Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

Abstract

Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36μM) for 18-96h. Every 24h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96h, and the expression of cell cycle regulators at 18h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles.

Keywords: Follicle; Genistein; Ovary; Steroidogenesis.

Figure 1. Effect of genistein on follicle growth over time

After isolation, antral follicle growth was measured daily along perpendicular axes and percent change in growth was determined from 24 to 96 h. The graph represents the means ± SEM of percent change in follicle growth from 3 separate experiments. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3; p≤ 0.05).

Figure 2. Effects of genistein on apoptotic factors and cell cycle regulators at 18 h

After culture of antral follicles for 18 h, follicles were collected and subjected to RNA extraction. The RNA was reverse transcribed to cDNA and used to measure gene expression of Bax, Bcl2, Cdkn1a, Ccna2, Ccnb1, Ccnd2, Ccne1, and Cdk4 by quantitative polymerase chain reaction (qPCR). Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=4; p≤ 0.05).

Figure 3. Effects of genistein on estradiol production over time

After culture of antral follicles for 24-96 h, media were collected and subjected to enzyme-linked immunosorbent assays for estradiol. The graph represents the means ± SEM from 3 separate experiments. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3; p≤ 0.05).

Figure 4. Effects of genistein on estrone production over time

After culture of antral follicles for 24-96 h, media were collected and subjected to enzyme-linked immunosorbent assays for estrone. The graph represents the means ± SEM from 3-5 separate experiments. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3-5; p≤ 0.05).

Figure 5. Effects of genistein on testosterone production over time

After culture of antral follicles for 24-96 h, media were collected and subjected to enzyme-linked immunosorbent assays for testosterone. The graph represents the means ± SEM from 3 separate experiments. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3; p≤ 0.05).

Figure 6. Effects of genistein on androstenedione production over time

After culture of antral follicles for 24-96 h, media were collected and subjected to enzyme-linked immunosorbent assays for androstenedione. The graph represents the means ± SEM from 3 separate experiments.

Figure 7. Effects of genistein exposure on DHEA production over time

After culture of antral follicles for 24-96 h, media were collected and subjected to enzyme-linked immunosorbent assays for DHEA. The graph represents the means ± SEM from 3-5 separate experiments. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3-5; p≤ 0.05).

Figure 8. Effects of genistein on progesterone production over time

After culture of antral follicles for 24-96 h, media were collected and subjected to enzyme-linked immunosorbent assays for progesterone. The graph represents the means ± SEM from 3 separate experiments. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3; p≤ 0.05).

Figure 9. Effects of genistein on the gene expression of steroidogenic enzymes over time

After culture of antral follicles for 24-96 h, follicles were collected and subjected to RNA extraction. The RNA was reverse transcribed to cDNA and used to measure gene expression of key steroidogenic enzymes by qPCR. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3-5; p≤ 0.05).

Figure 10. Effects of genistein on the gene expression of Esr1 and Esr2

After culture of antral follicles for 24-96 h, follicles were collected and subjected to RNA extraction. The RNA was reverse transcribed to cDNA and used to measure gene expression of Esr1 and Esr2 by qPCR. Asterisks (*) indicate a significant difference between control groups and genistein at each time point (n=3-5; p≤ 0.05).