A novel two-step strategy for in vitro culture of early-stage ovarian follicles in the mouse

Abstract

Objective: To develop an in vitro strategy to support the growth of early-stage follicles and produce mature oocytes competent for fertilization.

Design: Whole ovaries from 8-day-old mice were cultured for 4 days, and then secondary follicles were isolated and cultured for 12 days in a three-dimensional alginate or fibrin-alginate (FA) hydrogel matrix.

Setting: University-affiliated laboratory.

Animals: Mice.

Intervention(s): None.

Main outcome measures: Histologic evaluation of follicle development, steroid hormone production, and rates of oocyte maturation, oocyte fertilization, and embryo formation.

Result(s): Culture of 8-day-old mouse ovaries for 4 days resulted in transition of the follicle population from primordial and primary follicles to secondary follicles, similar to that seen in a 12-day-old ovary. Isolated secondary follicles cultured for 12 days showed larger increases in oocyte diameter and more frequent antrum formation and theca cell differentiation in the FA-hydrogel matrix compared with the alginate matrix. Steroid hormone secretion patterns were consistent with the changes in follicle morphology and cell differentiation observed in the cultured follicles. Compared with oocytes from alginate follicle cultures, a greater number of oocytes retrieved from the FA-based follicle cultures progressed to metaphase I, reached metaphase II, and could be fertilized and cleaved to two-cell embryos. The organ culture plus FA-hydrogel follicle culture strategy produced a very high rate of oocyte progression to metaphase II (88 +/- 8.7% [mean +/- SEM]) and formation of two-cell embryos (54 +/- 4%).

Conclusion(s): A strategy combining whole ovary culture of early-stage follicles and subsequent FA hydrogel in vitro follicle culture produced a high percentage of oocytes competent for fertilization; this might provide new options for fertility preservation in women and prepubertal girls facing fertility-threatening diseases or treatments.

Fig. 1

Representative photomicrographs of H&E stained paraffin sections of whole ovaries before and after culture. (A) Control, uncultured 8-day-old mouse ovary. (B) 8-day-old mouse ovary after 4 days of organ culture. (C) H&E staining of uncultured 8-day-old mouse ovary, which contains mainly primordial follicles with a few primary and secondary follicles. (D) H&E staining of 8-day-old mouse ovary after 4 days of organ culture. More secondary follicles were observed. (E) H&E staining of uncultured 12-day-old mouse ovary. (F) Follicle distribution in mouse ovaries before and after 4-day organ culture. 0 indicates primordial follicle; 1 indicates primary follicle; 2 indicates secondary follicle. The scale bars in A and B are 150 µM. Other scale bars are 200 µM. Letters indicate a statistically significant difference between groups (P<0.05).

Fig. 2

Development and differentiation of representative secondary follicles cultured in vitro. (A) Secondary follicles with centrally located immature oocytes isolated from cultured ovarian tissues. (B, C) Follicles maintained their 3-D structure with proliferation of granulosa cells, antrum formation (white arrowhead), and development of theca cell layers (black arrowhead) after 12 days of culture in 0.25% alginate or AF. (D) Follicle diameter in both culture systems increased significantly during the culture period. (E) Oocyte size increased significantly over the culture period. Statistically significant differences were observed between groups as indicated with different letters (P<.05). The scale bar represents 100 µM. (F, G) Average values of E₂ (F) and P₄ (G) secretion were measured in conditioned culture media from secondary follicle cultures.

Fig. 3

Meiotic and fertilization competence of oocytes from follicles cultured for 12 days in alginate (A–D) or FA (E–H) were assessed by IVM and IVF. CEOs isolated from antral follicles retrieved from alginate (A) or FA (E) culture systems were induced with hCG for 18 h in vitro. (B, F) In both environments, cumulus cells around the oocytes expanded. (C, G) Oocytes resumed meiosis and extruded the first polar body (arrowhead). (D, H) Two-cell embryos were obtained by IVF of MII oocytes. The scale bar represents 50 µM.