Vitrification of Ovarian Cortex from Prepubertal and Adult Cats Induces Less Damage to the Primordial Follicles than Slow Freezing.

Abstract

Due to certain similarities in reproductive mechanisms, the domestic cat has become a valuable model for exploring fertility preservation strategies for young girls and women. The objective of this study was to assess the survival of primordial follicles in prepubertal and adult ovarian tissues during slow freezing or vitrification. Freshly-excised ovaries from prepubertal (4-6 mo; n = 12 total ovaries; 6 replicates) and adult (>2 yr; n = 12 total ovaries; 6 replicates) females were transported at 4°C before processing. In each replicate, the cortex of each ovary (n = 2 from prepubertal and n = 2 from adult cats) was dissected and cut into 18 pieces (1-2 mm3) that were allocated to different treatments. Three pieces of each cortex were directly fixed (4% paraformaldehyde) to determine primordial follicle density in histological sections (i.e., number of follicles with visible oocyte nuclei/mm2 of section) as well as the proportion of intact primordial follicles (i.e., number of oocytes and surrounding granulosa cells without pycnotic nuclei, shrunken cytoplasm or vacuoles relative to a total of 60 follicles examined per ovary). Three other cortical pieces were dissociated (1 mg/ml collagenase) and stained (2 μM calcein-AM and 5 μM ethidium) to evaluate the proportion of viable primordial follicles (i.e., number of oocytes with surrounding granulosa cells stained only with calcein-AM relative to a total of 60 follicles examined per ovary). Six cortical pieces were exposed to 1.5 M ethylene glycol (EG; 15 min) and frozen using a slow cooling device (−1°C/min to −80°C ) before plunging into liquid nitrogen. The remaining six pieces were exposed to 1.5 M EG + 1.5 M DMSO + 0.5 M sucrose (5 min) before vitrification in liquid nitrogen. After 1 week of storage, all samples were rapidly warmed to 38°C and diluted stepwise in decreasing sucrose concentrations before fixation for histological assessment or dissociation for viability staining (as above). Primordial follicle density was higher (P<0.05; ANOVA) in the ovarian cortex of prepubertal (mean ±; SD; 93.0 ± 15.8 follicles/mm2) compared to adult (43.0 ± 13.6) females. Proportions of intact and viable follicles were similar (P>0.05) in fresh controls from prepubertal and adult ovarian tissues (intact follicles, 90.0 ± 5.8 and 92.5 ± 8.7%, respectively; viable follicles, 86.0 ± 2.7 and 82.5 ± 3.5%, respectively). Although cryopreservation decreased (P<0.05) the proportions of intact follicles compared to fresh controls, vitrification was superior (P<0.05) to slow freezing for preserving follicular structure in prepubertal (vitrification, 70.0 ± 3.4% intact follicles; slow freezing, 57.5 ± 8.7%) and adult ovarian tissues (vitrification, 73.5 ± 3.3%; slow freezing, 56.3 ± 4.8%). A similar pattern was observed for proportions of viable follicles, with vitrification exceeding (P<0.05) slow freezing in prepubertal (vitrification, 59.8 ± 4.6%; slow freezing, 37.8 ± 2.8%) and adult (vitrification, 62.5 ± 3.5%; slow freezing, 36.3 ± 4.5%) tissues. Interestingly, higher follicle densities in prepubertal versus adult ovaries had no influence (P>0.05) on follicle structure and viability post-thawing. Results demonstrate, for the first time, that vitrification permits retaining ≈80% of the original structure and ≈70% of the original viability of cat primordial follicles regardless of their density in the ovarian cortex. (Supported by the National Institutes of Health K01 RR020045). (platform)