Prominin-1 glycosylation changes throughout early pregnancy in uterine epithelial cells under the influence of maternal ovarian hormones

2017 ◽  
Vol 29 (6) ◽  
pp. 1194 ◽  
Author(s):  
Samson N. Dowland ◽  
Romanthi J. Madawala ◽  
Connie E. Poon ◽  
Laura A. Lindsay ◽  
Christopher R. Murphy
Keyword(s):  
Epithelial Cells ◽  
Early Pregnancy ◽  
Core Protein ◽  
Apical Plasma Membrane ◽  
Apical Region ◽  
Uterine Epithelial Cells ◽  
Oestrogen Treatment ◽  
Extracellular Loops ◽  
Progesterone Treatment ◽  
Ovariectomised Rats

In preparation for uterine receptivity, the uterine epithelial cells (UECs) exhibit a loss of microvilli and glycocalyx and a restructuring of the actin cytoskeleton. The prominin-1 protein contains large, heavily glycosylated extracellular loops and is usually restricted to apical plasma membrane (APM) protrusions. The present study examined rat UECs during early pregnancy using immunofluorescence, western blotting and deglycosylation analyses. Ovariectomised rats were injected with oestrogen and progesterone to examine how these hormones affect prominin-1. At the time of fertilisation, prominin-1 was located diffusely in the apical domain of UECs and 147- and 120-kDa glycoforms of prominin-1 were identified, along with the 97-kDa core protein. At the time of implantation, prominin-1 concentrates towards the APM and densitometry revealed that the 120-kDa glycoform decreased (P < 0.05), but there was an increase in the 97-kDa core protein (P < 0.05). Progesterone treatment of ovariectomised rats resulted in prominin-1 becoming concentrated towards the APM. The 120-kDa glycoform was increased after oestrogen treatment (P < 0.0001), whereas the 97-kDa core protein was increased after progesterone treatment (P < 0.05). Endoglycosidase H analysis demonstrated that the 120-kDa glycoform is in the endoplasmic reticulum, undergoing protein synthesis. These results indicate that oestrogen stimulates prominin-1 production, whereas progesterone stimulates the deglycosylation and concentration of prominin-1 to the apical region of the UECs. This likely presents the deglycosylated extracellular loops of prominin-1 to the extracellular space, where they may interact with the implanting blastocyst.

312. THE DISTRIBUTION OF PROMININ-1 IN THE RAT UTERUS DURING EARLY PREGNANCY

2010 ◽  
Vol 22 (9) ◽  
pp. 112
Author(s):  
S. N. Dowland ◽  
L. A. Lindsay ◽  
C. R. Murphy
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Early Pregnancy ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Uterine Epithelial Cells ◽  
Uterine Epithelial Cell ◽  
Terminal Web ◽  
Blastocyst Implantation

Prominin-1 is a recently discovered pentaspan membrane protein present in characteristic cholesterol-based vesicles and associated with microvilli. These vesicles are used to deliver prominin-1 to the apical plasma membrane in a number of cell types. Previous work on uterine epithelial cells has demonstrated a loss of microvilli and the presence of large, cholesterol-based vesicles at the time of implantation. Thus this study aims to determine a role for prominin-1 in rat uterine epithelial cells during early pregnancy. Immunofluorescence microscopy reveals punctate and diffuse prominin-1 staining below the apical plasma membrane on day 1 of pregnancy. At the time of blastocyst implantation (day 6) however, prominin-1 appears concentrated at the apical surface of the cell. Western blotting of isolated uterine epithelial cell lysate revealed a change in prominin-1 glycosylation during early pregnancy. Prominin-1 was determined to be glycosylated on day 1 of pregnancy, but these carbohydrate side chains were lost by the time of attachment. Results seen in the present study indicate that prominin-containing vesicles may be prevented from reaching the apical plasma membrane by the terminal web on day 1 of pregnancy. On day 6, the loss of the terminal web may allow the vesicles to approach and incorporate into the apical plasma membrane, as seen with other uterine vesicles. The deglycosylation of prominin-1 at this time is suggested to allow the protein to bind its ligand and activate downstream signalling pathways that permit implantation. This study constitutes the first reported observation of prominin in endometrial lumenal epithelial cells. These preliminary results, in consideration with previous reports of prominin expression in trophoblast cells, suggest an important role for this protein in early pregnancy.

237. Aquaporins in rat uterine epithelial cells during early pregnancy and in response to progesterone

2005 ◽  
Vol 17 (9) ◽  
pp. 93
Author(s):  
L. A. Lindsay ◽  
C. R. Murphy
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Early Pregnancy ◽  
Water Movement ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Uterine Lumen ◽  
Epithelial Barriers ◽  
Luminal Epithelial Cells ◽  
Ovariectomised Rats

