Androgens promote the acquisition of maturation competence in bovine oocytes

Abstract

Recent studies in mice suggest that androgens are important for normal follicle development. However, there have been few reports concerning the action of androgens in the growth of oocytes from large animals. The purpose of this study was to determine the roles of androgens in bovine oocyte growth in vitro. Oocyte-granulosa cell complexes (OGCs) collected from 0.4-0.7 mm early antral follicles were cultured for 14 days with 17β-estradiol (E2) and a non-aromatizable androgen, dihydrotestosterone (DHT). We also examined the ability of an androgen receptor (AR) inhibitor, hydroxyflutamide, to antagonize the effect of androgens on the oocytes. During growth culture, the OGC structures collapsed in the medium with DHT alone, while in the presence of E2, the OGC structures were maintained. In the medium with both androgens and E2, the mean diameter of oocytes was increased from 95 μm to around 120 μm, larger than those grown with E2 alone (115 μm). Also in the maturation culture, oocytes grown with androgens (A4 or DHT) and E2 showed higher percentages of metaphase II oocytes (63% or 69%, respectively) than those grown with E2 alone (32%). Moreover, these maturation rates were decreased by hydroxyflutamide in a dose-dependent manner. Immunostaining showed that ARs were expressed in oocytes and granulosa cells in early antral follicles, and the nuclei of granulosa cells showed intense AR expression. In conclusion, although E2 supports the OGC structure, additional androgens promote oocyte growth and their acquisition of meiotic competence via AR during in vitro growth culture.

Fig. 1.

Typical morphology of bovine oocyte-granulosa cell complexes (OGCs) during growth culture. OGCs were cultured for 14 days in the medium with steroid hormones alone (A) or with combinations of steroid hormones and OHF (B). E₂, 17β-estradiol; A₄, androstenedione; DHT, dihydrotestosterone; OHF, hydroxyflutamide; 10E₂, 10 ng/ml E₂; 10A₄, 10 ng/ml A₄; 10DHT, 10 ng/ml DHT; 10E₂ + 10A₄, 10 ng/ml E₂ plus 10 ng/ml A₄; and 10 E₂+10DHT, 10 ng/ml E₂ plus 10 ng/ml DHT. Scale bars represent 500 μm for each panel.

Fig. 2.

Integrity of bovine OGCs during growth culture with steroid hormones alone (A) or with combinations of steroid hormones and OHF (B). On days 0, 7 and 14, OGCs that showed degenerative signs, such as cytoplasmic degeneration of oocytes and/or complete detachment of granulosa cells from oocytes, were classified as degenerative complexes. See the footnotes in Fig. 1 for abbreviations. ^a,b Values with different superscripts differ significantly (P < 0.05).

Fig. 3.

Comparison of the diameters of bovine oocytes after 14 days of growth culture. For each panel, the white box on the left represents the diameter of oocytes isolated from early antral follicles (0.4−0.7 mm). The gray, striped and dotted boxes indicate the diameters of oocytes cultured for 14 days in the medium with or without steroid hormones and OHF. The white box on the right represents the diameter of oocytes obtained from antral follicles (4−6 mm). The numbers of oocytes examined are shown below each box (n), and the numbers above the boxes indicate the mean diameters of oocytes (μm). See the footnotes in Fig. 1 for the abbreviations. ^a−d Values with different superscripts differ significantly (P < 0.05). * Values were significantly different from those of oocytes before culture (white box on the left, P < 0.05).

Fig. 4.

Meiotic competence of bovine oocytes cultured for growth after in vitro maturation. The percentages of MII oocytes out of total oocytes initially used for in vitro growth culture are shown in both panels. For each panel, the white bars on the left and the right represent maturation rates of oocytes collected from early antral follicles (0.4−0.7 mm) and antral follicles (4−6 mm), respectively. The gray, striped and dotted bars indicate the maturation rates of oocytes used for growth culture. The numbers above the bars indicate the numbers of oocytes used for growth culture (n). The percentages of MII oocytes are shown at the bottom of each bar. ^a−e Values with different superscripts differ significantly (P < 0.05).

Fig. 5.

Expression of androgen receptors in bovine early antral follicles and OGCs. (A) Cryosections of 0.4−0.7 mm follicles were treated with anti-androgen receptor antibody and Alexa Fluor 488-labeled immunoglobulin antibody. Alexa Fluor 488 staining marks androgen receptors in green (a−c). DAPI staining marks the chromatin in blue (b). A magnified image of the cumulus oophorus in (a) is shown in (c). Scale bars represent 100 μm. (B) OGCs isolated from 0.4−0.7 mm follicles were stained. Alexa Fluor 488 staining marks androgen receptors in green (a, b). DAPI staining marks the chromatin in blue (b). A bright-field image is shown in (c). Scale bars represent 20 μm.