Limits...
Regulation of Akt expression and phosphorylation by 17beta-estradiol in the rat uterus during estrous cycle.

Dery MC, Leblanc V, Shooner C, Asselin E - Reprod. Biol. Endocrinol. (2003)

Bottom Line: Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus.Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells.Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Chimie-Biologie, Section Biologie Médicale, Université du Québec à Trois-Rivières, C,P, 500, Trois-Rivières, Québec, Canada G9A 5H7. Marie-Claude_Dery@uqtr.ca

ABSTRACT
Molecular and intra-cellular mechanisms involved in the regulation of apoptosis processes in endometrial cells are poorly understood and documented. We have investigated the possibility that Akt survival pathway might be involved in the regulation of apoptosis in the uterus during the estrous cycle. Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Uteri were collected and fixed for immunohistochemical staining (IHC) and apoptotic cell death detection by [TdT]-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) or endometrial protein extracts collected for Western analysis. TUNEL analysis revealed that apoptosis was mainly found at estrus compared to other day of estrous cycle. TUNEL positive cells were apparent in luminal epithelial cells only. No apoptotic cells were observed at proestrus. In contrast, proliferation was maximal at proestrus as confirmed with the expression of CDC47/MCM7 (a cell proliferation marker). Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus. Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells. PTEN protein, a phosphatase involved in the regulation of Akt phosphorylation, was present at all days of estrous cycle and showed no significant regulation in relation to cycle. Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus. Akt protein expression was maximal at estrus. IHC revealed that Akt expression was high in both stromal and epithelial cells at estrus. Further studies using ovariectomized rats demonstrated that 17beta-estradiol increased endometrial cell proliferation which was accompanied by an increase of both Akt expression and phosphorylation. These results suggest that increased Akt expression and activity in response to estradiol may be an important mechanism to protect endometrial cells from apoptotic triggering and to induce endometrial cell proliferation, whereas inhibition of Akt activity leads to caspase-3 activation and apoptosis in endometrial cells.

Show MeSH

Related in: MedlinePlus

Expression of CDC47/MCM7 (A), Akt (B) and Phospho-Akt (C) in response to 17β-estradiol (E2) in rat endometrium. Animals were ovariectomized for at least 10 days and then injected daily with E2 (or vehicle for control group) for 3 days. Rats were killed and endometrial proteins were collected for Western analysis. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent western blots densitometrical analysis and are the mean ± SEM of five independent experiments (5 different rats/endometrial extract per treatment group). *Significantly different from control (p < 0.001).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC161822&req=5

Figure 6: Expression of CDC47/MCM7 (A), Akt (B) and Phospho-Akt (C) in response to 17β-estradiol (E2) in rat endometrium. Animals were ovariectomized for at least 10 days and then injected daily with E2 (or vehicle for control group) for 3 days. Rats were killed and endometrial proteins were collected for Western analysis. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent western blots densitometrical analysis and are the mean ± SEM of five independent experiments (5 different rats/endometrial extract per treatment group). *Significantly different from control (p < 0.001).

Mentions: To further determine if 17β-estradiol might be involved in the regulation Akt phosphorylation/activity and expression, ovariectomized rats were treated with 17β-estradiol for 3 days. The results indicated that 17β-estradiol increased endometrial cell proliferation significantly (as determined by the expression of CDC47/MCM7) (Fig. 6). Furthermore, 17β-estradiol significantly induced both total Akt protein expression and phosphorylation. IHC revealed that 17β-estradiol induced both Akt expression and phosphorylation and was found at the cellular membrane as observed at estrus. IHC studies confirmed in ovariectomized rats treated by 17β-estradiol that both Akt expression and phosphorylation was increased in endometrial cells (Fig. 7). Increased endometrial cell proliferation (epithelial and stromal) in response to 17β-estradiol was confirmed using CDC47/ MCM7 proliferation marker (Fig. 7).


