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Technical Briefs |
1
Oncology Diagnostic Unit, Biochem. Dept.,
2
Dept. of Gyn., Ain Shams Faculty of Med., Abbassia, Cairo, Egypt, and
3
Fayoum General Hospital, Egypt;
a address for correspondence: fax 202-285-9928
Although progesterone and estrogens are essential for maintaining human pregnancy after implantation, few reports have investigated the localization of their specific receptors in different uterine cell types throughout pregnancy (1)(2). Wu et al. detected estrogen receptors (ER) in low concentrations in decidua in early pregnancy but not in term decidua (2). ER concentrations in term decidua of pregnancies with complications (in particular, preeclampsia) have not been investigated.
Classical biochemical assay methods estimate ER either in cytosol fraction or in the high-salt extracted nuclei (3)(4)(5). These assays need two subcellular fractionation steps and two ER assays in two fractions: cytosol and nuclear extract. To measure total ER (cytosolic and nuclear) in one fraction using one single assay in decidual samples, we modified the assay method by using a homogenization buffer containing KCl. The effective KCl concentration for maximum ER extraction without inhibition of the ER enzyme immunoassay (EIA) ranged from 0.4–0.8 mol/L KCl.
All steps of sample preparation in our laboratory were carried out at 4 °C, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO), and all women included in the study gave informed consent. We used scrapped decidual samples from placental bed from women with uncomplicated pregnancies who underwent cesarean section for obstetrical causes (n = 10) and from women with preeclampsia (n = 20): mild (n = 4), moderate (n = 6), and severe (n = 10). Tissues were immediately washed in ice-cold saline and homogenized at 1 g/10 mL in ice-cold homogenization buffer (10 mmol/L Tris buffer, pH 7.5, containing 10 mmol/L K2EDTA, 100 mL/L glycerol, 5 mmol/L benzamidine, 10 mmol/L 2-mercaptoethanol, 0.39 mmol/L phenylmethylsulfonyl fluoride, and 5 mg/L aprotinin) with Ultraturax T-25 homogenizer for five bursts of 1 min each, separated by a 1-min pause. The homogenate was filtered and divided into two parts. The first part was centrifuged in a Beckman CS-6R centrifuge (Brea, CA) at 800g for 15 min at 4 °C to obtain the crude nuclear pellet. The supernatant fluid was recentrifuged at 100 000g for 1 h with a Beckman L7 ultracentrifuge to obtain the cytosol. The crude nuclear pellet was dissolved in 10 mmol/L ice-cold phosphate buffered saline (pH 7.2) by sonication for three 30-s bursts and recentrifuged at 800g for 15 min at 4 °C. The washed nuclear pellet was incubated with 5 volumes of ice-cold high-salt extraction buffer (homogenization buffer containing 0.4 mol/L KCl) on ice for 30 min with vortex-mixing every 10 min. Then it was ultracentrifuged in a Beckman L7 ultracentrifuge at 100 000g for 30 min at 4 °C. The nuclear extract (supernatant) was obtained.
The second part of the homogenate was incubated with 0.4 mol/L KCl on ice for 30 min with vortex-mixing every 10 min, then ultracentrifuged in a Beckman L7 ultracentrifuge at 100 000g for 30 min at 4 °C. The supernatant was isolated. After quantifying the protein concentration in the cytosol, nuclear extract, and tissue extract by using Bradford's method (6) with bovine serum albumin as the calibrator, we applied the samples directly to the ELISA plate for assay of ER with EIA kit from Abbott Laboratories (Chicago, IL) (7).
ER concentrations estimated by our modified method strongly correlated
to those calculated as a sum of ER concentration in cytosol plus those
in nuclear extract (y = -0.98 + 1.128x,
r = 0.91, P = 0.005). Cytosolic,
nuclear, and total ER were not detectable (lower detection limit, 1
fmol/mg protein) in term decidua from women with uncomplicated
pregnancies or from women with mild or moderate preeclampsia, but were
detected in term decidua from women with severe preeclampsia (Fig. 1
) (cytosolic ER, 1.4–9 fmol/mg protein, mean 4.98 fmol/mg protein;
nuclear ER, 1.5–11 fmol/mg protein, mean 4.68 fmol/mg protein; total
ER 2.6–19.2 fmol/mg protein, mean 9.1 fmol/mg protein). This
difference was statistically significant by Student's
t-test (P = <0.01).
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Although our results suggest a significant association between ER detection in term decidua and severe preeclampsia, the cause–effect relationship cannot be determined in this study. Establishing a cause–effect relationship may improve understanding of the biological relationship and may have potential clinical implications through development of diagnostic or therapeutic markers for these lesions. We recommend the routine use of this modified assay for quantification of total ER in the clinical laboratory.
Acknowledgments
We thank A. Khalifa for his unlimited support.
References
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