Effects of Nicotine on Human Luteal Cells In Vitro: MATERIALS AND METHODS

Chemicals

The following chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO): nicotine and M-nicotine, progesterone, collagenase type IV, antibiotics, glutamine, and Hepes. The hCG was obtained from Serono (Milan, Italy), Nutrient Mixture Ham F-12 from Flow Laboratories (Milan, Italy), and fetal calf serum from Biological Industries (Kibbutz Beit Hae-mek, Israel). Trizol and reverse transcription-polymerase chain reaction (RT-PCR) kit reagents were purchased from Invitrogen, Life Technologies (Carlsbad, CA). The VEGF and GAPD primers were obtained from Pro-ligo, Primers and Probes (La Jolla, CA). The RIA kits were obtained by Radim (Rome, Italy). The [3H]PGE2 and [3H]PGF2„ were from NEN Life Science Products, Inc. (Boston, MA).

Luteal Cell Culture Preparation and Experimental Procedure

Corpora lutea (CL) were obtained during hysterectomy performed for nonendocrine gynecological disease (leiomyomatosis) in the midluteal phase of the menstrual cycle (Days 5-6 from ovulation). A total of 12 patients (age, 30-43 yr) were included in the present study. All had a history of regular menstrual cycles. Informed consent was obtained from each patient, and the present study was approved by the ethical committee internal to ‘‘Universita Cattolica Del Sacro Cuore’’ review board. canadian neighbor pharmacy online

The age of these CL was determined as follows: All patients were monitored until ovulation by daily measurement of basal body temperature and ultrasound examination of follicular growth. Once the maximal follicular diameter reached 18 mm, daily determination of plasma progesterone values was made. The time of ovulation (Day 0) was detected by the biphasic pattern of basal body temperature, the typical ultrasound disappearance of the dominant follicle or the ultrasound detection of CL, and the rise in plasma progesterone concentrations. At surgery, plasma samples were collected immediately before anesthesia to determine plasma progesterone concentrations.

The removed luteal tissue was immediately freed from blood vessels and ovarian stroma under a dissecting microscope, dissected, and minced. Human CL cultures were performed as described previously with some modifications. The luteal tissue was placed in 10 ml of prewarmed Ham F-12/Hepes medium containing type IV collagenase (200 U/ml), then incubated at 37°C in a shaking water bath for 45 min. The medium containing the cell suspension was filtered through a 40-|xm nylon mesh, and the collected cells were centrifuged and suspended twice in fresh medium. This procedure was repeated once with the remaining undigested tissue to purify luteal cells. Cells were stained for lipids with oil red O14 and counted; more than 90% of the luteal cells stained positive for lipids. The cells that did not stain for lipids were occasional vascular cells, such as erythrocytes and leukocytes. To estimate the leukocyte contamination, flow-cytometric characterization of our cell cultures was performed.

At the end of the isolation procedure, cells were counted in a hemo-cytometer, and viability was determined by the trypan blue exclusion test. The cells were diluted to a final concentration of 250 000 live cells/ml medium supplemented with 2 mM L-glutamine, 100 IU of penicillin, 100 mg/ml of streptomycin, and 10% fetal calf serum and cultured in 48-well plates for 24 h in 5% CO2/95% air at 37°C. After this time, the cells were attached to the wells.

Next, the medium was removed and replaced with fresh serum-free medium for untreated luteal cells (controls) or with serum-free medium containing the following treatments: nicotine or M-nicotine either alone (from 10—11 to 10—6 M) or in combination with hCG (25 ng/ml; 125 IU/ L). The hCG (100 ng/ml; 500 IU/L) and interleukin-1 p (0.1 ng/ml) have been used as positive control for production of progesterone and PGs, respectively. The medium was harvested after 24 h of culture and stored at —20°C until assayed by PGE2, PGF2„ or progesterone RIA.

In another group of experiments, the cells were cultured in T25 flasks and grown in fresh serum-free medium for untreated luteal cells (controls) or in serum-free medium containing the following treatments: hCG (100 ng/ml), nicotine (10—7 M), or M-nicotine (10—7 M). After 24 h of incubation, the cells were homogenized using 1 ml of Trizol reagent per flask. Total RNA extraction followed by RT-PCR was used to investigate VEGF expression.

Flow Cytometry of Cell Cultures

Single-color fluorescence flow cytometry was performed using fluorescein isothiocyanate monoclonal antibody to CD45 obtained from BD Biosciences (Franklin Lakes, NJ). Cytometric evaluation was performed using a FACScan (BD Biosciences) equipped with LYSIS II software (BD Biosciences). The percentage of contaminating leukocytes in luteal cell preparations was calculated by the combined use of side scatter and CD45 expression. Analysis was performed within the region defined by light scatter to avoid including cell debris and clumps from the analysis. The proportion of contaminating leukocytes ranged between 3% and 7%.

Analytical Methods

Commercial progesterone RIA kits were used. The intra- and interassay coefficients of variation were 4% and 10%, respectively. The RIA sensitivity regarding progesterone was 5 pg/tube.

