156 ENZYMATIC DISSOCIATION AND VITRIFICATION OF PREANTRAL FOLLICLES FROM THREATENED CARNIVOROUS MARSUPIALS

Abstract

The mammalian ovary contains a rich resource of immature preantral follicles present regardless of cycle stage or seasonality. These follicles can be harvested enzymatically and are preserved more successfully than larger, more mature follicles. Thus, the collection and storage of preantral follicles is an applicable technique for the opportunistic harvesting of ovarian tissue from wildlife for the purpose of genebanking. This study examined the potential of such tools for the conservation of two endangered Australian carnivorous marsupials (dasyurids): the northern quoll, Dasyurus hallucatus, and the Tasmanian devil, Sarcophilus harrisii. The fat-tailed dunnart, Sminthopsis crassicaudata, is a representative model dasyurid with the typical attributes of large gametes (220 μm oocytes) and short polyovular reproductive cycles. Ovarian tissue was dissociated with 0.05–0.1% collagenase and 0.01–0.02% DNase in DMEM (all Sigma-Aldrich, St. Louis, USA) for up to 30 min before being neutralized with DMEM +10% fetal calf serum (FCS, Trace Biosciences, Castle Hill, Australia). Survival was determined using membrane permeability stains targeted to the nucleus (SYBR14 and propidium iodide, Molecular Probes, Carlsbad, CA, USA) or cytoplasm (trypan blue, Sigma) and assessed with a four-level survival score (1 = 100% live, 2 = 50–99% live, 3 = 1–49% live, 4 = 0% live) for each stage of follicular maturation: primordial (PF), early (EF), and late (LF) primary and secondary (SF) follicles (n = 100 each). Dissociated EF and LF (n = 45) from S. crassicaudata were vitrified with a three-step medium composed of up to 18% DMSO and ethylene glycol in 1 m sucrose (all Sigma; Vajta G et al. 1998 Mol. Reprod. Dev. 51, 53–58), and post-thaw viability was confirmed with trypan blue. All experiments were carried out on at least three individuals; values are presented as mean ± SEM and differences were determined with ANOVA and post hoc Tukey tests. In all species, 100% of PF were highly viable (score 1) and there was a significant decrease (P < 0.05) in the percentage of highly viable PF, EF, and LF when compared with SF, which were most commonly observed with minor damage (score 2). An incomplete layer of granulosa cells was observed in 11.92 ± 1.08% of freshly dissociated S. crassicaudata follicles. This increased to 30.34 ± 12.22% after preservation. Of the surrounded follicles, 48.40 ± 14.09% were classified as score 1 or 2 after vitrification. Nonviable staining of the oocyte was observed only in naked oocytes. Although these protocols demonstrate a reliable method for follicle collection and preliminary evidence for tools allowing the preservation of female gametes, further optimization is recommended. The demonstrated techniques are highly applicable to genebanking because they utilize material from unstimulated ovaries, as is regularly seen with tissues collected opportunistically from anestrus, ill, or old wildlife. This study was made possible by the cooperation and collection of tissues by the staff from Territory Wildlife Park, Northern Territory, and Tamara Keeley from Western Plains Zoo, New South Wales.