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Perturbing the cellular levels of steroid receptor coactivator-2 impairs murine endometrial function.

Szwarc MM, Kommagani R, Jeong JW, Wu SP, Tsai SY, Tsai MJ, O'Malley BW, DeMayo FJ, Lydon JP - PLoS ONE (2014)

Bottom Line: As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes.Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality.This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility.

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Expression levels of SRC-2 in the murine endometrium are not significantly altered during the estrous cycle.(A) Left panels show immunostaining of SRC-2 in the murine endometrium during the estrus (top panel) and diestrus (bottom panel) phase of the estrous cycle; S, GE, and LE denote stroma, glandular epithelium, and luminal epithelium respectively. Note the slightly edematous stromal compartment and columnar luminal epithelial cells typical of an endometrium during the estrus stage of the murine cycle [67]. Conversely, luminal epithelial cells are more cuboidal and the sub-epthelial stroma is more condensed in the endometrium at diestrus. Middle panels show the corresponding vaginal cytology (cornified squamous epithelial (CE) cells and polymorphonuclear leukocytes (L)) which confirm the stage of the cycle [47], [52]. Right panels show corresponding mammary gland whole mounts; SB indicates side-branches. Note: a transient increase in the number of ductal side branches in the mammary gland is known to occur during the diestrus phase of the mouse estrous cycle [51]. Scale bar in top panels apply to corresponding bottom panels. (B) Comparative SRC-2 transcript levels in the murine endometrium at each of the four stages of the estrous cycle; n.s. denotes non-specific. (C) Immunostaining of SRC-2 in the human endometrium biopsied during the proliferative (left panel) and secretory (right panel) stages of the menstrual cycle. White arrowhead indicates glandular epithelial cells and stromal cells positive for SRC-2 expression; black arrowhead in left panel shows mitotic figures whereas the grey arrowhead in both panels indicates stromal cells negative for SRC-2 expression. Note the tortuosity typically exhibited by the glandular epithelium during the secretory phase of the menstrual cycle [68] (Fig. 1C (right panel)). Scale bar in the left panel applies to the right panel.
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pone-0098664-g001: Expression levels of SRC-2 in the murine endometrium are not significantly altered during the estrous cycle.(A) Left panels show immunostaining of SRC-2 in the murine endometrium during the estrus (top panel) and diestrus (bottom panel) phase of the estrous cycle; S, GE, and LE denote stroma, glandular epithelium, and luminal epithelium respectively. Note the slightly edematous stromal compartment and columnar luminal epithelial cells typical of an endometrium during the estrus stage of the murine cycle [67]. Conversely, luminal epithelial cells are more cuboidal and the sub-epthelial stroma is more condensed in the endometrium at diestrus. Middle panels show the corresponding vaginal cytology (cornified squamous epithelial (CE) cells and polymorphonuclear leukocytes (L)) which confirm the stage of the cycle [47], [52]. Right panels show corresponding mammary gland whole mounts; SB indicates side-branches. Note: a transient increase in the number of ductal side branches in the mammary gland is known to occur during the diestrus phase of the mouse estrous cycle [51]. Scale bar in top panels apply to corresponding bottom panels. (B) Comparative SRC-2 transcript levels in the murine endometrium at each of the four stages of the estrous cycle; n.s. denotes non-specific. (C) Immunostaining of SRC-2 in the human endometrium biopsied during the proliferative (left panel) and secretory (right panel) stages of the menstrual cycle. White arrowhead indicates glandular epithelial cells and stromal cells positive for SRC-2 expression; black arrowhead in left panel shows mitotic figures whereas the grey arrowhead in both panels indicates stromal cells negative for SRC-2 expression. Note the tortuosity typically exhibited by the glandular epithelium during the secretory phase of the menstrual cycle [68] (Fig. 1C (right panel)). Scale bar in the left panel applies to the right panel.