Both PGF2„ and PGE2 RIAs were characterized for measurement of prostanoids in human urine and later used successfully to measure PGs produced and released by several cell types in vitro, including cells from human ovaries. For each assay, incubation mixtures of 1.5 ml were prepared in disposable plastic tubes in which 50 |xl (for PGE2 or PGF2„) of incubation medium were diluted to 250 |xl with 0.025 M phosphate buffer (pH 7.5). Tritiated (3H) PGE2 or PGF2„ (2500-3500 cpm) and appropriately diluted antisera were added together to a final volume of 1.5 ml. The antisera (provided by Prof. G. Ciabattoni, Universita Cattolica del Sacro Cuore, Roma, Italia) were used at a final dilution of 1:120 000 or 1: 150 000 (for PGE2 or PGF2„y, respectively). A duplicate standard curve, ranging from 2 to 400 pg/tube, was run for each assay. All tubes were incubated for 24 h at 4°C. Separation of antibody-bound prostanoids was obtained with 2.5 mg of charcoal (Norit-A, Norit Americas, Inc. Marshall, TX), which absorbs 95%-98% of free PGs; a charcoal suspension (2.5 mg/50 |xl) in 0.025 M phosphate buffer (pH 7.5) was added to each tube after the addition of 100 |xl of 5% BSA. The tubes were shaken briefly and then centrifuged for 10 min at 4°C. Supernatants were decanted into 10 ml of scintillation liquid. Radioactivity was measured by liquid scintillation counting. The detection limit of the assay was 2 pg/tube in all cases. The inter- and intra-assay coefficients of variability were 2.7% and 2.9%, respectively, for PGE2 and 3.2% and 2.8%, respectively, for PGF2„.

RNA Extraction and Semiquantitative RT-PCR

Luteal cells grown under different treatments conditions were directly homogenized in the culture flask by adding 1 ml of Trizol reagent and by passing cell lysate several times through a pipette before sample collection in 1.5-ml Eppendorf tubes. These cells were then stored at —80°C.

The RNA isolation followed a three-step protocol. The ‘‘Phase Separation’’ was obtained adding 0.2 ml of chloroform, shaking the tubes vigorously by hand, and incubating at room temperature for few minutes. Centrifugation of samples at 12 000 rpm for 15 min at 4°C allowed mixture separation into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase that contained RNA and that was transferred to a fresh tube. The ‘‘RNA Precipitation’’ occurred by mixing the aqueous phase with 0.5 ml of isopropyl alcohol, incubating samples at room temperature for 10 min, and centrifuging at 12 000 rpm for 10 min at 4°C. The RNA precipitate forms a gel-like pellet on the side and bottom of the tube. After removal of the supernatant, the pellet was washed with 1 ml of 75% ethanol. The ‘‘RNA R-dissolving’’ in RNase-free water took place after centrifugation at 7500 rpm for 5 min at 4°C and a brief air-drying of the pellet.

Spectrophotometric analysis was used to determine RNA concentration by measuring the samples optical density (OD) at wavelength of 260 nm. Pure, isolated RNA samples have an OD260:OD280 ratio of greater than 1.6.

Single-strand cDNA for a PCR template was synthesized from 1 |xg of total RNA using a primer oligo (dT) and Moloney murine leukemia virus reverse transcriptase under the conditions indicated by the manufacturer. Each cDNA was amplified by PCR; the reactions were carried out in a 50-|xl volume containing cDNA, PCR buffer, MgCl2, dNTP, 1 |xM of both forward and reverse primers, and Thermus Aquaticus (Taq) DNA polymerase (2.5 units). Specific primers were designed for VEGF gene amplification (forward: 5′-CTTTTCGTCCAACTTCTGGG-3′; reverse: 5′-GGCTTGTCACATCTGCAAGT-3′) and for human glyceraldehyde-3-phosphate dehydrogenase (GAPD) control gene amplification (forward: 5′-CTTCACCACCATGGAGAAGG-3′; reverse: 5′-TGAAGTCAGAGGAG ACCACC-3′), leading to 939- and 807-base pair (bp) fragments for VEGF (corresponding to VEGF 121 and VEGF 165 mRNA) and to a 557-bp fragment for GAPD.

The PCR reaction conditions were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 60°C (for VEGF) or 58°C (for GAPD) for 1 min, and 72°C for 90 sec, and then a final extension at 72°C for 7 min. Starting experiments were performed to ensure being in the linear range of the PCR amplification curve.

Five microliters of each PCR product were visualized by ethidium bromide staining on a 1% agarose gel, and the bands were quantified using an image densitometer (Bio-Rad). The VEGF data are presented as the mean ± SD and were calculated after normalization to the GAPD data.

Data Analysis

Statistical analysis was performed using ANOVA followed by the Tu-key-Kramer test for comparisons of multiple groups or the Student f-test when appropriate.

The RNA isolation followed a three-step protocol. The ‘‘Phase Separation’’ was obtained adding 0.2 ml of chloroform, shaking the tubes vigorously by hand, and incubating at room temperature for few minutes. Centrifugation of samples at 12 000 rpm for 15 min at 4°C allowed mixture separation into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase that contained RNA and that was transferred to a fresh tube. The ‘‘RNA Precipitation’’ occurred by mixing the aqueous phase with 0.5 ml of isopropyl alcohol, incubating samples at room temperature for 10 min, and centrifuging at 12 000 rpm for 10 min at 4°C. The RNA precipitate forms a gel-like pellet on the side and bottom of the tube. After removal of the supernatant, the pellet was washed with 1 ml of 75% ethanol. The ‘‘RNA R-dissolving’’ in RNase-free water took place after centrifugation at 7500 rpm for 5 min at 4°C and a brief air-drying of the pellet.


Category: Cell

Tags: cell, corpus luteum, progesterone