Elsevier

Cryobiology

Volume 60, Issue 2, April 2010, Pages 129-137
Cryobiology

Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation

https://doi.org/10.1016/j.cryobiol.2009.10.003Get rights and content

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 (Me2SO) 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.

Introduction

The number of genetically modified mouse lines has rapidly expanded in the last decade and has become a challenge to mouse facilities in terms of cost and space to maintain the lines by breeding. Lines that have been published should be available for other researchers, so they cannot be terminated shortly after completion of a project. Moreover, important lines can be lost due to disease, genetic drift, microbiological or genetic contamination, impaired reproductive performance, or a large-scale environmental disaster such as fire or flood [23]. Therefore, preservation of germ cells has been applied more and more to maintain or propagate these increasing mutant mouse lines.

Cryopreservation of mouse embryos is now state of the art and thousands of mouse lines have been banked as frozen embryos, in centralized repositories or research institutes around the world [15], [19], [30]. However, quite a large number of animals and a substantial amount of labor are required to provide sufficient embryos to safeguard a line, rendering cryopreservation of mouse embryos an expensive and time consuming method to preserve lines. Superovulation and fertilization, either in vivo or in vitro, are mandatory steps prior to starting freezing sessions, and multiple freezing sessions are needed to acquire a frozen stock of sufficient size (particularly when also storage at other institutions is considered). Therefore, the method is not suitable for crisis intervention or rescuing a “last-of-line” mouse [15], [30]. Cryopreservation of sperm would be a suitable approach, as the freezing procedures are rather simple and do not require prior breeding of animals. Large numbers of sperm cells can be collected from even one male, which makes this an attractive preservation approach, even to rescue a line. Although cryopreservation of sperm has been applied to preserve many mutant mouse lines, obstacles remained for years in retrieving genetically modified offspring from frozen sperm of common genetic background strains such as C57BL/6, BALB/c or 129 by in vitro fertilization [30], [38], [54], [57]. In vitro fertilization (IVF) by co-incubation of frozen–thawed mutant sperm with congenic oocytes was very susceptible to failure until recently improved sperm cryopreservation and IVF procedures provided acceptable IVF results [42]. However, the intricate logistics for superovulating oocyte donors and synchronizing recipients make recovering offspring using frozen sperm more expensive and time consuming than recovering frozen embryos. Ovary cryopreservation could be a valid alternative for sperm or embryo preservation. Mouse ovarian tissue is easily collected from pre-selected donors and the freezing requires much less preparative work than embryo freezing. Only basic laboratory facilities and few animals are needed for archiving and retrieving a line by ovary cryopreservation since no IVF procedure is required. In addition, validation of frozen ovarian tissue is relatively cheap and simple.

Mouse ovary cryopreservation was first reported by Parrott in 1960 [43], who has obtained live offspring after orthotopic transplantation of sliced frozen mouse ovarian tissue. Fecundity of frozen mature mouse ovaries from different outbred strains was restored by autologous transplantation as live offspring were obtained [18], [21], [48]. Cryopreserved juvenile ovarian tissue from C57BL/6 and few hybrid strains yielded restoration of fertility after grafting thawed ovaries to the ovarian bursa of mature recipients [6], [7], [56], [58]. However, genetic modification is typically done in a limited number of inbred strains (C57BL/6, FVB, 129, BALB/c) that require further development of the method for practical application. Similarly, the study of ovary cryopreservation as a biomedical model depends on these strains to benefit from the wide choice of genetically modified mouse models. Vitrification, avoiding crystal formation by extreme elevation in viscosity of a fluid during rapid cooling, was applied as a method of cryopreservation in preserving biological material [13]. This relatively simple and effective cryopreservation procedure was also successfully applied to mouse ovarian tissue and preserving their fecundity by obtaining live offspring [3], [8], [20], [22], [27], [36], [37], [63]. Cryopreserved juvenile and adult ovaries from two mutant lines have resulted in live offspring after orthotopic transplantation [3], [20], [36], [37]. These reports indicate that cryopreservation of ovarian tissue is a potentially useful technology to preserve mutant mouse lines. However, in most published studies that evaluate freezing methods and the viability of post-thaw ovaries, inbred or hybrid mice were used as ovary donors and recipients. Reports on the application of this technology in different genetically modified mouse lines on congenic backgrounds are lacking. As genotype and genetic background affect the ability of thawed embryos to develop in vivo [11], [15], [30], [44], [49] as well as the viability of thawed sperm [30], [38], [42], [57], the performance of frozen–thawed ovarian tissue may also be hampered. An advantage of cryopreservation of ovaries is that recipient animals that produce mutant offspring may be repeatedly bred. However, the long-term functionality of the grafted thawed ovaries is still unknown.