Mentions: Immunohistochemistry and quantitative real-time PCR analysis were performed to determine whether endometrial SRC-2 expression levels change during the murine estrous cycle and whether its expression shifts from one endometrial cell-type to another with cycle progression. As observed in Fig. 1A, the spatial expression profile and levels of SRC-2 expression are indistinguishable in the endometrium of a wild type mouse at the estrus and diestrus stage of the estrous cycle; vaginal cytology and mammary gland morphology confirmed each stage of the cycle [47], [51], [52] (proestrus and metestrus stages showed similar results (data not shown)). In the case of the estrus phase of the cycle, SRC-2 expression is pronounced in the luminal and glandular epithelium as compared to the edematous stromal compartment. Whereas the majority of epithelial cells score positive for SRC-2 expression, not every stromal cell is immunopositive for SRC-2 (Fig. 1A (left top panel)). A similar spatial profile and level of expression for endometrial SRC-2 is observed at the diestrus stage of the cycle (Fig. 1A (left bottom panel)). These results indicate that endometrial SRC-2 protein levels are not significantly modulated by cycling hormone (i.e. E2 and P4) levels which occur during the cycle [53]. However, it is important to note that these results do not address whether post-translational modifications (i.e. phosphorylation) of the coregulator change with cycle progression; post-translational modifications are known regulators of SRC activity in other physiological systems [54]. In Fig. 1B, quantitative real-time PCR analysis demonstrates that endometrial SRC-2 transcript levels do not change with each stage of the cycle in the wild type mouse, further supporting conclusions drawn from results shown in Fig. 1A. Importantly, similar findings are obtained by immunohistochemical analysis of human endometrial samples biopsied during the proliferative and mid-secretory phase of the menstrual cycle (Fig. 1C). Like the mouse, SRC-2 expression is predominantly expressed in the epithelium of the human endometrium with equivalent expression levels of SRC-2 during the proliferative and secretory phases of the cycle; these findings confirm previous clinical observations [29]. Together, these results support the conclusion that transcript and protein levels of endometrial SRC-2 are not markedly altered during murine cycle progression, with a similar SRC-2 expression profile observed in the human endometrium.


Perturbing the cellular levels of steroid receptor coactivator-2 impairs murine endometrial function.

Szwarc MM, Kommagani R, Jeong JW, Wu SP, Tsai SY, Tsai MJ, O'Malley BW, DeMayo FJ, Lydon JP - PLoS ONE (2014)

