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Changes in Mouse Uterine Transcriptome in Estrus and Proestrus

ABSTRACT: The potential for xenoestrogens and other environmental chemicals to alter female reproductive function calls for the study of their affects on endogenous hormone-regulated gene expression pathways during uterine tissue remodeling which occurs as part of the menstrual/estrus cycle. However, our knowledge of these pathways is limited. Here, we characterize changes in the CD-1 mouse uterine transcriptome during proestrus and estrus, which are regulated by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 wk of age and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis identified 2,429 genes differentially expressed in estrus compared to proestrus, indicating that the mouse uterus undergoes remarkable remodeling during the estrus cycle, affecting ~10% of all protein-coding genes. Changes in gene expression associated with structural alteration of the uterus include remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis, MHC class II presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and several transcription factors with poorly understood roles in reproductive tissues, include several genes and gene networks that have been implicated in pathological states. The information presented here builds a background for understanding of mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases.

ORGANISM(S): Mus musculus

PROVIDER: GSE43064 | GEO | 2013/06/07

SECONDARY ACCESSION(S): PRJNA184202

REPOSITORIES: GEO

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Project description:The potential for xenoestrogens and other environmental chemicals to alter female reproductive function calls for the study of their affects on endogenous hormone-regulated gene expression pathways during uterine tissue remodeling which occurs as part of the menstrual/estrus cycle. However, our knowledge of these pathways is limited. Here, we characterize changes in the CD-1 mouse uterine transcriptome during proestrus and estrus, which are regulated by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 wk of age and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis identified 2,429 genes differentially expressed in estrus compared to proestrus, indicating that the mouse uterus undergoes remarkable remodeling during the estrus cycle, affecting ~10% of all protein-coding genes. Changes in gene expression associated with structural alteration of the uterus include remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis, MHC class II presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and several transcription factors with poorly understood roles in reproductive tissues, include several genes and gene networks that have been implicated in pathological states. The information presented here builds a background for understanding of mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases. Beginning on postnatal day 42, female CD-1 mice were monitored for reproductive stage cycle for 2 consecutive cycles and then total RNA was isolated from uterine horns at estrus or proestrus. Samples were pooled for microarray as follows. Six litters were used to generate 2 tissue pools for estrus and 2 tissue pools for proestrus, with estrus pool 1 and proestrus pool 1 comprised of mice from the same 3 litters, and estrus pool 2 and proestrus pool 2 comprised of mice from the other 3 litters. Each pool was comprised of uterine tissue RNA from n= 4-6 individual mice. Fluorescent labeling of RNA and hybridization of the Alexa 555-labeled (green) and Alexa 647-labeled (red) RNA samples to Agilent-026655 Whole Mouse Genome Microarray 4x44K v2 (Agilent Technology, Palo Alto, CA; catalog # G4846A) were carried out, with dye swapping for each estrus cycle stage comparison.

Project description:The uterus, a female reproductive organ regulated by the sex hormones estrogen and progesterone, undergoes periodic cyclical changes. The estrous cycle refers to the reproductive cycle in non-primate mammalian females. During the mouse estrous cycle, the uterus undergoes various physiological changes as a result of dynamic hormonal changes. Accurate regulation of these changes is crucial for the establishment of a successful pregnancy. Notably, estrogen plays an important role in the regulation of the proestrus and estrus stages of the estrous cycle. Family with sequence similarity 3 (Fam3) is a cytokine-like gene family with four members: Fam3a, Fam3b, Fam3c, and Fam3d. Expression and regulation of the Fam3 family members in mouse uterine physiology remain largely unknown. Therefore, this study aimed to investigate the expression of Fam family members in the uterus during the estrous cycle and evaluate its regulation by estrogen using a mouse model. Analysis of mouse uterine RNA sequencing data revealed upregulated expression of Fam3b, Fam3c, and Fam3d during the proestrus and estrus stages. Fam3d expression was dynamically regulated during the estrous cycle, with high expression levels during the proestrus and estrus stages. To investigate whether Fam3d expression is regulated by estrogen, we administered estradiol (E2) to ovariectomized mice at different time points. Fam3d expression was highest 24 h after E2 injection, suggesting that estrogen plays a crucial role in regulating Fam3d expression. Furthermore, inhibition experiments using the estrogen receptor alpha (ERα) antagonist ICI revealed that estrogen regulates Fam3d expression through the ERα-mediated pathway. Immunofluorescence staining demonstrated that FAM3D was exclusively expressed in the luminal and glandular epithelia but not in the stroma. Additionally, FAM3D was predominantly localized in the cytoplasm, particularly in the apical region, and not in the nucleus. These findings provide valuable insights into the potential role of Fam3d in the uterus and lay the groundwork for future research on its function and significance in uterine physiology.

Project description:The emergence of fully antimicrobial resistant Neisseria gonorrhoeae has led global public health agencies to identify a critical need for next generation anti-gonococcal pharmaceuticals. The development and success of these compounds will rely upon valid pre-clinical models of gonorrhoeae infection. We recently developed and reported the first model of upper genital tract gonococcal infection. During initial characterization, we observed significant reproductive cycle-based variation in infection outcome. When uterine infection occurred in the diestrus phase, there was significantly greater pathology than during estrus phase. The aim of this study was to evaluate transcriptional profiles of infected uterine tissue from mice in either estrus or diestrus phase in order to elucidate possible mechanisms for these differences. Genes and biological pathways with phase-independent induction during infection showed a chemokine dominant cytokine response to Neisseria gonorrhoeae. Despite general induction being phase-independent, this common anti-gonococcal response demonstrated greater induction during diestrus phase infection. Greater activity of granulocyte adhesion and diapedesis regulators during diestrus infection, particularly in chemokines and diapedesis regulators, was also shown. In addition to a greater induction of the common anti-gonococcal response, Gene Set Enrichment Analysis (GSEA) identified a diestrus-specific induction of type-1 interferon signaling pathways. This transcriptional analysis of murine uterine gonococcal infection during distinct points in the natural reproductive cycle provided evidence for a common anti-gonococcal response characterized by significant induction of granulocyte chemokine expression and high proinflammatory mediators. The basic biology of this host response to N. gonorrhoeae in estrus and diestrus is similar at the pathway level, but varies drastically in magnitude. Overlaying this, we observed type-1 interferon induction specifically in diestrus infection where greater pathology is observed. This supports recent work suggesting this pathway has a significant, possibly host-detrimental, function in gonococcal infection. Together these findings lay the groundwork for further examination of the role of interferons in gonococcal infection. Additionally, this work enables the implementation of the diestrus uterine infection model using the newly characterized host response as a marker of pathology and its prevention as a correlate of candidate vaccine efficacy and ability to protect against the devastating consequences of N. gonorrhoeae-associated sequelae. Murine microarrays were used to examine transcriptional differences underlying significantly different phenotypes associated with uterine N. gonorrhoeae infection in the estrus versus diestrus phases of the natural reproductive cycle.

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Changes in Mouse Uterine Transcriptome in Estrus and Proestrus

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