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Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus.

ABSTRACT: Cluster of differentiation 73 (CD73, also known as ecto-5'-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.

SUBMITTER: Lee J

PROVIDER: S-EPMC8431015 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:BACKGROUND: SERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent serpins that modulates the activity of the plasminogen activator (PA) and was implicated in tissue remodeling. In this study, we investigated the expression patterns of SERPINE2 in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation. METHODS: SERPINE2 was purified from mouse seminal vesicle secretion using liquid chromatography (LC) and identified by LC/tandem mass spectrometry. The antiserum against the SERPINE2 protein was raised in rabbits. To reveal the uterine and placental expression of SERPINE2, tissues at various stages were collected for real-time PCR quantification, Western blotting, and immunohistochemical staining. RESULTS: Serpine2 mRNA was the major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation, although Serpine1 mRNA had higher expression levels than Serpine2 mRNA in the placenta. Plat seemed to be the major PA in the mouse uterus and placenta. Antiserum against the SERPINE2 protein specifically recognized two forms of SERPINE2 and an extra 75-kDa protein, which was probably a complex of SERPINE2 with a certain protease, from among thousands of protein components in the tissue extract as demonstrated by Western blotting. In the uterus, SERPINE2 was primarily localized in luminal and glandular epithelial cells but it also was detected in circular and longitudinal smooth muscle cells during the estrous cycle and lactation. It was prominently expressed in decidual stroma cells, the metrial gland, and endometrial epithelium of the pregnant uterus. In the placenta, SERPINE2 was expressed in trophoblasts of the labyrinth and spongiotrophoblasts. However, its expression was remarkably reduced in giant cells which existed in the giant cell-decidual junction zone. In contrast, prominent expression of SERPINE2 seemed to be detected on clusters of glycogen cells near the junction zone. In addition, yolk sac membranes also showed high expression of SERPINE2. CONCLUSIONS: These findings indicate that SERPINE2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. It may participate in the PA-modulated tissue remodeling process in the mouse placenta and uterus.

Project description:The objective was to investigate the expression of bone morphogenetic protein (BMP) family members in the mouse uterus during the estrous cycle by real-time polymerase chain reaction (PCR) and immunohistochemistry. Uterine samples from Swiss ICR mice were collected and dissected free of surrounding tissue. One uterine horn was snap frozen in liquid nitrogen immediately after collection and stored at -80 °C for RNA extraction, and the other was fixed in 40 mg/ml paraformaldehyde at room temperature for immunolocalization of BMP2 protein. Real-time PCR analysis showed that the expression level of Bmp2 was significantly higher at proestrus than at estrus and metestrus (P<0.05). The relative abundance of Bmp4 exhibited significant fluctuations, but there were no statistically significant differences between the expression levels of Bmp2 and Bmp4 (P>0.05). The expression levels of Bmpr1a and Bmpr2 remained unchanged during estrous cycles. However, the level of Bmpr1b mRNA decreased significantly at estrus (P<0.05), increasing subsequently at metestrus. Furthermore, the level of Bmpr1b mRNA was significantly lower than those of Bmpr1a and Bmpr2 mRNA at the corresponding stages (P<0.05). All three receptor-regulated Smads (R-Smads) detected were differentially expressed in the mouse uterus and the expression levels of Smad1 and Smad5 were significantly higher than that of Smad8 (P<0.05). In addition, the expression level of Smad4 did not change substantially throughout the estrous cycle. Immunohistochemical experiments revealed that BMP2 protein was differentially expressed and localized mainly in the uterine luminal and glandular epithelial cells throughout the estrous cycle. In conclusion, our results provide information about the variation in the mRNA levels of Bmp2 and Bmp4 and related components of the BMP signaling pathway. The data provide quantitative and useful information about the roles of endometrial BMP proposed and demonstrated by others, such as the degradation and remodeling of the endometrium.

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Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus.

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