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Cattedra Di Fisiopatologia Della Riproduzione Umana,3 Università Cattolica Del Sacro Cuore, 00168 Roma, Italy
Istituto Scientifico Internazionale (ISI) "Paolo VI,"4 Università Cattolica Del Sacro Cuore, 00168 Roma, Italy
OASI Institute For Research,5 94018 Troina, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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release from luteal cells, with a dose-related efficacy for M-nicotine. On the contrary, PGE2 release was significantly inhibited by both nicotine and its metabolite. Finally, nicotine was able to increase VEGF mRNA expression significantly, whereas M-nicotine was not. In conclusion, nicotine and M-nicotine can induce a sort of luteal insufficiency by inhibiting progesterone release, probably through modulation of the PG system.
corpus luteum, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cigarette tobacco contains several substances. Carbohydrates and proteins are the most representative components, but alkaloids are significantly present as well. Nicotine, in particular, represents 90%–95% of total alkaloids. Nicotine is a highly toxic substance and is absorbed quickly through the respiratory tract, mouth mucosa, and skin. Approximately 80%–90% of nicotine is metabolized by the liver, but the kidneys and lungs are involved as well [5]. Cotinine, one of the nicotine metabolites, has been detected in human ovarian follicular fluid and in granulosa-luteal cells [6]. All this evidence indicates that nicotine can affect gamete cell functions.
In the present study, we investigated if nicotine and its methylated metabolite, N-methyl-nicotine (M-nicotine), could affect progesterone production by human luteal cells. Because the cyclooxygenase pathway is strictly involved in the regulation of luteal steroidogenesis, we also looked for a possible influence of nicotine on the release of prostaglandins (PG). Finally, we tested the effect of nicotine and its metabolite on gene expression of vascular endothelial growth factor (VEGF), an angiogenic factor whose expression is directly correlated with a normal luteal function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 Haemek, 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 Proligo, 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 "Università Cattolica Del Sacro Cuore" review board.
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 [7] 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-µm 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 [8]. 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 hemocytometer, 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-1ß (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 [9] 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 µl (for PGE2 or PGF2) of incubation medium were diluted to 250 µl 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, Universitá Cattolica del Sacro Cuore, Roma, Italia) were used at a final dilution of 1:120 000 or 1: 150 000 (for PGE2 or PGF2y, 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 µl) in 0.025 M phosphate buffer (pH 7.5) was added to each tube after the addition of 100 µl 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 µg 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-µl volume containing cDNA, PCR buffer, MgCl2, dNTP, 1 µM 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'-TGAAGTCAGAGGAGACCACC-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 Tukey-Kramer test for comparisons of multiple groups or the Student t-test when appropriate.
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RESULTS |
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The next step was to evaluate the effect of nicotine and its metabolite (10–11 to 10–6 M) on production of PGs. As shown in Figure 4, all tested doses of both substances were able to increase significantly PGF2 release by the cells; in particular, a steady-state level was reached with the lowest nicotine concentration (10–11 M), because no significant variation in PGF2 medium content was observed in the presence of higher nicotine doses. However, the effect of M-nicotine on the PG release was dose-related, and its stimulatory effect was slightly more potent than that of nicotine.
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On the other hand, both alkaloids were able to decrease PGE2 release by luteal cells, following a trend similar to that previously described for PGF2 increase. In fact, nicotine significantly inhibited PGE2 release at all tested doses without a dose-dependent trend, whereas M-nicotine significantly inhibited PGE2 release in a dose-dependent manner (Fig. 5).
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Finally, we tested the possibility for both substances to affect VEGF expression in cultured human luteal cells. As shown in Figure 6, nicotine was able to increase significantly the expression of VEGF in our cells. Interestingly, this effect was similar to that exerted by hCG, a powerful inducer of VEGF mRNA expression in this cell type. However, M-nicotine did not exert any effect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nicotine and its major metabolites have been found in follicular fluids of women undergoing assisted conception [6, 16] in a dose-related association with cigarette consumption [17, 18]. Interestingly, cotinine, a nicotine metabolite, was found in ovarian granulosa-luteal cells in a dose-dependent relationship with its follicular fluid levels. Binding of this alkaloid to nuclear and cytoplasmatic proteins could affect the developmental potential of maturing follicles and lead to perturbation in meiotic maturation of oocytes [6].
Whereas the developing follicle as well as the oocyte have been extensively indicated as possible targets of nicotine, the effects of nicotine on the luteal phase remain mostly unexplored. Because CL is the main endocrine gland supporting both the luteal phase and early pregnancy, we wanted to investigate the possible effect of nicotine on human luteal steroidogenesis. Interestingly, we found that both nicotine and M-nicotine were able to affect steroidogenesis significantly by inhibiting the release of progesterone by human luteal cells. This effect was demonstrated statistically even at the lowest tested concentrations. In these experiments, nicotine and M-nicotine concentrations ranged between the cotinine levels observed in the blood and follicular fluid of smoking women [16]. However, neither nicotine nor M-nicotine was able to affect the hCG-induced production of progesterone.
The mechanism through which hCG stimulates progesterone production is well known and widely described. Hormone/receptor interaction results in cAMP formation and arachidonic acid release; these molecules act in a synergistic manner to induce steroid production by increasing steroidogenic acute regulatory protein (StAR) gene expression [19–21].
