Comparison of Follicle Isolation Methods for Mouse in vitro Ovarian Follicle Culture

Abstract

Objective: Ovarian tissue cryopreservation can be considered as a fertility preservation option for pre-pubertal girls or single woman. However, transplantation of cryopreserved ovarian tissue carries a critical risk which is the possibility of re-implantation of malignant cell to fully cured cancer survivors. Ovarian follicle in vitro culture is a promising fertility preservation option to avoid risk of re-introduction of malignant cell. However, in vitro ovarian follicle culture protocol is still in an experimental phase. For the success of in vitro ovarian follicle culture, proper ovarian follicle isolation is preceded before beginning in vitro culture. Based on this background, the objective of this study is to compare four different follicle isolation methods from ovarian tissue and evaluate the effect of follicle isolation on further in vitro follicle culture and oocyte competency.

Materials and methods: This study was conducted by using 11 to 14 days-old female BDF-1 mouse. Ovaries were dissected and randomly divided into 4 groups according to follicle isolation method

1) mechanical isolation using 30G syringe needle (mechanical, MCH), 2) mechanical isolation using cell dissociation kit (mincing, MNC), 3) enzymatically digestion using collagenase type I (COL) and 4) enzymatically digestion using liberase (LIB). Follicles classified as early secondary follicle, 100~110 μm of diameter, were immediately cultured under culture media, which was consisted of 10 mIU/ml FSH, 1% ITS, 1% PS and 5% FBS in glutaMAX-αMEM. The media was refreshed every 4 days and concentrations of estradiol, progesterone and testosterone in spent media were measured by ELISA on day 4, 8 and 10. On day 10, ovulation was induced by adding 1.5 IU/ml hCG and 5 ng/ml EGF in the media. Ovulated oocyte was checked after 18 hours. Follicular diameter was measured every 2 days. On day 10, follicular survival rate, pseudo-antrum formation rate were examined. The 18 hours after ovulation induction, the cumulus oocyte complexes (COCs) rate and the number of mature oocyte (MII oocyte) was counted. The diameter of retrieved MII oocyte was measured. To evaluate competence of mature oocyte derived from in vitro follicle culture, spindle and chromosome alignment and mitochondrial activity in mature oocyte were measured by immunofluorescence staining.

Results: The yields of follicles per one ovary were significantly higher in COL and LIB group. Follicular viability after isolation is significantly lower in MCH group, 80.57%, compared with the other groups (90.27%, 89.35% and 92.49%, MNC, COL and LIB group respectively). On day 10, follicular diameter was significantly greater in MCH and MNC group, 496.84 ± 11.56 μm and 488.53 ± 12.74 μm respectively, than COL and LIB group, 430.41 ± 11.65 μm and 430.03 ± 12.00 μm respectively. After 10 days of in vitro culture, follicle survival rate was significantly higher in MCH and MNC group, 88.7% and 92.1% respectively, compared with COL and LIB group, 70.4% and 71.2% respectively. The pseudoantrum formation rate was significantly higher in MNC group, 83.0%, than MCH, COL and LIB group, 78.0%, 72.7% and 61.1% respectively. The COCs rate was statistically higher in MCH group, 69.1%, than MNC group, 52.5%. Then enzymatic digestion groups, COL and LIB, showed significantly lower COCs rate, 35.8% and 28.0% than MCH and MNC groups. The MNC group had made a result that significantly improved the mature oocyte rate, 73.6%, than MCH, COL and LIB group, 59.6%, 43.8% and 42.2% respectively. The estradiol level on day 10 in MCH group, 1.44 ± 0.37 ng/ml, was significantly higher than COL group, 0.53 ± 0.04 ng/ml. The progesterone and testosterone level on day 10, there was no significant difference among the experimental groups. The diameter of MCH and MNC group, 70.21 ± 0.54 μm and 69.33 ± 0.58 μm respectively, showed no significant difference compared with in vivo control group, 72.03 ± 0.77 μm. Normal meiotic spindle and chromosome rate is significantly higher in MCH and MNC group, 85.1% and 91.7%, than COL and LIB group, 42.9% and 50.0%. The MNC group showed the highest level of mitochondrial activity, 980.30 ± 69.01, among the other groups, 489.32 ± 25.78, 824.01 ± 27.32, 722.11 ± 25.31 and 405.56 ± 78.24 that control, MCH, COL and LIB group respectively.

Conclusion: MNC method showed significantly improved rate of follicle diameter, survival, pseudoantrum formation, a mature oocyte after in vitro culture. Furthermore, a normal meiotic spindle and chromosome rate was significantly higher than COL and LIB group. Based on results of this study, MNC method can be alternative method for MCH method that laborious and time consuming. The enzymatic digestion group, COL and LIB, were inappropriate method for successful in vitro follicle culture on the basis of the result of present study. However, the fertilization ability of retrieved oocyte, pre-implantation and post-implantation competency of embryo from in vitro follicle culture should be evaluated in further study.