In the present study, we investigated the application of cryopreservation of juvenile ovarian tissue to different genetically modified mouse lines in three genetic backgrounds, using modified high concentrations of cryoprotectants with an instrumented ultra-rapid freezing method. To confirm the quality of the cryopreserved mutant ovaries, we investigated the fertility of thawed ovary recipients, wild-type but congenic with the ovary donor, which had undergone orthotopic transplantation following natural mating by congenic wild-type males. The recipients were maintained under breeding conditions for several months to evaluate the functionality of the cryopreserved ovaries as a function of time after transplantation.

Section snippets

Animal care and husbandry

Animals were kept and used in compliance with the European guidelines [9] and The Netherlands legislation for the protection of animals used for research, including ethical review. Mice were housed in sterilized 435 cm2 cages (TECNIPLAST Sealsafe™ Individually Ventilated Cage, Italy), with wood chips (Lignocel®, J. RETTENMAIER & SÖHNE GmbH, Rosenberg, Germany) as bedding and paper tissue as nesting material. Room conditions were 19–22 °C and 53–63% relative humidity, and artificial illumination

Fertility of recipients transplanted with frozen–thawed ovaries from different mutant lines

Validation of the frozen ovary stocks was carried out by transplanting thawed mutant ovaries to a total of 111 recipients; four to ten recipients were used for each mutant line. Among the recipients, 96 recipients delivered at least 1 litter (96/111, 86.5%) and 45 recipients delivered genetically modified offspring within their first to fourth litters (45/111, 40.5%). In total, 53 ovary recipients (53/111, 47.7%) delivered only wild-type pups. Table 1 summarizes the reproduction capacity

Discussion

Cryopreservation of ovarian tissue has proven to be a feasible and useful technology to preserve germ cells from mice while preserving the genetic background; its use for genetically modified lines was developed and evaluated only in recent years. The method is easy to plan and manage, and can be done using relatively simple research infrastructure and skills. The collection of tissue from donors can be planned at the time of birth, any material from non-modified donors can be selected later by

Acknowledgments

We thank Dr. Niels Galjart, Dr. Gosia Oklejewicz, Dr. Willy Baarends, Dr. Ingrid van der Pluijm, Dr. Cathy Bakker, Dr. Tatjana Nikolic, Dr. Rudi Hendriks, Prof. Riccardo Fodde, Dr. Claudia Gaspar, Nanda Keijzer, Hanneke Korsten, Ingeborg Nieuwenhuizen (Erasmus University Medical Centre) for providing mutant mouse lines and PCR genotyping; Vincent Vaes, Helen Kock, Iris Janssen, John Mahabier (Erasmus Laboratory Animal Science Centre) for organizing, breeding and care of the animals; Pei-Shiue

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      The proportions of the different categories of normal ovarian follicles in the different experimental groups are shown in Figure 3. Most follicles found in the grafts were primordial or primary, probably because of their low metabolic rates that make them more resistant to post-transplant ischemia [23,24]; in addition, they have a simple morphology [25] and are concentrated in the periphery of the ovarian cortex, and are the first follicles to benefit from revascularization [26]. However, it is still unknown for how long this environment remains because grafts can remain viable for several months [6,7], but complete follicular consumption, with the presence of fibrosis areas where once was ovarian parenchyma, has been observed after 1 year [27].

    This work was funded by Department of Farm Animal Health of Faculty of Veterinary Medicine of Utrecht University and Erasmus Laboratory Animal Science Centre of Erasmus University Medical Centre.

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