Effect of different culture systems and 3, 5, 3'-triiodothyronine/follicle-stimulating hormone on preantral follicle development in mice

Abstract

The mechanical method to isolate preantral follicle has been reported for many years. However, the culture systems in vitro are still unstable. The aim of this study was to analyze the effect of the culture system of mice preantral follicles on the follicular development in vitro. The results showed that the 96-well plate system was the most effective method for mice follicle development in vitro (volume change: 51.71%; survival rate: 89%, at day 4). Follicle-stimulating hormone (FSH) and Thyroid hormone (TH) are important for normal follicular development and dysregulation of hormones are related with impaired follicular development. To determine the effect of hormone on preantral follicular development, we cultured follicle with hormones in the 96-well plate culture system and found that FSH significantly increased preantral follicular growth on day 4. The FSH-induced growth action was markedly enhanced by T₃ although T₃ was ineffective alone. We also demonstrated by QRT-PCR that T₃ significantly enhanced FSH-induced up-regulation of Xiap mRNA level. Meanwhile, Bad, cell death inducer, was markedly down-regulated by the combination of hormones. Moreover, QRT-PCR results were also consistent with protein regulation which detected by Western Blotting analysis. Taken together, the findings of the present study demonstrate that 96-well plate system is an effective method for preantral follicle development in vitro. Moreover, these results provide insights on the role of thyroid hormone in increasing FSH-induced preantral follicular development, which mediated by up-regulating Xiap and down-regulating Bad.

Figure 1. Representative examples of preantral follicle and oocyte in different phase. (A)

Follicle isolated mechanically (day 0): central germinal vesicle (GV)-stage oocyte (arrow head) with a thin zona pellucida, surrounded by two layers of cuboidal granulosa cells (arrow), a basal membrane and a single layer of theca cells (original magnification ×200). (B) Preantral follicle after 4 days of culture with FSH (10 ng/ml) had intact basal membrane with proliferated granulosa cells (original magnification ×200). (C) Preantral follicle after 4 days of culture had shrunk with smaller oocyte (original magnification ×200). (D) The follicle culture on day 4 was punctured and healthy oocyte was released. (E, F) The oocytes from those darken and or shrunk follicles showed retrogression with many particles or big gap between oocyte and zona pellucida (original magnification ×200). Bars  =  40 µm.

Figure 2. Effect of different culture system on preantral follicular development in vitro.

Mouse preantral follicles (100–130 µm in diameter) were isolated mechanically and cultured in different systems. M1 group was developed in culture dishes (35 mm) with 2 ml medium. In the second group (Method 2, M2), medium was made into 10×20 µl culture droplet, and covered with 3 ml of washed mineral oil. Follicles were placed in the culture droplets, 10 follicles per droplet. Two other groups were cultured in 4-well (Method 3, M3) and 96-well tissue culture plates (Method 4, M4) respectively. Follicular diameter was measured daily and results were expressed as change in follicular volume. Note that follicles grew faster in M4 group than M1 and M3 groups although the statistic analysis was not significant among others groups. ⁺P<0.05 vs. M1 at the same culture duration, *P<0.05, ***P<0.001, vs. M3 at the same culture duration.

Figure 3. Effect of FSH and T₃ on preantral follicular growth in vitro.

Preantral follicles were cultured for 4 days with or without FSH (10 ng/ml) and T₃ (1.0 nM). Follicles cultured in the absence of FSH and T₃ was named as CTL group. Follicular diameter was measured daily and results were expressed as change in follicular volume and represented as means ± SEM of a total of 120 follicles from four independent experiments. Note that T₃, while ineffective alone, significantly increased FSH-induced preantral follicular growth at Day 4 (P<0.001). ⁺P<0.05, ⁺⁺⁺P<0.001 vs. FSH alone at the same culture duration, *P<0.05, **P<0.01, vs. CTL at the same culture duration (Three way ANOVA).

Figure 4. Effect of FSH and T₃ on follicular Xiap expression in vitro.

Results are represented as means±SEM of four independent experiments. Note that T₃, although ineffective alone, significantly increased FSH-induced Xiap expression (T₃ 1.0 nM; P<0.01). *: P<0.05 vs CTL. +: P<0.05, ++: P<0.01vs FSH alone (Two way ANOVA)

Figure 5. Effect of FSH and T₃ on follicular Bad expression in vitro.

Results were represented as means±SEM of four independent experiments. FSH significantly down-regualted Bad expression (P<0.05), the down-regulation effect was dramatically enhanced by T₃ (P<0.01) although T₃ alone was ineffective. *: P<0.05 vs CTL; +, P<0.05; ++, P<0.01 vs FSH (Two way ANOVA).