Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles

Abstract

Background: Premature ovarian insufficiency is a common complication of anticancer treatments in young women and girls. The ovary is a complex, highly regulated reproductive organ, whose proper function is contingent upon the bidirectional endocrine, paracrine, and autocrine signaling. These factors facilitate the development of the follicles, the functional units of the ovary, to progress from the gonadotropin-independent, paracrine-controlled early stage to the gonadotropin-dependent, endocrine-controlled later stage. We hypothesized that the low survival rate of individually cultured early-stage follicles could be improved with co-culture of adipose-derived stem cells (ADSCs) that secrete survival- and growth-promoting factors.

Materials and methods: Ovarian follicles ranging from 85 to 115 μm in diameter, from 10- to 12-day-old B6CBAF1 mice were mechanically isolated and co-encapsulated with ADSCs within alginate-based 3D culture system. The follicles were cultured for 14 days, imaged using light microscopy every 2 days, and matured at the end. Follicle media were changed every 2 days and collected for hormone measurements. Follicle diameter, morphology, number of transzonal projections, and survival and maturation rates were recorded. Statistical analyses using one- and two-way ANOVA were performed to compare hormone levels, survival of the follicles and ADSCs, oocyte maturation rates, and follicle growth.

Results: The co-encapsulation of the follicles with ADSCs increased follicle survival, ranging from 42.4% for the 86-95 μm to 86.2% for the 106-115-μm follicle size group. Co-culture also improved the follicle growth, the rate of antrum formation and oocyte maturation compared to the follicles cultured alone. The levels of androstenedione, estradiol, and progesterone of co-encapsulated follicles increased progressively with time in culture.

Conclusions: To our knowledge, this is the first report of an in vitro system utilizing mouse adipose-derived stem cells to support the development of the mouse follicles. Our findings suggest that co-encapsulation of ADSCs with early-stage follicles supports follicular development, through secretion of cytokines that promote follicular survival, antrum formation, and meiotic competence. The unique 3D culture system that supports the survival of both cell types has translational implications, as ADSCs could be used as an autologous source for in vitro maturation of early-stage human follicles.

Keywords: 3D culture; Adipose-derived stem cells; Ovarian follicle.

Fig. 1

Encapsulation of ADSC in alginate. a Representative live/dead images of ADSCs in alginate over 14 days of culture in follicle culture conditions. Scale bars = 500 μm. b No significant difference was found in ADSC survival in when compared to traditional two-dimensional culture over the 14-day culture period. Sample size, n = 3. c Representative image of ADSC cultured in traditional tissue culture-treated flask. Scale bars = 100 μm. d Representative image of ADSC cultured in alginate. Scale bars = 100 μm

Fig. 2

Representative images of the follicles in culture. Arrows = individual ADSCs in alginate. a–c Representative images of the follicles in vitro from the three diameter groups (A = 85–95 μm, B = 96–105 μm, C = 106–115 μm). Scale bars = 50 μm (100 μm for inserts). d–f Representative images of follicle growth and follicle development on days 2 (d), 4 (e), and 12 (f). Scale bars = 100 μm. g–i Representative images of various modes of follicular death in vitro. g Extruded oocyte. h An atretic-looking follicle. i A dark and dense looking follicle. Scale bars = 100 μm

Fig. 3

Survival and growth of the follicles with and without co-encapsulation with ADSCs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Survival and growth of the follicles in the ranges of 85–95 μm (a, b), 96–105 μm (c, d), and 106–115 μm (e, f) cultured alone (gray) or co-encapsulated with ADSCs (black). Sample sizes of growth curves: 85–95 μm: n = 2 (follicle alone) and 25 (co-encapsulated); 96–105 μm: n = 5 (follicle alone) and 59 (co-encapsulated); and 106–115 μm: n = 9 (follicle alone) and 50 (co-encapsulated). Data in growth curves presented as mean ± SEM

Fig. 4

Hormone secretion profiles of the follicles co-encapsulated with ADSCs and ADSCs only. Increasing hormone production during culture suggests follicular function. Top row demonstrates the levels of A4, second row E2, and third row P4. Statistical significance was marked by different letters. The hormone secretion profiles of ADSCs when encapsulated alone in alginate are demonstrated in the figure on the bottom left. Sample size: n = 3 per follicle size group. Data presented as mean ± SEM. A4/E2 ratios on the last day of culture are summarized in the table

Fig. 5

Representative images of oocyte stages after in vitro follicle maturation. GV germinal vesicle in an immature oocyte, GVBD germinal vesicle breakdown, MII oocytes arrested at meiosis II stage

Fig. 6

TZP counts in the follicles cultured alone and co-encapsulated with ADSCs. Significantly higher numbers of TZPs are found between the oocyte and cumulus granulosa cells of the follicles co-encapsulated compared to control after 2 days of culture. Scale bars = 10 μm. Sample size: n = 10 per follicle size group per condition