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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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Spatial expression pattern of hSRC-2 in the endometrium of the SRC-2:OE mouse.(A) and (B) represent low magnification images of transverse sections of the uterine mid-horn immunostained for SRC-2 from the SRC-2LSL and SRC-2:OE mouse respectively. (C and D) display higher magnification images of uterine sections shown in (A and B) respectively. Corresponding uterine sections stained for the Myc-epitope tag are shown in Fig. S2. (E) and (F) show high magnification images of uterine mid-horn sections stained for BrdU incorporation from the SRC-2LSL and SRC-2:OE mouse respectively. (G) Bar graph shows the percentage of BrdU incorporation in the LE and GE cellular compartments of the SRC-2:OE endometrium compared to the corresponding SRC-2LSL control (***denotes p<0.001 (n = 5 mice per genotype)). Scale bars in (A, C, and E) apply to (B, D, and F) respectively; LE, GE, and S denote luminal epithelium, glandular epithelium, and stroma respectively.
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pone-0098664-g004: Spatial expression pattern of hSRC-2 in the endometrium of the SRC-2:OE mouse.(A) and (B) represent low magnification images of transverse sections of the uterine mid-horn immunostained for SRC-2 from the SRC-2LSL and SRC-2:OE mouse respectively. (C and D) display higher magnification images of uterine sections shown in (A and B) respectively. Corresponding uterine sections stained for the Myc-epitope tag are shown in Fig. S2. (E) and (F) show high magnification images of uterine mid-horn sections stained for BrdU incorporation from the SRC-2LSL and SRC-2:OE mouse respectively. (G) Bar graph shows the percentage of BrdU incorporation in the LE and GE cellular compartments of the SRC-2:OE endometrium compared to the corresponding SRC-2LSL control (***denotes p<0.001 (n = 5 mice per genotype)). Scale bars in (A, C, and E) apply to (B, D, and F) respectively; LE, GE, and S denote luminal epithelium, glandular epithelium, and stroma respectively.

Mentions: To confirm that the hSRC-2 transgene is expressed in all endometrial cell types that express PR and SRC-2 [27], immunohistochemistry was applied to endometrial sections obtained from 3-month old SRC-2:OE and SRC-2LSL siblings (Fig. 4). Using antibodies against hSRC-2, high levels of hSRC-2 expression was detected in the luminal epithelium, glandular epithelium, and stromal cellular compartments of the SRC-2:OE endometrium. As seen at low power magnification, the endometrium of the SRC-2:OE mouse is consistently larger than that of its SRC-2LSL sibling (compare Fig. 4A with 4B; also Fig. S2). Although the size of the myometrium is equivalent in the SRC-2:OE and SRC-2LSL uterus, the SRC-2:OE stromal and glandular epithelial compartments are consistently larger than seen in the SRC-2LSL uterus. At higher power magnification, it is clear that the SRC-2:OE endometrium displays larger epithelial glands than the SRC-2LSL endometrium (compare Fig. 4C with 4D; also Fig. S2.). The majority of the glandular epithelial cells in the SRC-2:OE endometrium are positive for high levels of hSRC-2 expression (Fig. 4D; specificity of SRC-2 antibody was confirmed using uterine tissue obtained from the SRC-2 knockout mouse (Fig. S3)). Furthermore, immunohistochemical analysis of BrdU incorporation revealed a significant increase in the number of luminal and glandular epithelial cells that score positive for BrdU in the SRC-2:OE endometrium when compared to the SRC-2LSL endometrium (Fig. 4 G–I; also Fig. S2). A similar result was observed in six month old SRC-2:OE mice (Fig. S2). Given the observed increase in the number of proliferating epithelial cells in the SRC-2:OE endometrium, we next asked whether this increase in proliferation would be enough to influence the fertility status of the mouse.


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)

Spatial expression pattern of hSRC-2 in the endometrium of the SRC-2:OE mouse.(A) and (B) represent low magnification images of transverse sections of the uterine mid-horn immunostained for SRC-2 from the SRC-2LSL and SRC-2:OE mouse respectively. (C and D) display higher magnification images of uterine sections shown in (A and B) respectively. Corresponding uterine sections stained for the Myc-epitope tag are shown in Fig. S2. (E) and (F) show high magnification images of uterine mid-horn sections stained for BrdU incorporation from the SRC-2LSL and SRC-2:OE mouse respectively. (G) Bar graph shows the percentage of BrdU incorporation in the LE and GE cellular compartments of the SRC-2:OE endometrium compared to the corresponding SRC-2LSL control (***denotes p<0.001 (n = 5 mice per genotype)). Scale bars in (A, C, and E) apply to (B, D, and F) respectively; LE, GE, and S denote luminal epithelium, glandular epithelium, and stroma respectively.
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pone-0098664-g004: Spatial expression pattern of hSRC-2 in the endometrium of the SRC-2:OE mouse.(A) and (B) represent low magnification images of transverse sections of the uterine mid-horn immunostained for SRC-2 from the SRC-2LSL and SRC-2:OE mouse respectively. (C and D) display higher magnification images of uterine sections shown in (A and B) respectively. Corresponding uterine sections stained for the Myc-epitope tag are shown in Fig. S2. (E) and (F) show high magnification images of uterine mid-horn sections stained for BrdU incorporation from the SRC-2LSL and SRC-2:OE mouse respectively. (G) Bar graph shows the percentage of BrdU incorporation in the LE and GE cellular compartments of the SRC-2:OE endometrium compared to the corresponding SRC-2LSL control (***denotes p<0.001 (n = 5 mice per genotype)). Scale bars in (A, C, and E) apply to (B, D, and F) respectively; LE, GE, and S denote luminal epithelium, glandular epithelium, and stroma respectively.
Mentions: To confirm that the hSRC-2 transgene is expressed in all endometrial cell types that express PR and SRC-2 [27], immunohistochemistry was applied to endometrial sections obtained from 3-month old SRC-2:OE and SRC-2LSL siblings (Fig. 4). Using antibodies against hSRC-2, high levels of hSRC-2 expression was detected in the luminal epithelium, glandular epithelium, and stromal cellular compartments of the SRC-2:OE endometrium. As seen at low power magnification, the endometrium of the SRC-2:OE mouse is consistently larger than that of its SRC-2LSL sibling (compare Fig. 4A with 4B; also Fig. S2). Although the size of the myometrium is equivalent in the SRC-2:OE and SRC-2LSL uterus, the SRC-2:OE stromal and glandular epithelial compartments are consistently larger than seen in the SRC-2LSL uterus. At higher power magnification, it is clear that the SRC-2:OE endometrium displays larger epithelial glands than the SRC-2LSL endometrium (compare Fig. 4C with 4D; also Fig. S2.). The majority of the glandular epithelial cells in the SRC-2:OE endometrium are positive for high levels of hSRC-2 expression (Fig. 4D; specificity of SRC-2 antibody was confirmed using uterine tissue obtained from the SRC-2 knockout mouse (Fig. S3)). Furthermore, immunohistochemical analysis of BrdU incorporation revealed a significant increase in the number of luminal and glandular epithelial cells that score positive for BrdU in the SRC-2:OE endometrium when compared to the SRC-2LSL endometrium (Fig. 4 G–I; also Fig. S2). A similar result was observed in six month old SRC-2:OE mice (Fig. S2). Given the observed increase in the number of proliferating epithelial cells in the SRC-2:OE endometrium, we next asked whether this increase in proliferation would be enough to influence the fertility status of the mouse.

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