Implantation of the rat blastocyst is a highly regulated process, involving transformation of the uterine environment into one which is receptive to an implanting blastocyst. At the time of implantation, in response to progesterone, there is a dramatic decrease in the amount of uterine luminal fluid leading to close apposition between the luminal epithelium and trophoblastic cells. The rat blastocyst also always implants at the antimesometrial pole of the uterine lumen and currently mechanisms regulating this process are unknown. Aquaporins, a family of transmembrane water channels, are involved in the regulation of water movement across epithelial barriers. We investigated several aquaporins in the rat uterus during early pregnancy using reverse transcriptase PCR. Immunofluorescence and immunogold electron microscopy techniques were then used to investigate the localisation of particular aquaporins including AQP5 in the uterine epithelium during early pregnancy and in ovariectomised rats treated with progesterone. There was an increase in AQP5 molecules in the apical plasma membrane of luminal epithelial cells at the time of implantation, with a greater increase at the mesometrial compared to antimesometrial pole. A similar result was seen in luminal epithelial cells from ovariectomised rats treated with progesterone, however there was no differential concentration between mesometrial and antimesometrial poles, as there was during early pregnancy. It is suggested that the increase in AQP5 protein expression in the apical plasma membrane of luminal epithelial cells is involved in reabsorption of luminal fluid at the time of implantation. Furthermore, the differential concentration of AQP5 on luminal epithelial cells at the time of implantation could lead to the establishment of a fluid gradient within the uterine lumen and hence lead to the asymmetrical implantation position of the rat blastocyst.

310. CAVEOLIN 1 AND 2 IN THE UTERUS DURING EARLY PREGNANCY

2010 ◽  
Vol 22 (9) ◽  
pp. 110
Author(s):  
R. J. Madawala ◽  
C. R. Murphy
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Early Pregnancy ◽  
Basolateral Membrane ◽  
Membrane Curvature ◽  
Major Protein ◽  
Uterine Epithelial Cells ◽  
Caveolin 1 ◽  
Blastocyst Implantation

Rat uterine epithelial cells undergo many changes during early pregnancy in order to become receptive to blastocyst implantation. These changes include basolateral folding and the presence of vesicles of various sizes which are at their greatest number during the pre-implantation period. The present study investigated the possible role that caveolin 1 and 2 plays in this remodelling specifically days 1, 3, 6, 7, and 9 of pregnancy. Caveolin is a major protein in omega shaped invaginations of the plasma membrane called caveolae that are considered to be specialised plasma membrane subdomains. Caveolae are rich in cholesterol, glycosphingolipids, and GPI anchored proteins and are involved in endocytosis and membrane curvature. Immunofluorescence microscopy has shown caveolin 1 and 2 on day 1 of pregnancy are localised to the cytoplasm of luminal uterine epithelial cells, and by day 6 of pregnancy (the time of implantation), it concentrates basally. By day 9 of pregnancy, expression of both caveolin 1 and 2 in luminal uterine epithelia is cytoplasmic as seen on day 1 of pregnancy. A corresponding increase in protein expression of caveolin 1 on day 6 of pregnancy in luminal uterine epithelia was observed. Interestingly however, caveolin 2 protein expression decreases at the time of implantation as found by western blot analysis. Both caveolin 1 and 2 were localised to blood vessels within the endometrium and myometrium and also the muscle of the myometrium in all days of pregnancy studied. In addition, both caveolin 1 and 2 were absent from glandular epithelium, which is interesting considering that they do not undergo the plasma membrane transformation. The localisation and expression of caveolin 1 and 2 in rat luminal uterine epithelium at the time of implantation suggest possible roles in trafficking of cholesterol and/or various proteins for either degradation or relocation. Caveolins may contribute to the morphology of the basolateral membrane seen on day 6 of pregnancy. All of which may play an important role during successful blastocyst implantation.

445. Focal adhesions disassemble during early pregnancy in rat uterine epithelial cells

2008 ◽  
Vol 20 (9) ◽  
pp. 125
Author(s):  
Y. Kaneko ◽  
L. A. Lindsay ◽  
C. R. Murphy
Keyword(s):  
Epithelial Cells ◽  
Western Blotting ◽  
Early Pregnancy ◽  
Active Form ◽  
Focal Adhesions ◽  
Proteolytic Fragment ◽  
Uterine Epithelial Cells ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Calpain 2

Successful blastocyst implantation require uterine epithelial cells (UECs) to undergo the 'plasma membrane transformation' followed by the removal of these cells around the implantation sites. The present study investigated the distribution and expression of two principal focal adhesion proteins talin and paxillin in rat UECs during early pregnancy and their role in the loss of these cells at the time of implantation. Results from immunofluorescence microscopy have demonstrated a major distributional change of talin and paxillin in UECs where an intense basal staining of these proteins on day 1 of pregnancy was lost at time of implantation. This was consistent with the significant decrease in paxillin seen through western blotting analysis. Interestingly the amount of talin was not significantly different between day 1 of pregnancy and at the time of implantation with a calpain 2 mediated proteolytic fragment of talin seen on both of these days of pregnancy. Calpain 2 activity was further investigated through western blotting analysis and increases in the active form of calpain 2 at the time of implantation. These observations suggest that talin and paxillin have disassembled from the site of focal adhesions where talin is undergoing cleavage by active calpain 2 at the time of implantation. This allows UECs to become less adherent to the underlying basal lamina facilitating their removal during blastocyst invasion. Hence, disassembly of focal adhesions at the time of implantation is a critical event for successful implantation and placentation.

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