279 IMPROVEMENT OF RAT IN VITRO FERTILIZATION PROTOCOL USING CRYOPRESERVED SPERM AND ITS APPLICATION TO OTHER STRAINS: A PRELIMINARY STUDY

Abstract

In rats, the success of in vitro fertilization (IVF) was reported almost 40 years ago. Although it had been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported. Very recently, we reported establishment of a successful IVF system using frozen-thawed spermatozoa treated with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), in Wistar rats (Seita et al. 2009 Biol. Reprod.). The objectives in this study were (1) improvement of the IVF system to a more convenient and simple protocol and (2) a preliminary study for applying our IVF system to inbred rat strains Fischer 344 (F344) and Brown-Norway (BN). In experiment 1, we examined the effect of preincubation time for frozen-thawed sperm on fertilization (2 pronuclei formation). Frozen-thawed sperm were preincubated up to 6 h and then used for IVF according to our previous report. Data showed that sperm preincubated for 5 h contributed to higher fertility than those for other preincubation times. In experiment 2, we examined the effect of co-culture time up to 10 h for IVF on fertility and embryonic development in vitro. The oocytes co-cultured with sperm beyond 6 h showed higher fertilization and blastocyst formation rates than those in 2 and 4 h. In experiment 3, we examined the effect of initial culture period in fertilization medium (310 mOsm modified R1ECM; O h et al. 1998 Biol. Reprod.) on embryonic development in vitro. After IVF, oocytes were cultured in fertilization medium for 6, 12, or 24 h and further cultured in R1ECM up to 120 h. Cleavage rates were not affected by initial culture time in fertilization medium. However, oocytes cultured in fertilization medium for 12 h showed higher blastocyst formation rate than those for 0, 6 and 24 h. In experiment 4, we examined whether the IVF protocol can be applied to F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Our data demonstrate that (1) a more efficient IVF system using frozen-thawed rat sperm was developed and (2) the IVF system can be applied to at least F344 strain. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.