytes activated before and after ROSI (55%) was comparable with that of ICSI oocytes (60%), and significantly higher than those of ROSI oocytes activated either before or after ROSI (29-39%; P < 0.05). No offspring were produced by transfer of the cleaving ROSI oocytes, while 8% of the cleaving ICSI oocytes transferred gave birth to offspring. In the second series, oocytes were exposed to 5, 10, or 20 microM ionomycin, followed by ROSI, 5 microM ionomycin treatment, and incubation with 5 microg/ml cycloheximide (CHX) + 2 mM 6-dimethylaminopurine (DMAP). Significantly higher cleavage rates were derived from oocytes activated with 10 and 20 microM ionomycin before ROSI (91% and 82%, respectively; P < 0.05) compared to those activated with 5 microM ionomycin before ROSI (53%). Live offspring were obtained when the cleaving ROSI oocytes with the initial ionomycin treatment at 5 and 10 microM were transferred (offspring rate 2% and 4%, respectively). These activation regimens, however, were not valid for the ROSI using cryopreserved round spermatids. In conclusion, rabbit ROSI oocytes were capable of developing into full-term when the oocytes were activated with a combined treatment of ionomycin and CHX/DMAP.

Molecular Cloning and Characterization of Phospholipase C Zeta in Equine Sperm and Testis Reveals Species-Specific Differences in Expression of Catalytically Active Protein1

“…For instance, in the mouse, rabbit, rat, and pig, round spermatids do not yet express PLCZ [15,[34][35][36][37][38][39]47], because the expression of the protein is first evident at the elongated spermatid stage [15,34,36]. Whereas in the mouse normal offspring have been produced by the injection of these round cells, an additional activation mechanism is necessary to initiate [Ca 2þ ] i rises and, thus, the embryonic development program [34,36].…”

“…Indirect evidence suggests interspecies differences based upon injection of round spermatids into mouse oocytes and their ability (or inability) to trigger [Ca 2þ ] i oscillations and/or oocyte activation [34][35][36][37][38][39]. Immunohistochemistry was performed to characterize the ontogeny of PLCZ protein expression in equine testis tissue Values are mean 6 SEM.…”

Derivation of Embryonic Stem Cell Lines from Parthenogenetically Developing Rat Blastocysts

“…The oocytes were chemically activated by incubation with 5 mM ionomycin (SigmaAldrich) for 5 min and 5 mg/mL cycloheximide + 2 mM 6-dimethylaminopurine (Sigma-Aldrich) for 4 h at 37°C with 5% CO 2 as described previously [15,16] with slight modifications. At 6 h after activation, parthenogenetic zygotes with two visible pronuclei were transferred into the oviducts of recipient rats that had been previously mated with a vasectomized male rat.…”

Germ cells and fertilization: why I studied these topics and what I learned along the path of my study

“…The centrosome in round spermatids may be too 'immature' to serve as the centre of microtubular aster formation necessary for orderly movement of spermatozoa and egg chromosome (Tachibana et al, 2009). However, full term foetuses were obtained by treating eggs (rabbit) with ionomycine before and after round spermatid injection (Hirabayashi et al, 2009). I do not believe in 'exception'.…”