Dexamethasone Restores the Repressive Effect of Tumor Necrosis Factor-α on Follicular Growth and E2 Secretion During the In Vitro Culture of Preantral Follicles

Abstract

As a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) is central to the female reproductive tract and affects various phases of follicular development and uterine cycles. High levels of TNF-α play a vital role in the pathogenesis of polycystic ovary syndrome (PCOS) in patients. Clinicians know that dexamethasone can inhibit the induction of androgen by suppressing the adrenal glands which improves the status of the endocrine system in PCOS patients. We hypothesize that dexamethasone has much more functionality and can exert a therapeutic effect by antagonizing TNF-α. We added TNF-α to the follicular culture medium to simulate the high TNF-α levels observed in the endocrine environment of PCOS patients. Dexamethasone was added to the medium to determine if it could counteract the inhibitory effect of TNF-α on follicular growth and 17β-estradiol (E₂) secretion. Follicular diameter, E₂ concentration, follicle survival, antral-like cavity formation, and ovulation were measured to assess the effects of dexamethasone. In our work, TNF-α inhibited in vitro follicular growth and E₂ secretion in a dose-dependent manner. Based on the results of the present research, we concluded that the addition of dexamethasone partially counteracts the repressive effect of TNF-α on follicle growth and E₂ secretion during in vitro culture of the preantral follicles of mice. Thus, the findings in this paper suggest that dexamethasone may act as a therapy by counteracting the effects of TNF-α in PCOS patients. These results provide a new foundation for exploring the treatment of PCOS patients with dexamethasone.

Keywords: Dexamethasone; In vitro culture; Polycystic ovary syndrome; Preantral follicle; TNF-α.

Fig. 1

Timeline of the in vitro culture of preantral follicles

Fig. 2

An illustration of the control and experimental groups to determine the effects of FSH, TNF-α, dexamethasone, and their interactions on follicle growth and E2 secretion

Fig. 3

Microphotographs showing in vitro growth of mouse preantral follicles with and without FSH. A Examples of follicular growth with or without FSH (100mIU/mL). Most of the follicles in the FSH− group were degraded and broken (c), but follicles treated with FSH became larger and formed antral-like cavities after 8 to 12 days in culture. Follicles formed obvious antral-like cavities after 12 days in culture. Arrowhead shows an antral-like cavity (g). Supplementation with 5 IU/mL of HCG and 5ng/mL of EGF causes the COC to be released from the follicle after 20 h. Dual arrows refer to released COC (h) (bar =50μm). B A picture of an ovulated COC with extension of the cumulus (bar =50μm).C In the FSH+ group, using 80IU/mL of hyaluronidase to remove surrounding cumulus cells, we can observe a group of metaphase II oocytes (bar =50μm)

Fig. 4

Influence of FSH on follicle diameter and E₂ secretion. a Diameter changes of follicles in the FSH+ group during in vitro growth. b Changes of E₂ levels in culture medium in the FSH+ group during in vitro growth

Fig. 5

Influence of TNF-α on follicle growth and E₂ secretion. Follicles were treated with 5, 10, 15, and 20ng/mL TNF-α, and follicle development was assessed under an inverted microscope. A Influence of different doses of TNF-α on follicular survival after 12 days in culture. Numbers inside of the bars demonstrate surviving follicles/total follicles. B Influence of different doses of TNF-α on follicular antral-like cavity formation rates after 12 days in culture. Numbers inside of the bars demonstrate follicles with an antral-like cavity/total follicles. C Influence of different doses of TNF-α on follicular diameter after 12 days in culture. Data are represented as means ±SD. D Influence of different doses of TNF-α on follicular E₂ secretion after 12 days in culture. Data are represented as means ±SD. All tests comprised fewer than 3 independent tests. The FSH− group without FSH and TNF-α served as negative controls. The different letters on the bars indicate statistical significance (P<0.05). E In the FSH+ group, granular cell layers grew more densely (bar =50μm). F In the T₁₅ group, granular cell layers were sparse (bar =50μm)

Fig. 6

Dexamethasone restores the inhibitory effect of TNF-α on follicular growth and E₂ secretion. Follicles were treated with 0.1, 1, or 10μg/mL of dexamethasone in the presence of 15ng/mL of TNF-α. a The influence of various compounds on follicular survival after 12 days in culture. Numbers inside of the bars demonstrate surviving follicles/total follicles. b The influence of various compounds on follicular antral-like cavity formation after 12 days in culture. Numbers inside of the bars demonstrate follicles with an antral-like cavity/total follicles. c The influence of various compounds on follicular diameter after 12 days in culture. Data are represented as means ±SD. d Influence of various compounds on follicular E₂ secretion after 12 days in culture. Data are represented as means ±SD. All tests comprised fewer than 3 independent tests. Follicles were treated with absolute ethyl alcohol as the only vehicle control (e group). The different letters on the bars indicate statistical significance (P<0.05)

Fig. 7

Influence of dexamethasone (1μg/mL) on follicular growth and E₂ secretion. Follicles treated with and without 1μg/mL dexamethasone. a Influence of 1μg/mL dexamethasone on follicle survival after 12 days in culture. Numbers inside of the bars demonstrate surviving follicles/total follicles. b Influence of 1μg/mL dexamethasone on follicular antral-like cavity formation after 12 days in culture. Numbers inside of the bars demonstrate follicles with an antral-like cavity/total follicles. c Influence of 1μg/mL dexamethasone on follicular diameter after 12 days in culture. Data are represented as means ±SD. d Influence of 1μg/mL dexamethasone on follicular E₂ secretion after 12 days in culture. Data are represented as means ±SD. All tests comprised fewer than 3 independent tests. The e group was treated with absolute ethyl alcohol only. Controls were treated with FSH only. Different letters on the bars indicate statistical significance (P<0.05)