Regulation of Akt expression and phosphorylation by 17beta-estradiol in the rat uterus during estrous cycle.

Dery MC, Leblanc V, Shooner C, Asselin E - Reprod. Biol. Endocrinol. (2003)

Expression of CDC47/MCM7 (A), Akt (B) and Phospho-Akt (C) in response to 17β-estradiol (E2) in rat endometrium. Animals were ovariectomized for at least 10 days and then injected daily with E2 (or vehicle for control group) for 3 days. Rats were killed and endometrial proteins were collected for Western analysis. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent western blots densitometrical analysis and are the mean ± SEM of five independent experiments (5 different rats/endometrial extract per treatment group). *Significantly different from control (p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161822&req=5

Figure 6: Expression of CDC47/MCM7 (A), Akt (B) and Phospho-Akt (C) in response to 17β-estradiol (E2) in rat endometrium. Animals were ovariectomized for at least 10 days and then injected daily with E2 (or vehicle for control group) for 3 days. Rats were killed and endometrial proteins were collected for Western analysis. β-actin blots shown were used as controls to correct for loading in each lane. Blots shown are from one representative experiment. Graphics represent western blots densitometrical analysis and are the mean ± SEM of five independent experiments (5 different rats/endometrial extract per treatment group). *Significantly different from control (p < 0.001).
Mentions: To further determine if 17β-estradiol might be involved in the regulation Akt phosphorylation/activity and expression, ovariectomized rats were treated with 17β-estradiol for 3 days. The results indicated that 17β-estradiol increased endometrial cell proliferation significantly (as determined by the expression of CDC47/MCM7) (Fig. 6). Furthermore, 17β-estradiol significantly induced both total Akt protein expression and phosphorylation. IHC revealed that 17β-estradiol induced both Akt expression and phosphorylation and was found at the cellular membrane as observed at estrus. IHC studies confirmed in ovariectomized rats treated by 17β-estradiol that both Akt expression and phosphorylation was increased in endometrial cells (Fig. 7). Increased endometrial cell proliferation (epithelial and stromal) in response to 17β-estradiol was confirmed using CDC47/ MCM7 proliferation marker (Fig. 7).

Bottom Line: Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus.Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells.Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Chimie-Biologie, Section Biologie Médicale, Université du Québec à Trois-Rivières, C,P, 500, Trois-Rivières, Québec, Canada G9A 5H7. Marie-Claude_Dery@uqtr.ca

ABSTRACT
Molecular and intra-cellular mechanisms involved in the regulation of apoptosis processes in endometrial cells are poorly understood and documented. We have investigated the possibility that Akt survival pathway might be involved in the regulation of apoptosis in the uterus during the estrous cycle. Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Uteri were collected and fixed for immunohistochemical staining (IHC) and apoptotic cell death detection by [TdT]-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) or endometrial protein extracts collected for Western analysis. TUNEL analysis revealed that apoptosis was mainly found at estrus compared to other day of estrous cycle. TUNEL positive cells were apparent in luminal epithelial cells only. No apoptotic cells were observed at proestrus. In contrast, proliferation was maximal at proestrus as confirmed with the expression of CDC47/MCM7 (a cell proliferation marker). Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus. Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells. PTEN protein, a phosphatase involved in the regulation of Akt phosphorylation, was present at all days of estrous cycle and showed no significant regulation in relation to cycle. Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus. Akt protein expression was maximal at estrus. IHC revealed that Akt expression was high in both stromal and epithelial cells at estrus. Further studies using ovariectomized rats demonstrated that 17beta-estradiol increased endometrial cell proliferation which was accompanied by an increase of both Akt expression and phosphorylation. These results suggest that increased Akt expression and activity in response to estradiol may be an important mechanism to protect endometrial cells from apoptotic triggering and to induce endometrial cell proliferation, whereas inhibition of Akt activity leads to caspase-3 activation and apoptosis in endometrial cells.

Show MeSH
Related in: MedlinePlus