Increased Expression of the Relaxin Receptor (LGR7) in Human Endometrium during the Secretory Phase of the Menstrual Cycle

Plasma membrane Ca2+-pumping ATPases (PMCA) play a critical role in maintaining cellular Ca2+ homeostasis. The PMCA mRNA are encoded on 4 genes, designated PMCA1 to PMCA4. In a previous study, we found that both PMCA1 and PMCA4 are expressed at similar levels in astrocytes and in neurons. Although PMCA1b is expressed in the uterus of rats during the oestrous cycle, the expression of PMCA1 and its potential roles has not been elucidated during the menstrual cycle in the human endometrium. Thus, in the current study, the expression pattern of PMCA1 was examined to predict its roles in the human endometrium during the menstrual cycle. Human uterine tissues (total n = 40) were separated into 3 groups according to menstrual cycle phase: menstrual phase, proliferative phase (early, mid, late), and secretory phase (early, mid, late). Using real-time PCR and Western blot analysis, uterine expression of PMCA1 mRNA and protein increased to 1.5-fold in the early-, mid- and late-proliferative phases in the endometrium of the human uterus, compared with other menstrual phases. In addition, uterine PMCA1 was abundantly localised in the cytoplasm of the luminal and glandular epithelial cells in the menstrual phases, indicating that this protein may participate in the uterine Ca balance of the human endometrium during the menstrual cycle. Taken together, these results suggest that a high level of uterine PMCA1 expression may be involved in reproductive functions during the menstrual cycle of humans.

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Expression of Functional Prolactin Receptors in Nonpregnant Human Endometrium: Janus Kinase-2, Signal Transducer and Activator of Transcription-1 (STAT1), and STAT5 Proteins Are Phosphorylated after Stimulation with Prolactin

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.7.4989 ◽

1998 ◽

Vol 83 (7) ◽

pp. 2545-2553 ◽

Cited By ~ 27

Author(s):

H. N. Jabbour ◽

H. O. D. Critchley ◽

S. C. Boddy

Keyword(s):

Menstrual Cycle ◽

Stromal Cells ◽

Human Endometrium ◽

Janus Kinase ◽

Glandular Epithelium ◽

Ribonuclease Protection Assay ◽

Signal Transducer ◽

Janus Kinase 2 ◽

Secretory Phase ◽

Stat Proteins

PRL is synthesized by decidualized endometrial stromal cells from the midsecretory phase in a nonconception cycle and throughout pregnancy. The exact role of PRL in the human endometrium remains to be elucidated; however, the pattern of expression supports a role for PRL during implantation and placentation. This study investigated the site and pattern of expression of PRL receptors in the nonpregnant human endometrium. In situ hybridization and immunohistochemistry localized expression of the receptor in the glandular epithelium and a subset of stromal cells of the endometrium. As judged by the intensity of staining, expression of the receptor was dramatically up-regulated during the secretory phase. Expression of the PRL receptor gene in the endometrium from the secretory phase of the menstrual cycle was confirmed by ribonuclease protection assay using 50μ g total ribonucleic acid. Phosphorylation of Janus kinase-2 (JAK2), STAT1 (signal transducer and activator of transcription-1), and STAT5 proteins in response to PRL was investigated to establish the signaling pathway of PRL in the human endometrium. Endometrial tissue was collected during the secretory phase of the menstrual cycle and incubated in the presence of 100 ng/mL human PRL for 0, 5, 10, and 20 min. JAK2 phosphorylation was induced by PRL at 5 min, whereas STAT1 and STAT5 phosphorylation was apparent 20 min after stimulation with PRL. Immunohistochemistry localized the JAK/STAT proteins in the glandular epithelial cells and a subset of stromal cells, as was observed for the PRL receptor. Secretory phase stromal and glandular cells cultured separately and in the presence or absence of 100 ng/mL PRL confirmed the PRL-induced phosphorylation of JAK2/STAT proteins, at least in the glandular compartment. These studies demonstrate an up-regulation of expression of functional PRL receptors during the secretory phase of the menstrual cycle. Further, decidual PRL through a paracrine mechanism may influence glandular epithelial function/secretions and direct gene transcription through the JAK/STAT pathway. The target genes activated by PRL in the glandular epithelium of the nonpregnant human endometrium remain to be elucidated.

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241. Changes in the expression of Annexin IV mRNA and protein in human endometrium

Reproduction Fertility and Development ◽

10.1071/srb05abs241 ◽

2005 ◽

Vol 17 (9) ◽

pp. 95

Author(s):

A. P. Ponnampalam ◽

P. A. W. Rogers

Keyword(s):

