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Estrogen Regulates Local Cysteine Metabolism in Mouse Myometrium

  • Reproductive Biology: Original Article
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Abstract

Sulfur amino acid metabolism influences reproductive physiology, and transsulfuration in particular may be critical for normal cellular function. The sex hormone estrogen (E2) modulates gene expression and redox balance in some tissues by inducing the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The role of sex hormones in sulfur amino acid metabolism by uterine smooth muscle is not known. Here, we show that CBS and CSE proteins increase in the mouse myometrium during estrus and diestrus, respectively, suggesting that E2 reciprocally regulates myometrial CBS and CSE expression. In ovariectomized mice, exogenous E2 upregulates CBS and downregulates CSE levels. E2 promotes CBS mRNA and protein expression but attenuates CSE protein expression without affecting CSE mRNA. This pattern of E2-stimulated changes in transsulfuration enzyme expression is specific to the uterine smooth muscle. E2 does not change vaginal or cervical expression of CBS or CSE significantly, and E2 decreases expression of CSE in the liver without affecting CBS. E2 also downregulates myometrial cysteinesulfinic acid decarboxylase (CSAD) and decreases myometrial biochemical synthesis of the gaso-transmitter hydrogen sulfide (H2S). These findings suggest that myometrial sulfur amino acid metabolism may regulate uterine redox homeostasis, with implications for the source and metabolism of myometrial cysteine in high E2 states such as estrus and pregnancy.

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All data and material are available upon request to the corresponding author.

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Acknowledgments

The authors thank Brian W. Wakefield for technical assistance.

Funding

This work was supported by a Perinatology-Neonatology T32 training grant (5T32HD007186-37, to DDG), a Basil O’Connor Starter Scholar Award (5-FY12-57), and a Society for Maternal Fetal Medicine/American Association of Obstetricians and Gynecologists Foundation Scholar Award (to KJH).

KNM is supported by the William R. Hummel Homocystinuria Research Fund and the Ehst-Hummel-Kaufmann Family Endowed Chair in Inherited Metabolic Disease.

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Authors and Affiliations

  1. Department of Biology , University of Louisville , 2301 South 3rd Street, Louisville, Kentucky, 40292, USA

    Damian D. Guerra

  2. Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, Aurora, CO, 80045, USA

    Rachael Bok, Kelsey Breen, Vibhuti Vyas & K. Joseph Hurt

  3. Section of Clinical Genetics and Metabolism, Department of Pediatrics, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, Aurora, CO, 80045, USA

    Hua Jiang & Kenneth N. MacLean

  4. Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue, Mail Stop 8613, Aurora, CO, 80045, USA

    K. Joseph Hurt

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  1. Damian D. Guerra
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Summary sentence: Estrogen upregulates cystathionine-β-synthase (CBS) and downregulates cystathionine-γ-lyase (CSE) and cysteinesulfinic acid decarboxylase (CSAD) in a myometrium-specific manner suggesting local regulation of uterine cysteine metabolism.

Electronic Supplementary Material

ESM 1

Validation of CBS and CSE antibodies. Immunoblots of liver lysates confirming antibody specificity. CSE KO: CSE-null. CBS KO: CBS-null. GAPDH: loading control. (PPTX 56 kb)

ESM 2

Isolated mouse primary uterine smooth muscle cells exhibit expected morphology and immunomarkers. We assessed expression of CBS and CSE and various markers in uterine smooth muscle cells and control cell lines: α-SMA (myocytes), vimentin (fibroblasts), cytokeratin (epithelial cells), VWF (endothelial cells), CBS, and CSE. Control cells were: PHM1–41 (human myometrial myocytes), MS1 (mouse pancreatic islet endothelium), Ishikawa (human endometrial adenocarcinoma), and HeLa cells (cytokeratin only). Primary myometrial cells and PHM1–41 cells do not express significant cytokeratin, VWF, or vimentin. They exhibit fusiform morphology characteristic of myocytes. Upper left insets: primary antibody excluded (negative controls). Blue channel: DAPI. Scale bars: 20 μm. (PPTX 1206 kb)

ESM 3

Estrogen induces uterine hypertrophy and alters vaginal lavage cytology in ovariectomized mice. A. E2 increases uterine volume (alone or in combination with P4), while P4 slightly reduces volume. Scale bars: 5 mm. B. E2 and P4 increase the relative abundance of cornified cells and leukocytes, respectively. Scale bars: 100 μm. (PPTX 1367 kb)

ESM 4

Estrogen and progesterone effects on CBS and CSE protein expression do not vary by uterine location in ovariectomized mice. A. Upper and lower uterus CBS and CSE respond identically to E2 and P4. GAPDH: loading control. B-C. Quantification of A. n.s.: not significant by two-way ANOVA (lower vs. upper uterus). N: mice. (PPTX 156 kb)

ESM 5

Estrogen and progesterone do not affect vaginal or cervical CBS or CSE protein levels. A. E2 and P4 do not affect CBS or CSE protein levels in ovariectomized mouse cervix or vagina. Veh: sesame oil vehicle. β-actin: loading control. B-C. Quantification of A. n.s.: not significant by Kruskal-Wallis test. N: mice. (PPTX 158 kb)

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Guerra, D.D., Bok, R., Breen, K. et al. Estrogen Regulates Local Cysteine Metabolism in Mouse Myometrium. Reprod. Sci. 28, 79–90 (2021). https://doi.org/10.1007/s43032-020-00284-6

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