Seasonal expressions of androgen receptor, estrogen receptors and cytochrome P450 aromatase in the uteri of the wild Daurian ground squirrels (Spermophilus dauricus)

Abstract

The reproductive tissues including the uterus undergo dramatic changes in seasonal breeders from the breeding to non-breeding seasons. Classically, sex steroid hormones play important roles in the uterine morphology and functions. To clarify the relationship between sex steroid hormones and seasonal changes in the uterine morphology and functions, the wild Daurian ground squirrels (Spermophilus dauricus) were used as seasonal breeder model. And the immunolocalizations and expression levels of androgen receptor (AR), estrogen receptors α and β (ERα and ERβ) and cytochrome P450 aromatase (P450arom) were investigated in the uteri of the wild Daurian ground squirrels in the breeding (April) and the non-breeding (June) seasons via immunohistochemistry, Western blot and RT-PCR. Histologically, the uterine weight, the thickness of endometrium and the glandular density were significantly higher in the uteri of the breeding season than those of the non-breeding season. In both seasons, the immunostaining of AR was only presented in stromal cells of the uteri; the positive staining of ERα and ERβ were localized in stromal cells and glandular cells; P450arom was merely immunolocalized in glandular cells. The protein and mRNA expression levels of ERα, ERβ and P450arom were higher in the uteri of the breeding season than those of the non-breeding season; conversely, the expressions of AR were higher in the uteri of the non-breeding season comparing with those of the breeding season in both protein and mRNA levels. The AR: ER ratio in the uteri of the non-breeding season exceeded the AR: ER ratio in the uteri of the breeding season in the wild Daurian ground squirrels. These results suggested that seasonal changes in the expression levels of AR, ERs and P450arom might be correlated with the uterine morphology and histology changes, and estrogen may play an important autocrine/paracrine role in regulating the uterine functions of the wild Daurian ground squirrels.

Keywords: Androgen receptor; P450arom; estrogen receptor α; estrogen receptor β; uteri; wild Daurian ground squirrel..

Figure 1.

Anatomy and morphology of the uteri in the wild Daurian ground squirrels. Morphological features of uteri were observed in the wild Daurian ground squirrels during the breeding season (a) and the non-breeding season (b). The weight of uteri in the wild Daurian ground squirrels during the breeding season and the non-breeding season (c). Data were shown as the mean ± SEM. ***P<0.001.

Figure 2.

Histological structure of the wild Daurian ground squirrels’ uteri by hematoxylin- eosin (HE). The myometrium and endometrium of uterine tissues were observed in the wild Daurian ground squirrels during the breeding (a) and non-breeding seasons (b). The mean thickness of endometrium in the wild Daurian ground squirrels during the breeding season and the non-breeding season (c). The mean density of uterine glands in the wild Daurian ground squirrels during the breeding season and the non-breeding season (d). In each period, n = 5. SC, stromal cell; GC, glandular cell; GL, glandular lumen; L, uterine lumen. Data were shown as the mean ± SEM. **P<0.01; ***P<0.001.

Figure 3.

Immunohistochemistry of AR, ER, ER and P450arom in the uteri of the wild Daurian ground squirrels during the breeding and non-breeding seasons. In the boxed area on the bottom left in x’, immunoreactive cells were shown at higher magnification. The left column (a, c, e, g, i) represented staining in the breeding season, and the right column (b, d, f, h, j) represented immunostaining in the non-breeding season, respectively. The positive immunoactivities for AR (a,b), ER (c,d) and ER (e,f ) were observed in the nucleus, while the positive immunoactivities for aromatase (g,h) was in the cytoplasm. Negative controls (i,j) were counterstained with haematoxylin. In each period, n = 5. SC, stromal cell; GC, glandular cell.

Figure 4.

Western blot analysis of the protein levels of AR, ER, ER and P450arom during the breeding and non-breeding seasons, respectively. -actin blots were used as controls to correct for loading in each lane. The expression levels of AR (a), ER (a), ER (c), P450 arom (d) in uteri of the wild Daurian ground squirrels in different seasons were determined by densitometric analysis. B, the breeding season; NB, the non-breeding season; NC, the negative control. In each period, n = 4. Data were shown as the mean ± SEM. **P<0.01; ***P<0.001.

Figure 5.

RT-PCR analysis of the mRNA levels of AR, ER, ER and P450arom during the breeding and non-breeding seasons. The expressions of genes AR (a), ER (b), ER (c) and CYP19 (d) showed the changes during the breeding and non-breeding seasons. B, the breeding season; NB, the non-breeding season; NC, the negative control. In each period, n = 4. Data were shown as the mean ± SEM. ***P<0.001.

Figure 6.

The ratio of AR to ERs in the uteri of the wild Daurian ground squirrels. The ratio of AR to ER (a) and ER (b) in the uteri of wild Daurian ground squirrels according to the mRNA expression levels of AR and ERs. Data were shown as the mean ± SEM. ***P<0.001.