On the other hand, a variety of nonneuronal tissues have been demonstrated recently to express nicotinic acetylcholine receptors (nAChRs) [22–24]. In human umbilical vein endothelial cells, nAChR-induced action has been described as being completely dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways [23]. Despite that report, the mechanisms underlying nAChR signaling and its downstream pathways remain mostly unknown. Therefore, new studies regarding signaling cascades are necessary to understand if the inhibitory effect of nicotine on progesterone production is not able to overcome the hCG-activated pathway or if nicotine and hCG act through two different, noninterfering metabolic pathways.
The next step was to investigate the mechanisms through which nicotine reduced progesterone production. The PGs are widely described as playing a key role in the complex regulation of CL physiology. Human CL produces both PGE2 and PGF2 in different ways during the distinct luteal phases [25, 26]. In a large number of species, including humans, PGF2 seems to be important for luteolysis, but accumulating evidence suggests a luteotropic effect of PGE2 [27, 28]. In the present study, we demonstrated that both nicotine and M-nicotine were able to significantly increase PGF2 release and to reduce PGE2 production by luteal cells, even at the lowest tested doses.
It has been demonstrated previously that PGF2 from 10–11 to 10–6 M is able to reduce significantly progesterone production by granulosa-luteal cells in vitro [29]. Release of PGF2 induced by nicotine and M-nicotine in human isolated luteal cells ranged between 10–9 and 10–8 M according to our results. Therefore, it is tempting to speculate that nicotine and its metabolite could influence luteal steroidogenesis by modulating the PG system.
Other in vitro studies have investigated the effect of smoking on human granulosa-luteal cells and produced conflicting final data. Barbieri et al. [30] demonstrated that in vitro exposure to low-molecular-weight constituents of tobacco-smoke extracts inhibited granulosa cell aromatase. In contrast, Weiss and Eckert [16] found that neither nicotine nor cotinine influenced progesterone or estradiol secretion by granulosa cells, whereas Bodis et al. [31] found a dose-dependent increase in estradiol secretion and a decrease in progesterone production by granulosa cells in response to nicotine treatment.
Our results, obtained using isolated human luteal cells, are in agreement with those of Bodis et al. The discrepancies with the other reports could depend on the different cell type used in each study. Although granulosa-luteal cells share some similarities with human luteal cells, these two cell types differ for both anatomical and biochemical properties.
Finally, we investigated VEGF mRNA expression in luteal cells in response to nicotine and M-nicotine treatments. In human ovaries, angiogenesis is associated with follicular development and CL formation. Among the several factors involved in angiogenesis, VEGF has been described to be a potent proangiogenic factor. In addition to its stimulating effect on endothelial cell growth in vitro [32], this cytokine is also a blood vessel permeability inducer in vivo [33]. The presence of VEGF and its specific receptor, the fms-like tyrosine kinase (Flt-1), has been demonstrated recently in human granulosa and theca-luteal cells. Concentrations of VEGF in luteal extracts were higher in the early and midluteal phases and tended to decrease toward the late luteal phase, suggesting a VEGF steroidogenic support through vascularization promotion [34]. Interestingly, VEGF correlation with cell hypoxic conditions has been widely reported [35], and Ferrara et al. [36] demonstrated VEGF mRNA up-regulation in rat hypoxic luteal cells. On the other hand, the hypoxic in vivo nicotine effect is well known, even though the mechanism by which it occurs is still not completely understood [37].
In the present study, we found that nicotine significantly increased VEGF mRNA expression in human luteal cells but that M-nicotine did not. How nicotine can actually induce VEGF expression on human isolated luteal cells has not yet been clarified; therefore, more details about molecular pathways are necessary to address the discrepancy between nicotine and M-nicotine regarding the induction of VEGF expression.
In conclusion, we demonstrated the ability of nicotine and M-nicotine to affect production of progesterone and PGs as well as expression of VEGF in human isolated luteal cells. Based on these results, we can speculate that nicotine and M-nicotine could influence CL physiology by modulating both its steroidogenic activity (probably through the PG system) and its vascularization. Furthermore, we can speculate that a CL deficiency may be one of several mechanisms through which nicotine can cause infertility and early pregnancy loss.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Rosanna Apa, Cattedra Di Fisiopatologia Della Riproduzione Umana, Università Cattolica Del Sacro Cuore, Largo A. Gemelli 8, 00168 Roma, Italia. FAX: 39 06 3051160; krimisa{at}libero.it
Received: 21 May 2004.
First decision: 10 June 2004.
Accepted: 29 October 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and E2 synthesis by human corpus luteum in vitro: a possible balance of luteotropic and luteolytic effects. J Clin Endocrinol Metab 1999 84:2507-2512 in human urine. J Endocrinol Invest 1979 2:173-179[Medline] and in the capacity for their in vitro formation in the human corpus luteum. Prostaglandins 1985 30:91-98[CrossRef][Medline] by corpora lutea, corpora albicantes and stroma from the human ovary. J Endocrinol 1996 68:401-407. Physiol Rev 1976 56:595-601 on the hCG-stimulated progesterone production by human corpora lutea. Prostaglandins 1984 27:465-470[CrossRef][Medline]-receptor mRNA in human granulosa-luteal cells by human Chorionic gonadotropin and prostaglandin F2alpha. Endocrine 1998 8:261-267[CrossRef][Medline]This article has been cited by other articles:
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  S. P. Ng, B. G. Steinetz, S. G. Lasano, and J. T. Zelikoff Hormonal Changes Accompanying Cigarette Smoke-Induced Preterm Births in a Mouse Model Experimental Biology and Medicine, September 1, 2006; 231(8): 1403 - 1409. [Abstract] [Full Text] [PDF]
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