Different Regulatory Pathways of Endometrial Connexin Expression: MATERIALS AND METHODS
Adult female Sprague-Dawley rats were housed under defined conditions with a temperature of 22 ± 1°C, an atmospheric humidity of 55% ± 10% (mean±SEM), and a 12L:12D photoperiod. They were fed standard pellet food and provided with water ad libitum. All animal experiments were approved by the institutional animal care committee of the government.
The ERa knockout (aERKO) and ERp knockout (pERKO) mice were obtained from the breeding colony at Taconic Farms (Germantown, NY). Mice were maintained in plastic cages under a 12L:12D photoperiod in a temperature-controlled room (21-22°C) and were fed NIH 31 mouse chow (National Institutes of Health, Bethesda, MD) and fresh water ad libitum. All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
17p-Estradiol (E2) and progesterone (both from Sigma, Taufkirchen, Germany) as well as the antiestrogen ICI 182780 (kindly provided by A. Wakeling, Zeneca Pharmaceuticals, Cheshire, U.K.) were dissolved in benzyl benzoate and administered s.c. in sesame oil (100 |xl for mice, 200 |xl for rats). Corresponding to estrogenic effects in pseudopregnant rats described previously, rats were injected with 1 |xg of E2 and 0.5 mg of ICI 182780, and mice were injected with 0.1 ^g of E2 and 0.1 mg of ICI 182780. Controls were given an equal volume of vehicle only. At least three animals were used for each experimental approach.
Mating was performed overnight with male animals, and the day of vaginal plug (mice) and sperm finding (rats) was designated as 0 days postcoitum (dpc). Delayed implantation was achieved in rats by ovariectomy on 2 dpc and supplying progesterone (4 mg rat-1 day-1) from the day of ovariectomy onward. In rats, pregnancy was confirmed up to 4 dpc and during delayed implantation by flushing the uterus and counting the number of blastocysts.
Pseudopregnancy and Artificial Decidualization
Pseudopregnancy was achieved in ovariectomized rats and mice by treating them with E2 (1 and 0.05 ^g/day, respectively) for 2 days, leaving them untreated for 2 days, and then substituting progesterone (5 and 1 mg/day, respectively) for 3 days. In ovariectomized rats, decidualization was obtained under anesthesia by scratching the antimesometrial luminal surface of one uterine horn with a hooked needle by way of a lateral abdominal incision and supplying E2 (0.1 ^g/day) and progesterone (5 mg/day) for 24 h. The nonscratched contralateral horn served as a control. In mice, decidualization was obtained by vaginally injecting 100 |xl of oil and supplying E2 (0.05 ^g/day) and progesterone (1 mg/day) for 24 h.
Rats were killed by ether and mice by cervical dislocation. Uterine horns were removed, and small pieces were frozen in liquid nitrogen for subsequent histochemical analysis. Rat uteri were opened longitudinally on an ice-cold glass plate, and the endometrium was carefully scraped off. Histological examination of the removed endometrium revealed no contamination with myometrial tissue (data not shown). The rat endometrial samples as well as the whole-mice uteri were frozen in liquid nitrogen and stored at -80°C.
For in vitro organ-culture experiments, uteri of pseudopregnant rats were opened longitudinally and incubated for 18 h in culture medium (Dulbecco modified Eagle medium [Gibco BRL, Eggenstein, Germany] + 10% heat-inactivated fetal calf serum [Biochrome, Berlin, Germany] + 100 U/ml of penicillin/streptomycin [Boehringer, Mannheim, Germany]) or in culture medium containing E2 (5 ng/ml ), ICI 182780 (5 |xg/ml ), actinomycin D (Sigma; 5 |xg/ml ), dbcAMP (Sigma; 0.5 mg/ml ), 12-O-tetradecanolyphorbol-13-acetate (TPA; Sigma; 20 ng/ml ), catechol estrogen (Sigma; 1 |xg/ml ), interleukin (IL)-1p (Sigma; 10 ng/ml ), or PGF2a (Sigma; 1 |xg/ml ). After incubation, endometrium was scraped off for RNA isolation, frozen in liquid nitrogen, and stored at -70°C.
Northern Blot Analysis
Total RNA was extracted from endometrial tissue using the RNeasy midi kit (Qiagen, Hilden, Germany). Five micrograms of total RNA (as estimated from optical absorbance measurements at 260 nm) were electro-phoresed on a denaturing agarose-formaldehyde gel and blotted onto nylon membranes (Hybond-M; Amersham-Bucher GmbH, Braunschweig, Germany). The cDNA probes were random prime-labeled with a [32P]dCTP and hybridized with the RNA blots overnight at 42°C in a solution containing 55% deionized formamide, 1 M NaCl, 1% SDS, 10% dextran sulfate, and 100 |xg/ml of salmon sperm DNA. The following connexin cDNAs were used for hybridization: a 1.1-kilobase (kb) cDNA corresponding to part of the coding region of rat Cx26 gene, a 1.4-kb cDNA corresponding to the coding region of rat Cx43 gene, and a rat actin-specific cDNA probe or 18S RNA probe. Blots were washed at 60°C in 1X SSC (0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS for 1 h, in 0.5X SSC and 0.1% SDS for 30 min, and in 0.2X SSC and 0.1% SDS for 30 min. Exposure to Kodak XAR-5 films (Eastman Kodak, Rochester, NY) took place at —70°C with intensifying screens.
Signals detected by autoradiography were quantified by densitometry. Densitometric values for connexin expression were calculated relative to the p-actin level of the corresponding lane for possible differences in RNA loading.
Analysis of variance was used for statistical testing, and when necessary, the Scheffe test was used as a post-hoc test. For Figure 5, the Mann-Whitney U-test was performed. The level of significance was set at P < 0.05.
Immunohistochemical staining was performed on cryostat sections (thickness, 4-6 |xm) as described previously using affinity-purified rabbit antibodies (1 |xg/ml) to Cx26 from mouse liver gap junctions and to the C-terminal 22 amino acids of rat Cx43, respectively, after rat heart (Cx43) and liver (Cx26) were tested for positive controls. For negative controls, preimmune serum was used instead of the primary antibody showing no staining. Photographs were taken with an Axiophot microscope (Zeiss, Jena, Germany) equipped for epifluorescence. The intensity of immunostaining was semiquantitatively divided into strong (+ + +), moderate (+ + ), low ( + ), or negative (—).