Immunoreactivities of androgen receptor, estrogen receptors, p450arom, p450c17 proteins in wild ground squirrels ovaries during the nonbreeding and breeding seasons

Abstract

The aim of this study was to elucidate the regulatory role of androgen in the follicular development of wild female ground squirrels. Immunohistochemical staining of FSHR, LHR, P450c17, P450arom, androgen receptor (AR), estrogen receptors (ERa and ERb) were executed in ovaries of female ground squirrels from both breeding and nonbreeding seasons. In addition, total ovarian proteins were extracted from the ovaries of squirrels from breeding and nonbreeding seasons, and Western blot analysis were performed in order to probe for FSHR, LHR, P450c17, P450arom, AR, ERa and ERb. The results of immunohistochemical staining and Western blotting of P450c17 showed that there was no significant difference between the breeding and nonbreeding seasons. It was found that granulosa cells expressed P450arom during the breeding season. In contrast, there was no positive staining of P450arom in the nonbreeding season. There was no significant difference in immunoreactivity of AR between the breeding and nonbreeding seasons. However, the immunoreactivities of ERa and ERb were both significantly reduced in the nonbreeding season compared to the breeding season. The positive stains of FSHR and LHR were found in the granulosa cells and theca cells of the ovaries of the breeding and nonbreeding seasons. In addition, the Western blotting results of FSHR and LHR showed a significant reduction in the nonbreeding season compared with the breeding season. These findings suggested that androgen might be predominantly converted into estrogen in order to regulate the follicular development via binding of estrogen receptors during the breeding season, whereas androgen might predominantly directly bind androgen receptor to regulate the follicular development during the nonbreeding season in the ovaries of wild female ground squirrels.

Figure 1

Seasonal changes in the ovary of wild female ground squirrels.a: The histological observation and diagram of the ovary in breeding season. b: The histological observation and diagram of the ovary in nonbreeding season. c: Histological ovarian pattern diagram of the wild ground squirrels during the breeding season. d: Histological ovarian pattern diagram of the wild ground squirrels during the nonbreeding season. e: Seasonal change of the number of primary follicles. f: Seasonal change of the number of secondary follicles. g: Seasonal change of the number of antral, post-antral follicles. h: Seasonal change of the number of corpus luteum. PF, Primary Follicle; SF, Secondary Follicle; AF, Antral Follicle; DF, Dominant Follicle; CL, Corpus Luteum. Data are shown as the mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Bars = 200μm.

Figure 2

The immunolocalization of P450c17 and P450arom in the ovary of wild female ground squirrels. The immunolocalization of P450c17 (a, b, c) and P450arom (d, e, f) in the ovary of wild female ground squirrels during the breeding and nonbreediang seasons. The breeding season divided into two stages, the large follicle stage (a, d) and the small follicle (b, e). g, negative control. IC, interstitial cell; TC, theca cell; GC, granulosa cell. Bars =50μm.

Figure 3

The immunolocalization of AR and ERs in the ovary of wild female ground squirrels. The immunolocalization of androgen receptor (a, b, c), ERa (d, e, f) and ERb (g, h, i) in the ovary of wild female ground squirrels during the breeding and nonbreediang seasons. The breeding season divided into two stages, the large follicle stage (a, d, g) and the small follicle (b, e, h). j, negative control. IC, interstitial cell; TC, theca cell; GC, granulosa cell. Bars =50μm.

Figure 4

The immunolocalization of FSHR and LHR in the ovary of wild female ground squirrels. The immunolocalization of FSHR (a, b) and LHR (c, d) in the ovary of wild female ground squirrels during the breeding and nonbreediang seasons. e, negative control. IC, interstitial cell; TC, theca cell; GC, granulosa cell. Bars =50μm.

Figure 5

Western blotting of FSHR, LHR, P450c17, P450arom, AR and ERs in the ovary of wild ground squirrels. The expression levels of FSHR (a), LHR (b), P450c17 (c), P450arom (d), AR (e), ERa (f) and ERb (g) were analyzed by optical density in the ovary of wild ground squirrels. The proteins were extracted from the whole ovary of the breeding season (lane 1), nonbreeding season (lane 2). b-actin (h) was used for endogenous control. The analysis of optical density of FSHR, LHR, P450c17, P450arom, AR, ERa and ERb were shown in histogram a’-g’, respectively. Data are shown as the mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 6

The ratio of AR to ERs in the ovary of wild ground squirrels. The ratio of AR to ERa (a) and ERb (b) in the ovary of wild ground squirrels according to the expression level of AR and ERs. Data are shown as the mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001.