title: Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation.
abstract: The rapid expansion of mutant mouse colonies for biomedical research has resulted in lack of space at laboratory animal facilities and increasing risks of losing precious lines. These challenges require cheap and effective methods in addition to freezing embryos and sperm to archive the expanding mutant mouse lines. Cryopreservation of mouse ovarian tissue has been reported, but the application in the diverse mutant lines and genetic backgrounds has not yet been studied. In this study, juvenile ovaries (10-day-old) collected from genetically modified mouse lines were cryopreserved using high concentrations of cryoprotectants (dimethyl sulfoxide (Me(2)SO) and ethylene glycol (EG)) and instrumented ultra-rapid freezing. The validation of the frozen ovary batches was assessed by orthotopically transplanting a thawed ovary into a nearly completely ovariectomized mature female (congenic with the ovary donor). After 2 weeks of recovery, the ovary recipient was continuously paired with a male (congenic with the ovary donor) to evaluate the fertility of the recipient and delivered offspring were genotyped to evaluate the continued functionality of the grafted ovary. The recipient females delivered genetically modified offspring starting 6 weeks after ovary transplantation and lasting up to 6 months. The presented cryopreservation and transplantation protocols enabled retrieval of the genetic modification in 20 (from 22) genetically modified mutant mouse models on a C57BL/6 (17), FVB (2), or BALB/c (1) background. The thawed ovaries functioned after successful orthotopic allotransplantation to congenic wild-type recipients and produced mutant offspring, which allowed recreation of the desired genotype as a heterozygote on the proper genetic background. The results indicate that cryopreservation of mouse ovaries is a promising method to preserve genetic modification of the increasing number of mutant mouse models and can be used as a model for ovary cryopreservation using a variety of mouse mutants.
authors.affiliation: Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Kuo-Yu
authors.initials: KY
authors.lastname: Huang
authors.forename: Suzanna A
authors.initials: SA
authors.lastname: de Groot
authors.forename: Henri
authors.initials: H
authors.lastname: Woelders
authors.forename: Gijsbertus T J
authors.initials: GT
authors.lastname: van der Horst
authors.forename: Axel P N
authors.initials: AP
authors.lastname: Themmen
authors.forename: Ben
authors.initials: B
authors.lastname: Colenbrander
authors.forename: J Martje Fentener
authors.initials: JM
authors.lastname: van Vlissingen
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D005260:Female; D005298:Fertility; D006085:Graft Survival; D051379:Mice; D020297:Mice, Congenic; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D010053:Ovary / Q000502:physiology / Q000637:transplantation; D011247:Pregnancy; D013045:Species Specificity; D014184:Transplantation, Homologous
chemical_list: D003451:Cryoprotective Agents; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide