Changes in the expressions of annexin A1, annexin A5, inhibin/activin subunits, and vitamin D receptor mRNAs in pituitary glands of female rats during the estrous cycle: correlation analyses among these factors

Abstract

Pituitary gonadotropin secretion is regulated by several pituitary factors as well as GnRH and ovarian hormones. To elucidate the regulatory mechanisms of pituitary gonadotropin secretions, we observed changes in the mRNA levels of pituitary factors, namely annexin A1 (Anxa1) and Anxa5, inhibin/activin subunits, follistatin (Fst), and vitamin D receptor (Vdr), in female rat pituitary glands during the estrous cycle. Additionally, levels of LHβ subunit (Lhb), FSHβ subunit (Fshb), and GnRH receptor (Gnrh-r) mRNA were examined. During proestrus, Anxa1, Anxa5, Vdr, and inhibin α-subunit (Inha) exhibited the lowest levels, while during estrus, activin βB-subunit (Actbb), Lhb, and Gnrh-r were the lowest. Moreover, Fshb exhibited the highest value during metestrus, whereas Fst did not differ significantly. Correlation analyses revealed 16 statistically significant gene combinations. In particular, four combinations, namely Anxa5 and Inha, Anxa5 and Actbb, Inha and Vdr, and Inha and Actbb, were highly significant (P<0.0001), while four combinations, Anxa1 and Anxa5, Anxa1 and Vdr, Anxa5 and Vdr, and Lhb and Gnrh-r, were moderately significant (P<0.001). The remaining eight combinations that exhibited statistical significance were Anxa1 and Inha, Anxa1 and Actbb, Vdr and Actbb, Anxa1 and Fshb, Inha and Lhb, Actbb and Fshb, Actbb and Lhb, and Fst and Fshb (P<0.05). These results highlight strong correlations among Anxa1, Anxa5, Vdr, Inha, and Actbb, thereby suggesting that an interaction among ANXA1, ANXA5, and VDR may lead to further communications with inhibin and/or activin in the pituitary gland.

Keywords: annexin; estrous cycle; gonadotropins; inhibin/activin; vitamin D receptor.

Fig. 1.

Changes in the mRNA levels of annexin A1 (Anxa1; A), annexin A5 (Anxa5; B), and vitamin D receptor (Vdr; C) in the pituitary glands of female rats during the estrous cycle. The anterior pituitaries were collected between 1000 and 1200 hr on metestrus (D1), diestrus (D2), proestrus (P), and estrus (E). Values are represented as the mean ± SEM (n=5). Each value is presented as a ratio to the value observed in proestrus. Data labeled with different letters differ significantly from each other (P<0.05, Tukey’s multiple comparison test).

Fig. 2.

Changes in the mRNA levels of inhibin α-subunit (Inha; A), inhibin/activin βB-subunit (Actbb; B), and follistatin (Fst; C) in the pituitary glands of female rats during the estrous cycle. The anterior pituitaries were collected between 1000 and 1200 hr on metestrus (D1), diestrus (D2), proestrus (P), and estrus (E). Values are represented as the mean ± SEM (n=5). Each value is presented as a ratio to the value observed in proestrus. Data labeled with different letters differ significantly from each other (P<0.05, Tukey’s multiple comparison test).

Fig. 3.

Changes in the mRNA levels of luteinizing hormone β subunit (Lhb; A), follicle stimulating hormone β subunit (Fshb; B), and gonadotropin-releasing hormone receptor (Gnrh-r; C) in the pituitary glands of female rats during the estrous cycle. The anterior pituitaries were collected between 1000 and 1200 hr on metestrus (D1), diestrus (D2), proestrus (P), and estrus (E). Values are represented as the mean ± SEM (n=5). Each value is presented as a ratio to the value observed in proestrus. Data labeled with different letters differ significantly from each other (P<0.05, Tukey’s multiple comparison test).

Fig. 4.

Scatterplots denoting the correlations between the mRNA levels of annexin A5 (Anxa5) and inhibin/activin βB-subunit (Actbb; A), Anxa5 and inhibin α-subunit (Inha; B), vitamin D receptor (Vdr) and Inha (C), and Actbb and Inha (D) in the pituitary glands of female rats during the estrous cycle. The anterior pituitaries were collected between 1000 and 1200 hr on metestrus (D1), diestrus (D2), proestrus (P), and estrus (E). Values are presented as a ratio to the mean value observed in proestrus, and a regression line is displayed. Statistical analyses were performed using Pearson’s product-moment correlation.

Fig. 5.

Scatterplots denoting the correlations between the mRNA levels of annexin A1 (Anxa1) and annexin A5 (Anxa5; A), Anxa1 and vitamin D receptor (Vdr; B), Anxa5 and Vdr (C), and luteinizing hormone β subunit (Lhb) and gonadotropin-releasing hormone receptor (Gnrh-r; D) in the pituitary glands of female rats during the estrous cycle. The anterior pituitaries were collected between 1000 and 1200 hr on metestrus (D1), diestrus (D2), proestrus (P), and estrus (E). Values are presented as a ratio to the mean value observed in proestrus, and a regression line is displayed. Statistical analyses were performed using Pearson’s product-moment correlation.

Fig. 6.

Diagram summarizing the correlations among different gene expressions, namely annexin A1 (Anxa1), annexin A5 (Anxa5), vitamin D receptor (Vdr), inhibin/activin βB-subunit (Actbb), inhibin α-subunit (Inha), gonadotropin-releasing hormone receptor (Gnrh-r), luteinizing hormone β subunit (Lhb), follicle stimulating hormone β subunit (Fshb) and follistatin (Fst), in the pituitary glands of female rats during the estrous cycle. The lines connecting the different factors indicate the correlations among them. The thickest line (red), second thickest line (yellow), third thickest line (green), and dotted line (blue) represent the correlations among the groups A, B, C, and D presented in Table 2, respectively.