Expression levels of SRC-2 in the murine endometrium are not significantly altered during the estrous cycle.(A) Left panels show immunostaining of SRC-2 in the murine endometrium during the estrus (top panel) and diestrus (bottom panel) phase of the estrous cycle; S, GE, and LE denote stroma, glandular epithelium, and luminal epithelium respectively. Note the slightly edematous stromal compartment and columnar luminal epithelial cells typical of an endometrium during the estrus stage of the murine cycle [67]. Conversely, luminal epithelial cells are more cuboidal and the sub-epthelial stroma is more condensed in the endometrium at diestrus. Middle panels show the corresponding vaginal cytology (cornified squamous epithelial (CE) cells and polymorphonuclear leukocytes (L)) which confirm the stage of the cycle [47], [52]. Right panels show corresponding mammary gland whole mounts; SB indicates side-branches. Note: a transient increase in the number of ductal side branches in the mammary gland is known to occur during the diestrus phase of the mouse estrous cycle [51]. Scale bar in top panels apply to corresponding bottom panels. (B) Comparative SRC-2 transcript levels in the murine endometrium at each of the four stages of the estrous cycle; n.s. denotes non-specific. (C) Immunostaining of SRC-2 in the human endometrium biopsied during the proliferative (left panel) and secretory (right panel) stages of the menstrual cycle. White arrowhead indicates glandular epithelial cells and stromal cells positive for SRC-2 expression; black arrowhead in left panel shows mitotic figures whereas the grey arrowhead in both panels indicates stromal cells negative for SRC-2 expression. Note the tortuosity typically exhibited by the glandular epithelium during the secretory phase of the menstrual cycle [68] (Fig. 1C (right panel)). Scale bar in the left panel applies to the right panel.
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pone-0098664-g001: Expression levels of SRC-2 in the murine endometrium are not significantly altered during the estrous cycle.(A) Left panels show immunostaining of SRC-2 in the murine endometrium during the estrus (top panel) and diestrus (bottom panel) phase of the estrous cycle; S, GE, and LE denote stroma, glandular epithelium, and luminal epithelium respectively. Note the slightly edematous stromal compartment and columnar luminal epithelial cells typical of an endometrium during the estrus stage of the murine cycle [67]. Conversely, luminal epithelial cells are more cuboidal and the sub-epthelial stroma is more condensed in the endometrium at diestrus. Middle panels show the corresponding vaginal cytology (cornified squamous epithelial (CE) cells and polymorphonuclear leukocytes (L)) which confirm the stage of the cycle [47], [52]. Right panels show corresponding mammary gland whole mounts; SB indicates side-branches. Note: a transient increase in the number of ductal side branches in the mammary gland is known to occur during the diestrus phase of the mouse estrous cycle [51]. Scale bar in top panels apply to corresponding bottom panels. (B) Comparative SRC-2 transcript levels in the murine endometrium at each of the four stages of the estrous cycle; n.s. denotes non-specific. (C) Immunostaining of SRC-2 in the human endometrium biopsied during the proliferative (left panel) and secretory (right panel) stages of the menstrual cycle. White arrowhead indicates glandular epithelial cells and stromal cells positive for SRC-2 expression; black arrowhead in left panel shows mitotic figures whereas the grey arrowhead in both panels indicates stromal cells negative for SRC-2 expression. Note the tortuosity typically exhibited by the glandular epithelium during the secretory phase of the menstrual cycle [68] (Fig. 1C (right panel)). Scale bar in the left panel applies to the right panel.
Mentions: Immunohistochemistry and quantitative real-time PCR analysis were performed to determine whether endometrial SRC-2 expression levels change during the murine estrous cycle and whether its expression shifts from one endometrial cell-type to another with cycle progression. As observed in Fig. 1A, the spatial expression profile and levels of SRC-2 expression are indistinguishable in the endometrium of a wild type mouse at the estrus and diestrus stage of the estrous cycle; vaginal cytology and mammary gland morphology confirmed each stage of the cycle [47], [51], [52] (proestrus and metestrus stages showed similar results (data not shown)). In the case of the estrus phase of the cycle, SRC-2 expression is pronounced in the luminal and glandular epithelium as compared to the edematous stromal compartment. Whereas the majority of epithelial cells score positive for SRC-2 expression, not every stromal cell is immunopositive for SRC-2 (Fig. 1A (left top panel)). A similar spatial profile and level of expression for endometrial SRC-2 is observed at the diestrus stage of the cycle (Fig. 1A (left bottom panel)). These results indicate that endometrial SRC-2 protein levels are not significantly modulated by cycling hormone (i.e. E2 and P4) levels which occur during the cycle [53]. However, it is important to note that these results do not address whether post-translational modifications (i.e. phosphorylation) of the coregulator change with cycle progression; post-translational modifications are known regulators of SRC activity in other physiological systems [54]. In Fig. 1B, quantitative real-time PCR analysis demonstrates that endometrial SRC-2 transcript levels do not change with each stage of the cycle in the wild type mouse, further supporting conclusions drawn from results shown in Fig. 1A. Importantly, similar findings are obtained by immunohistochemical analysis of human endometrial samples biopsied during the proliferative and mid-secretory phase of the menstrual cycle (Fig. 1C). Like the mouse, SRC-2 expression is predominantly expressed in the epithelium of the human endometrium with equivalent expression levels of SRC-2 during the proliferative and secretory phases of the cycle; these findings confirm previous clinical observations [29]. Together, these results support the conclusion that transcript and protein levels of endometrial SRC-2 are not markedly altered during murine cycle progression, with a similar SRC-2 expression profile observed in the human endometrium.

Bottom Line: As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes.Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality.This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility.

Show MeSH
Related in: MedlinePlus