Menstrual Cycle ◽

Cellular Localization ◽

Single Cells ◽

Human Endometrium ◽

Reproductive Age ◽

Histological Evaluation ◽

Secretory Phase ◽

Proliferative Phase ◽

Cyclic Changes ◽

Late Secretory Phase

In a previous study investigating global gene expression throughout the menstrual cycle,1 Annexin 4 (ANXIV) was identified as having significant cyclic changes in human endometrium. ANXIV belongs to a ubiquitous family of Ca2+-dependent phospholipid and membrane-binding proteins. The aims of this study were to investigate the cellular localization and regulation of ANXIV mRNA and temporal expression of ANXIV protein in human endometrium during the menstrual cycle. mRNA Expression: The menstrual cycle was divided into seven stages by histological evaluation. Curettings of endometrium were collected from 60 cycling women. For cellular localization, tissues from eight endometrial curettings were dissociated with collagenase into single cells, separated into epithelial and stromal cell fractions and snap frozen. Total RNA was extracted and ANXIV mRNA was quantified by real-time PCR. Immunohistochemistry: Full thickness endometrial tissue was collected from 50 reproductive age women undergoing hysterectomy. Tissue sections were formalin-fixed and paraffin-embedded. Goat polyclonal ANXIV antibody was used to localize ANXIV protein. ANXIV mRNA was significantly upregulated in the whole tissue during mid-late secretory phase of the cycle, and was predominantly expressed in epithelial cells. ANXIV protein was detected in the luminal and glandular epithelium in high levels throughout the menstrual cycle except in early secretory (ES) phase. The intensity of immunostaining was stronger in the glands of the basalis compared to functionalis in early proliferative phase, however, by the late secretory phase the functionalis glands showed higher expression levels. ANXIV mRNA data are consistent with a role for progesterone in upregulating the expression of ANXIV, although protein levels remain high through menstruation and into the proliferative phase. ANXIV can indirectly inhibit prostaglandin production, which is important for implantation. Hence the low levels of ANXIV protein at ES phase may relate to processes involved in implantation. (1)Ponnampalam et al. (2004). Mol. Hum. Reprod. 10(12), 879–893.

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Localization of Interferon Regulatory Factor-1 (IRF-1) in Nonpregnant Human Endometrium: Expression of IRF-1 Is Up-Regulated by Prolactin during the Secretory Phase of the Menstrual Cycle

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.84.11.6142 ◽

1999 ◽

Vol 84 (11) ◽

pp. 4260-4265 ◽

Cited By ~ 9

Author(s):

H. N. Jabbour ◽

H. O. D. Critchley ◽

L.-y. Yu-Lee ◽

S. C. Boddy

Keyword(s):

Menstrual Cycle ◽

Interferon Regulatory Factor ◽

Human Endometrium ◽

Regulatory Factor ◽

Secretory Phase ◽

Interferon Regulatory Factor 1

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126. Progesterone regulates CXCL14 (macrophage inflammatory protein 2γ) mRNA in human endometrium

Reproduction Fertility and Development ◽

10.1071/srb05abs126 ◽

2005 ◽

Vol 17 (9) ◽

pp. 75

Author(s):

N. M. Mokhtar ◽

S. K. Smith ◽

D. Charnock-Jones

Keyword(s):

Menstrual Cycle ◽

In Situ Hybridisation ◽

Transcript Level ◽

Human Endometrium ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Secretory Phase ◽

Ishikawa Cell ◽

Inflammatory Protein ◽

Macrophage Inflammatory Protein

The emergence of microarray technology has enabled a thorough study of the level of transcripts in the human body. A high density micoarray analysis revealed a comprehensive list of transcripts, which were significantly different between mid-proliferative and mid-secretory phase endometrium.2 An EST identified from the HG_U95B chip is identical to the 3′UTR of CXCL14 or macrophage inflammatory protein 2γ (MIP 2γ). The level is 19-fold higher in the mid-secretory compared to the mid-proliferative phase of menstrual cycle. This has suggested that the transcript level of CXCL14 may be directly regulated by progesterone. Northern hybridisation and in situ hybridisation confirmed that the transcript level of CXCL14 (MIP 2γ) was high in the mid-to-late secretory endometrium and its mRNA was localised in the glandular epithelium of this tissue.1 In silico analysis has predicted six progesterone response elements (PREs) within 2040 bp upstream from the ATG site. To investigate the possible functions of these PREs, a dual luciferase assay was performed on the ishikawa cell line transfected with five deletion constructs of the gene promoter. Cells were co-transfected with progesterone receptor B (PRB) and treated with 10–6 M progesterone. Luciferase activities of these constructs have localised two fragments that were most likely to contain the active PREs, i.e. PRE1 and PRE2. An electrophoretic mobility shift assay showed that PRE oligonulcleotides within these two regions were able to bind PRB that was synthesised in vitro, although there was a stronger signal seen in the PRE2 region. A dose competition study revealed PRE1/PRB and PRE2/PRB protein binding could be competed with different concentrations of cold wild-type competitor oligonucleotides. Mutagenesis of PRE1 and PRE2 analysed by luciferase reporter assay reduced the inductive effect of progesterone treatment. This study indicates that progestegen induced transcript encoding a chemokine in the human endometrium may likely act as a chemoattractant for leucocytes during the secretory phase of the menstrual cycle. (1)Mokhtar NM, Smith SK, Charnock-Jones DS. (2003). Characterisation of chemokine macrophage inflammatory protein 2gamma mRNA in human endometrium. 50th Society for Gynaecologic Investigation. Washington DC, USA. March 2003.(2)Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillip SC, Smith SK. (2003). Determination of the transcript profile of human endometrium. Mol. Hum. Reprod. 9, 19–33.

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