155 ESTABLISHMENT OF AN IN VITRO FERTILIZATION PROTOCOL USING CRYOPRESERVED EPIDIDYMAL AND EJACULATED RAT SPERMATOZOA

Abstract

In rats, successful IVF with cryopreserved rat sperm has never been reported. The objective of the present study was to establish and improve the IVF protocol using epididymal and ejaculated rat spermatozoa after cryopreservation. At first, we examined whether cryopreserved ejaculated spermatozoa would be useful for IVF in Wistar rats. Capacitation-associated tyrosine phosphorylation was accelerated in frozen-thawed ejaculated sperm in a time-dependent manner. These frozen-thawed spermatozoa were co-cultured with cumulus–oocyte complexes in modified R1ECM for 10 h. The putative zygotes were transferred to R1ECM and then cultured up to 144 h. Although the rates of insemination and 2 pronucleus (2PN) formation were low (26.5 and 23.0%, respectively), most of 2PN oocytes were developed to the 2-cell stage (91.0%). A total of 44 embryos at the 2-cell stage derived from frozen-thawed ejaculated sperm were transferred to 5 recipient females and 21 pups (47.7%) were delivered. Next, we used frozen-thawed epididymal spermatozoa for IVF in Wistar rats. After thawing, intracellular cyclic adenosine monophosphate (cAMP), free cholesterol levels and capacitation-associated protein tyrosine phosphorylation levels of the sperm were assayed. Intracellular cAMP and free cholesterol levels in frozen-thawed epididymal sperm were maintained at a low level, suppressing capacitation-associated tyrosine phosphorylation. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX) dramatically increased cAMP and capacitation-associated tyrosine phosphorylation levels in frozen-thawed epididymal sperm. When the IBMX-treated frozen-thawed sperm was used for IVF, the proportions of 2PN and the development to blastocysts were significantly higher (approximately 40 and 20%, respectively) than those of frozen-thawed epididymal sperm treated without IBMX (approximately 10 and 3%, respectively). These embryos were developed to term at a high success rate (49%) equivalent to the rate obtained with IVF using fresh sperm (58%). Moreover, we tried to apply our IVF system to inbred rat strains [Fischer 344 (F344) and Brown-Norway (BN)]. We examined whether the IVF protocol was available for 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, capacitation-associated 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. Taken together, we developed an IVF protocol using cryopreserved rat sperm and our data suggest that the IVF system can be applied not only to Wistar rats but also to the F344 strain.

This research was also partially supported by a research project grant awarded by the Azabu University Research Services Division.