title: Generation of live rats produced by in vitro fertilization using cryopreserved spermatozoa.
abstract: In rats, the success of in vitro fertilization (IVF) was reported 40 years ago. Although it has 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 to date. Here, we report establishment of a successful IVF system using frozen/thawed rat spermatozoa. Our data showed that intracellular cAMP and free cholesterol levels in frozen/thawed rat sperm were maintained low, suppressing capacitation-associated tyrosine phosphorylation. The treatment of methyl-beta-cyclodextrin improved removal of free cholesterol from the membrane in frozen/thawed sperm but not induction of capacitation-associated tyrosine phosphorylation in the sperm. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), dramatically increased cAMP and tyrosine phosphorylation levels in frozen/thawed rat sperm. When the IBMX-treated frozen/thawed sperm were used for IVF, the proportions of pronuclear formation and blastocyst formation were significantly higher than those of frozen/thawed sperm treated without IBMX (P < 0.05). The embryos were developed to term at a high success rate equivalent to the rate obtained with IVF using fresh sperm. Thus, we established for the first time a successful IVF system in rats using cryopreserved spermatozoa.
authors.affiliation: Laboratory of Animal Reproduction, Graduate School of Veterinary Science, Azabu University, Sagamihara, Japan.
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Seita
authors.forename: Shuhei
authors.initials: S
authors.lastname: Sugio
authors.forename: Junya
authors.initials: J
authors.lastname: Ito
authors.forename: Naomi
authors.initials: N
authors.lastname: Kashiwazaki
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D005298:Fertility / Q000502:physiology*; D005307:Fertilization in Vitro / Q000379:methods*; D008297:Male; D023421:Models, Animal; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D010726:Phosphodiesterase Inhibitors; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine