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Melatonin-induced stimulation of rat corpus epididymal epithelial cell proliferation
Life Sciences, Vol. 65, No. 10, pp. 1067-1076, 1999 Copyright 0 1999 Elsevier Sciace. Inc. Printed in the USA. All rights resaved 0024-3205/9!9/6see front matter
PIISOO24-3205(99)00337-9
ELSEVIER
MELATONIN-INDUCED
STIMULATION OF RAT CORPUS EPIDIDYMAL CELL PROLIFERATION
EPITHELIAL
Li Li’, Joseph T.Y. Wong 2, Shiu F. Pang’ and Stephen Y.W. Shiu’ ‘Department of Physiology, The University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong; and *Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
(Receivedin final form May 5, 1999) Summary Stimulation of rat epididymal epithelial cell proliferation by melatonin was demonstrated by thymidine incorporation and flow cytometric analyses. The stimulatory effect of melatonin was dependent on the hormone concentration and the duration of cell exposure to the hormone. Maximal stimulation of [3H]thymidine incorporation into epididymal epithelial cells by melatonin was observed at lx 1V9 M So-dihydrotestosterone in medium, while lower or higher concentrations of androgen attenuated the stimulatory effect of melatonin. Interestingly, a nuclear melatonin receptor agonist (1-[3-allyl-4-0x0thiazolidine-2-ylidene]-4-methyl-thiosemi-carbazone, CGP 52608) induced opposite effect on epithelial cell proliferation to that produced by melatonin. Our data suggest that melatonin-induced stimulation of rat epididymal epithelial cell proliferation is not likely to be mediated by nuclear receptor. Furthermore, sequential changes of cell cycle distribution with melatonin treatment also supports a stimulatory action of melatonin on epididymal epithelial cell proliferation. Key n%fs: melatonin, Sadihydrotestosterone, CGP 52608, cell proliferation, ~11 cycle distribution
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone synthesized and secreted predominantly by the pineal gland in the dark (1). Recent research has provided considerable evidence that this evolutionarily conserved signaling biomolecule subserves multiple functions in mammals, including the regulation of circadian rhythms and reproductive responses (1, 2). Intriguingly, both receptor and non-receptor mediated mechanisms have been proposed for the actions of melatonin (3). To date, specific G protein-coupled (MELBA/ mti, MEL& MT2, Mel,, and ML2/ MTj) and nuclear melatonin receptors (RZRo) have been reported (4, 5, 6) and the tissue distribution of some of these receptors have been mapped using pharmacological, immunological and molecular biological methods (7, 8, 9). Functionally, it has been shown that the G protein-coupled MELBA and MELis receptor subtypes provide the molecular basis for two
Correspondence to: Dr. SYW Shiu, Department of Physiology, The University Shu Fan Building, 5 Sassoon Road, Hong Kong, China
of Hong Kong, Li
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distinct separable effects of melatonin on the physiology of suprachiasmatic nucleus (SCN) in mice. and that MELlu receptor can possibly serve as a back-up for MEL,* receptor in mediating melatonin-induced phase shifts in mouse SCN (10). This highlights the importance of MEL,* receptor of the SCN in regulating the circadian and reproductive responses in seasonal mammal (11). The expression of specific melatonin receptors in tissues outside the central nervous system adds further weight to the long-believed direct regulatory action of the pineal neurohormone on peripheral tissue or cell biology (1). Indeed. it has recently been found that nuclear melatonin receptors may be responsible for repressing 5-lipoxygenase gene expression in human B lymphocytes (12). while enhancing cytokine production by human Thl cells and monocytes (13). Previous studies by our laboratories have demonstrated the expression of high affinity 2[‘2SI]iodomelatonin binding sites, satisfying the pharmacokinetic properties of a specific receptor, in the corpus epididymis of rats (14, 15). These high affinity binding sites in the rat epididymis have been identified by cDNA cloning to be G protein-coupled MEL!* and MELia receptors (7, 16). In situ hybridization studies have demonstrated that both MEL,* and MELia melatonin receptor mRNAs are expressed by epithelial cells of rat corpus epididymis (7). Although these high affinity melatonin receptors, whose activities are regulated by androgens, are negatively coupled to adenylyl cyclase via pertussis toxin-sensitive Gi proteins (7) the biological functions of these Gi-coupled melatonin receptors in epididymal epithelial cells are yet undefined. Given that the androgen can regulate the expression of these membrane receptors (14) and enhance the inhibition of cyclic AMP by melatonin (7), investigations were conducted to examine if melatonin can reciprocally modulate androgen-mediated cellular processes, such as cell proliferation, in the epididymis of male reproductive tissues. Materials and Methods Materials Chemicals for cell culture were purchased from GIBCO BRL chemical Co. (Grand Island, N.Y.). Melatonin and 5a-dihydrotestosterone (SIX-DHT) were supplied by Sigma Chemical Co. (St. Louis, MO). [3H]Thymidine (5 Ci/mmol) was purchased from Amersham International Co. (Aylesbury, UK) l-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-Methyl-thiosemi-c~b~one (CGP 52608) a nuclear melatonin receptor agonist (6) was kindly provided by Dr. I. Wiesenberg (Novartis Pharma Inc., Basel, Switzerland). Male Sprague-Dawley (SD) rats (6-week-old) were obtained from the Laboratory Animal Unit of The University of Hong Kong, and kept under a 12 h light, 12 h dark cycle (light on from 0300-1500 h) for 1 week. Light was provided by ceilingmounted fluorescent lamps with an intensity of about 150-200 lux at the top of the cage. Temperature was kept constant at 23 + 2 “C. Standard food and water were provided ad libitum. The epididymides of animals were collected after sacrifice at mid-light (0900 h). Rat corpus epididymal epithelial cell culture Primary culture of rat corpus epididymal epithelial cells has been previously reported (7). Briefly, rat corpus epididymides were cut into small pieces and placed in sterile Hank’s balanced salt solution (HBSS) containing 0.25% trypsin. Tissues were incubated for 30 min at 32 “C and separated by centrifugation at 800 g for 5 min. The pellet was resuspended in Eagle’s minimum essential medium (EMEM), containing collagenase IA (1 mg/ml) for 2 hr at 32 “C. Cells were separated by centrifugation at 800 g for 10 min. The pellet was then resuspended in EMEM containing nonessential amino acids, sodium pyruvate (1 x lo” M), glutamine (4x 10” M), 5adihydrotestosterone (lx lo-” M), 10% fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 pg/ml). The cell suspension was maintained at 32 “C in a humidified atmosphere of 5% CO2/95% air for 8 hr. During this period, non-epithelial cells such as fibroblasts and smooth muscle cells attached rapidly to the plastic surface of the flask, whereas
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epididymal cells remained suspended in the medium. The epithelial cells in the supematant were then cultured at a concentration of 1x10’ cells per ml at 32 “C. The epididymal epithelial cell monolayers became confluent after 3 days of culture. Cells in the second or third passages were used for determination of [3H]thymidine incorporation or cell cycle distribution. [3H]thymidine incorporation assay Cells (2~10~) in the second or third passages were plated in 6-well plastic dishes and maintained in medium containing 10% FBS at 32°C until they were approximately 70-80% confluent. Quantitation of pre-existing melatonin concentration in FBS-supplemented medium used for culturing cells for [3H]thymidine incorporation studies was performed by radioimmunoassay ( 17) and found to be less than 1O-” M. The cells were incubated for 48 hr in medium containing 10% FBS with melatonin (5~10‘~ M to 5x10-’ M), CGP 52608 (5x10_’ M, 1~10~~ M, 2~10~~ M) or an equal volume of vehicle (0.015% DMSO). For experiments involving melatonin pulse-treatment, cells were incubated with melatonin (5~10~~ M to 5x10“ M) for 6 or 12 hr per day for 3 consecutive days. For So-DHT experiments, cells were treated for 48 hr in medium added with 5x lo-” M So-DHT, 1~10~~ M So-DHT, lx 10m8M Sa-DHT or without Sa-DHT, in the presence of melatonin (5~10~~ M to 5x10-’ M) or vehicle (0.015% DMSO). After 48 hr incubation, [3H]thymidine (5 Ci/mmol, 1 @i/well) was added, and incubation was resumed for another 24 hr. Cells were incubated in 1 ml 10% (wt/vol.) trichloroacetic acid (TCA) for 30 min. The supematant was removed and the precipitate was solubilized in 0.42 ml 1N NaOH for 1 hr. After saving 10 pl aliquots in duplicates for later protein determination by Lowry’s method (18) 0.1 ml glacial acetic acid was added to neutralize the remaining solution. The content of each well was then transferred to a vial with 4 ml scintillation cocktail. The amount of radioactivity was determined by scintillation spectrometry (Beckman LS 6500 multi-purpose scintillation counter). Flow cytometric analysis of cell cycIe distribution Rat epididymal epithelial cells (4~10~) were seeded into 25 cm* flasks until they were approximately 70 - 80 % confluent. Cells were then incubated in medium added with melatonin (5~10.~ M) or an equal volume of vehicle (0.015% DMSO). After 8, 16 and 24 hr of incubation, cells were collected by centrifugation (1000 g x 5 min) at 4 “C after treatment with 0.25% trypsin in PBS solution. The cells were washed three times in PBS and fixed two times, for 5 min each, in 95% ethanol buffered with PBS at 4°C. Fixed cells were rehydrated with PBS and treated with RNase (100 ug/ml) for 20 min at 37 “C, prior to staining with propidium iodide (50 ug/ml) for 30 min in the dark. Using red propidium-DNA fluorescence, 20,000 events were acquired with a vantage cytometer for each sample and the percentage of cells in Go/r, S and Gz-M phases of the cell cycle was determined by the Cell-Fit analytical software (Becton-Dickinson, San Jose, CA, USA). Statistics Each set of experiment, repeated three or four times, was conducted with individual sample in triplicate or quadruplicate. Numerical results were expressed as mean f SE and expressed as a percentage of the mean vehicle-treated control level, which was arbitrarily defined as 100%. The data were analyzed with one way ANOVA and Bonferroni’s t test as appropriate. The level of significance was set at P < 0.05. r
Results
Effect of melatonin on r3H/thymidine incorporation into epididymal epithelial cells Melatonin induced an increase of cellular [3H]thymidine incorporation in a concentrationdependent manner (Fig. 1). Significant (P < 0.05) increase in [3H]thymidine incorporation was observed in cells which were incubated, continuously, with 5x 10m8M to 5x IO-’ M melatonin.
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Maximal stimulation to 181.0 f 5.1% of the control value was recorded when melatonin concentration was 5~10~~ M. Increase in [3H]thymidine incorporation into the cells which received pulse treatment of melatonin was found to depend on the pulse duration. No significant increase in [3H]thymidine incorporation was noted for cells which were pulsed-treated with melatonin for 6 hr per day for 3 consecutive days. For cells treated with melatonin for 12 hr per day consecutively for 3 days, [3H]thymidine incorporation into the cells increased significantly to 115.7 + 4.4% (P < 0.05) and 126.0 k 4.8% (P < 0.01) of control value when the respective concentrations of melatonin were 5x 10e6M and 5x 1O-’ M.
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Fig. 1 Effect of melatonin on [3H]thymidine incorporation into rat epididymal epithelial cells. Cells were incubated in medium containing 1~10~~ M SWDHT with melatonin for 6 hr (open circle), 12 hr (open quadrate) and 24 hr (open triangle) per day for 3 consecutive days. Data shown are the mean XLSE of 3 separate experiments assayed in triplicates or quadruplicates. Results are expressed as a percentage of the mean [3H]thymidine incorporation into vehicle-treated control (100%). *, P < 0.05; **, P < 0.01 vs. control. Effects of melatonin with and without 5 a-DHT on J3H]thymidine incorporation The levels of [3H]thymidine incorporation varied with the concentrations of Sa-DHT in the medium. [3H]Thymidine incorporation levels were 12837 + 1201 dpm per mg protein, 13289 + 1010 dpm per mg protein, 14010 + 454 dpm per mg protein, and 18720 f 742.2 dpm per mg protein (three separate experiments) when the cells were incubated with 0 M, 5x 1O-” M, 1x 1Oe9 M and 1x10-* M Sa-DHT, respectively. without melatonin addition (Fig. 2). In the absence of Sa-DHT, significant increases in cellular [3H]thymidine incorporation to 127.3 + 2.5% (P< 0.05) and 133.0 + 5.5% (P < 0.01) of the control value were recorded when the concentrations of melatonin were 5x 1Om6M and .5x1Om5M respectively. An increase of androgen concentration up to 1x 10m9 M Sa-DHT enhanced the sensitivity and magnitude of cellular [3H]thymidine incorporation to melatonin. The lower concentrations of melatonin that induced a significant (P< 0.05) increase in cellular [3H]thymidine incorporation were 5~10.~ M and 5x10-* M for cells that were, respectively, co-incubated with 5x 1OF” M and 1x 1OW9 M Sa-DHT. In the presence of 5x 1U5 M melatonin, [3H]thymidine incorporation was increased to 145.3 + 6.1% (P< 0.01) and 181 .O + 5.1% (P< 0.01) of the control value when the cells were respectively co-incubated with 5~10-‘~ M and lx low9 M Sa-DHT. Despite a 1.46-fold elevation of basal [3H]thymidine incorporation when Sa-DHT was increased from 0 M to 1x 10m8M, a decrease in the cellular responses to
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melatonin in the presence of l~lO_~ M So-DHT was recorded. While 5~10~~ M melatonin increased [3H]thymidine incorporation into cells to 139.6 k 7.1% (P < 0.01) of the control value, the lowest concentration of melatonin that could induce a significant (P < 0.05) increase in [3H]thymidine incorporation was 5~10~~ M.
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Fig. 2 Effect of melatonin on [3H]thymidine incorporation into rat epididymal epithelial cells incubated with different concentrations of So-dihydrotestosterone (So-DHT). Cells were incubated in medium containing increasing concentrations of melatonin (5~10~~ to 5~10~~) with 5x10-” M So-DHT (closed lozenge), 1x1w9 M So-DHT (open triangle), 1x 10m8M So-DHT (cross), or without So-DHT (open circle) for 3 days. Data shown are the mean f SE of 3 separate experiments assayed in triplicates or quadruplicates. *, P < 0.05; **, P < 0.01 vs. control. Effect of CGP 52608 on r3H]hymidine incorporation In contrast to the stimulatory effect of melatonin, [3H]thymidine incorporation was decreased, concentration-dependently, to 98.6 k 4.5%, 87.6 f 2.9% and 66.5 k 3 .O% of the control value when the cells were incubated with 5x10-’ M, 1x 10m6M and 2~10~~ M CGP 52608, respectively. Significant differences between CGP 52608-treated cells and controls were observed at 1x 10m6M (P < 0.05) and 2~10~~ M (P < 0.01) concentrations of the ligand (Fig. 3). Effect of melatonin on cell cycle distribution Flow cytometric analysis of cellular DNA content was conducted to study the effect of melatonin on epididymal epithelial cell cycle. The experiment was repeated four times and representative DNA profiles with melatonin or vehicle treatment are shown in Fig. 4. Compared with vehicletreated control, treatment of the cells with melatonin for 16 hours induced a significant decrease in Go/G, cells (61.7 Z!Z1.7% versus 46.2 f 3.1%, P < 0.05) and a coordinate increase in Gz+M cells (12.5 * 1.7% versus 25.2 k 2.4%, P < O.OS), while no significant changes in the proportion of cells in Go/Gi, S and Gz+M phases were detected when the cells were exposed to melatonin for 8 and 24 hours (Table 1). In addition, no significant changes in the DNA profiles were observed among control cells harvested at 0, 8, 16 and 24 hr and among melatonin-treated cells at 0, 8 and 24 hr after the start of the experiments.
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CGP 52608 concentration (M) Fig. 3 Effect of CGP 52608 on [‘Hlthymidine incorporation into rat epididymal epithelial cells. Cells were incubated with CGP 52608 (5x 1O-’ M, 1x 1Oe6M and 2x 1Om6M) for 3 days, *. P < 0.05; **, P < 0.01 vs. control. Data shown are the mean k SE of 3 separate experiments assayed in triplicates or quadruplicates. Results are expressed as a percentage of the mean [3H]thymidine incorporation into vehicle-treated control (100%).
Fig. 4 Representative DNA histograms of vehicle- and melatonin-treated rat epididymal epithelial cells. Cells were treated with vehicle (a, b, c, d, respectively) or 5x10-’ M melatonin (A, B, C, D, respectively) for 0, 8, 16 and 24 hrs. The relative fluorescence intensity (channel number) and the cell number per channel are represented by the x- and y-axis of the histogram respectively.
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Table 1. Effect of melatonin on cell cycle distribution corpus epididymal epithelial cells vehicle
ohr
8hr
64.3 + 2.9 22.2 f. 1.5 13.6 + 2.1
64.4 f 3.5 21.6 + 2.0 14.0 f 2.6
%GdG,
65.7 + 3.7 20.2 k 2.7 14.1 + 2.9
61.5 + 4.5 23.0 f 2.5 15.5 f 3.3
61.7 + 1.7 27.8 + 3.3 12.5 k 1.7
46.2 f 3.1* 28.4 * 3.7 25.2 f 2.4*
67.5 k 2.1 19.4 + 1.8 13.1 + 2.5
64.9 + 3.1 20.4 f 1.5 14.5 f 2.3
%Go/G, 16hr %S %G2+M
%Go/G1 24hr %S %G2+M
of rat
melatonin
%G,,/G, %S %G2+M
%S %G2+M
1073
The rat corpus epididymal epithelial cells were treated with 5 x 10m7M melatonin or vehicle for 0, 8, 16,24 hr. The data are expressed as mean f SE (n=4). *, P < 0.05, vs. vehicle. Discussion In the present investigation, epididymal epithelial cell proliferation was found to be stimulated by melatonin in a concentrationand time-dependent manner (Fig. 1). While exposure of the epitbelial cells to 6 hours of melatonin daily induced no effect on cell proliferation, increases in cellular proliferative responses to melatonin were observed when the cells were incubated with high concentrations (5~10~~ M and 5x10-’ M) of the pineal hormone daily for 12 hours. In addition, epididymal epithelial cell proliferation was augmented with daily exposure of the cells to melatonin continuously for 24 hr, as evidenced by a lowering in the effective concentration of melatonin in inducing a stimulatory action and an increase in the magnitude of cellular [3H]thymidine incorporation (Fig. 1). Similar to our findings, proliferation of other cell types, such as the lymphoid cells, has been reported to be stimulated concentration-dependently by supraphysiological concentrations of melatonin (19,20,21). Furthermore, our results suggest that the action of melatonin on cell proliferation is likely to be dependent on the time of exposure to the hormone. Being an anabolic sex steroid, testosterone stimulates cell proliferation of target male reproductive tissues including the epididymal epithelial cells. The cellular proliferative responses to melatonin were found to vary with the concentrations of So-DHT, the active metabolite of testosterone, in the culture medium. The epididymal epithelial cells were most sensitive to the stimulatory action of melatonin in the presence of 1~10~~ M So-DHT, while, lower or higher concentrations of 5~DHT reduced the sensitivity of the cells to melatonin (Fig. 2). Apparently, an optimal level of So.-DHT, possibly 1x 10A9M under in vitro conditions, is needed for maximal stimulatory effect of melatonin on epididymal epithelial cell proliferation. Interestingly, this optimal concentration of Sa-DHT is within the physiological range of androgen levels reported
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for rat blood plasma, which is 40-fold less than the concentration fluid entering the epididymis (22).
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of androgens
in the testicular
In the literature, G protein-coupled membrane receptors, nuclear receptors and cytosolic and nuclear sites have been reported to mediate the diverse cellular actions of melatonin (5, 6,23). Of the three G protein-coupled melatonin receptor subtypes (MELBA, MELia and Meli,) which have been cloned in vertebrates (5,24), two (MEL,* and MELia) of them have, so far, been reported in mammals (25, 26). While it is hitherto unknown whether nuclear melatonin receptors are expressed in the epididymis, expression of MELi* and MELta receptor mRNAs have recently been demonstrated in rat epididymal epithelial cells (7). In our study, epididymal epithelial cell proliferation was inhibited by micromolar CGP 52608, a nuclear melatonin receptor agonist (13, 6). Since the action of CGP 52608 on epididymal epithelial cell proliferation is opposite to that of melatonin, the observed stimulatory effect of melatonin on epithelial cell proliferation in the rat epididymis is less likely to be mediated by nuclear melatonin receptor. In light of many reports that have linked the CAMP signaling pathways to regulation of cell proliferation (27, 28, 29) and our previous observation that Gi-coupled MEL iA and MELin receptors are expressed in rat epididymal epithelial cells (7), G protein-coupled membrane receptors are possible candidate molecules for mediating the observed action of melatonin on cell proliferation. Apart from membrane receptors, melatonin has also been shown to interact with calmodulin (30) as well as protein kinase C (31), which are known to be important intracellular biomodulators involved in cell proliferation control (32). Melatonin has been demonstrated to inhibit rat cerebellar nitric oxide synthase and cyclic GMP production via complex formation with calmodulin (33). Besides binding to calmodulin, melatonin was reported to activate Ca2+-dependent protein kinase C activity in vitro (3 1). Apparently, G protein-coupled MELBA and MELta membrane receptors, as well as cytosolic proteins like calmodulin and protein kinase C are possible cellular targets, which are involved in the transduction of the stimulatory signal of melatonin on epididymal epithelial cell proliferation in rats. Further clues to the possible intracellular mechanisms of melatonin action on epididymal epithelial cell proliferation were provided by sequential flow cytometric analysis of cell cycle distribution with melatonin treatment for 0, 8, 16, and 24 hr (Fig. 4 and Table 1). Similar DNA profiles of the epididymal epithelial cell population treated with vehicle over 24 hr reflect a dynamic steady state of the control cells, with relatively constant proportion of the cells in different phases of the cell cycle at the 8 hourly time points sampled. Compared with the vehicletreated controls, no significant changes in the proportion of cells in different cell cycle phases were detected up to 8 hr of incubation with melatonin, whereas, a significant increase in G2+M and decrease in Go/G, cells were detected with continuous treatment of melatonin for 16 hr. It is apparent from the data that it takes on the order of 8 to 16 hr for epididymal epithelial cells to become stimulated by melatonin to progress from Go/G, to G2+M. The time span observed is consistent with that reported for growth factor-induced shift of fibroblasts from Go to Gi phases, which takes about 8 hr (34) and that for fibroblasts to transit from Ga to S phases, which takes at least 12 hr (35). It is also noteworthy that further incubation of the epididymal epithelial cells with melatonin up to 24 hr resulted in a drop of Gz+M and rise of GdGi cells back to the fractions observed after 8 hr of melatonin treatment, suggesting that the cycling time of epididymal epithelial cells in the presence of 5x 1O-’ M melatonin may be on the order of 16 hr. Interestingly, the estimated cycling time of epididymal epithelial cells is similar to that reported for exponentially proliferating 3T3 fibroblast cells (34). Our flow cytometric results, therefore, support a stimulatory action of melatonin on epididymal epithelial cell proliferation by increasing the number of proliferating cells, possibly through activation of quiescent Go cells to re-enter the Gi phase of the cell cycle. The flow cytometric analysis can not distinguish between cells in Go phase and cells in Gi phase, that is why the proportion of melatonin-treated cells in Go/G1 phase
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at 24 hr has no significant different from that of the vehicle-treated cells. It means although the proportion of the GI phase cells treated with melatonin may be increase, the proportion of the cells in G1 plus Go phase treated with melatonin may no change than it treated with vehicle at 24 hr. Given that control of switches in and out of GI is believed to be the main determinant of postembryonic cell proliferation rate in vivo (39, future investigations are warranted to examine whether melatonin, like the growth factors, may serve as a regulator of the molecular cell cycle machinery. In summary, our data showed that melatonin stimulates the proliferation of rat epididymal epithelial cells via mechanisms that probably do not involve the nuclear receptor. It is possible that the observed action of melatonin on cell proliferation may be transduced via membrane receptors, whose activities have been shown to be regulated by androgens (7, 14). Taking into account that aging leads to decrease in pineal melatonin secretion (36) as well as reduction in the number of principal and basal epididymal epithelial cells in rats (37), it is tempting to postulate that the observed stimulatory action of melatonin on cell proliferation may be important for maintaining the normal architectural organization of the functional epithelium in the rat corpus epididymis by replacing effete principal and basal cells in vivo. Acknowledgments This work was supported by CRCG research grant #10200288/18626/21400/301/01, Elaine GCF Tso Memorial Fund and Neuroendocrinology Research Fund of the University of Hong Kong. The authors acknowledge Dr. I. Wiesenberg (Novartis Pharma Inc.) for the gift of CGP 52608, Mr. F. T.W. Wong, Mr. Y.T. Wong and Miss Kimmy Tsang for their technical assistance. Ms. L. Li is a recipient of a postgraduate studentship of the University of Hong Kong and the data presented are derived from part of her PhD thesis work. References 1. S.F. PANG, P.P.N. LEE, Y.S. CHAN, and E.A. AYRE, Melatonin: Biosynthesis, Physiological effects and clinical applications, H.S. Yu and R.J. Reiter (Eds), 129-153, CRC Press, Boca Raton, (1993). 2. R.J. REITER, Endocr. Rev. 12 151-180 (1991). 3. R.J. REITER, Eur. J. Endocrinol. 134 412-420 (1996). 4. M.L. DUBOCOVICH, Trends Pharmacol. Sci. 16 50-56, 1995. 5. S.M. REPPERT, D.R. WEAVER, and C. GODSON, Trends Pharmacol. Sci. 17 loo-102 (1996). 6. I. WIESENBERG, M. MISSBACH, J.P. KAHLEN, M. SCHRADER, and C. CARLBERG, Nucleic Acids Res. 23 327-333 (1995). 7. L. Li, J.N. XU, Y.H. WONG, J.T.Y. WONG, SF. PANG, and S.Y.W. SHIU, J. Pineal Res. 25 219-228 (1998). 8. S.M. REPPERT, C. GODSON, C.D. MAHLE, D.R. WEAVER, S.A. SLAUGENHAUPT, and J.F. GUSELLA, Proc. Natl. Acad. Sci. U. S. A. 92 8734-8738 (1995). 9. Y. SONG, C.W. CHAN, G.M. BROWN, S.F. PANG, and M. SILVERMAN, FASEB J. 1193100 (1997). 10. C. LIU, D.R. WEAVER, X. JIN, L.P. SHEARMAN, R.L. PIESCHL, V.K. GRIBKOFF, and S.M. REPPERT, Neuron 19 91-102 (1997). 11. D.R. WEAVER, C. LIU, and S.M. REPPERT, Mol. Endocrinol. 10 1478-1487 (1996). 12. D. STEINHILBER, M. BRUNGS, 0. WERZ, I. WIESENBERG, C. DANIELSSON, J.P. KAHLEN, S. NAYERI, M. SCHRADER, and C. CARLBERG, J. Biol. Chem. 270 70377040 (1995). 13. S. GARCIA-MAURINO, M.G. GONZALEZ-HABA, J.R. CALVO, M. RAFII-EL-IDRISSI, V. SANCHEZ-MARGALET, R. GOBERNA, and J.M. GUERRERO, J. Immunol. 159 574-
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581 (1997) 14. S.Y.W. SHIU, P.H. CHOW, Z.H. YU, F. TANG, and S.F. PANG, Life Sci. 59 1165-1174 (1996). 15. Z.H. YU, P.H. CHOW, and S.F. PANG, J. Pineal Res. 17 195-201 (1994). 16. S.Y.W. SHIU, L. LI, J.T.Y. WONG, and S.F. PANG, Chin. Med. J. 110 648-655 (1997). 17. G.M. BROWN, J. SEGGIE and L.J. GROTA, Neuroendocrinology 41 3 l-35 (1985). 18. O.H. LOWRY, N.J. ROSEBROUGH. A.L. FARR, and R. J. RANDALL, J. Biol. Chem. 193 265-27.5 (195 1). 19. P. INSERRA, Z. ZHANG, S.K. ARDESTANI, M. ARAGHI-NLKNAM, B. LIANG, S. JIANG, D. SHAW, M. MOLITOR, K. ELLIOTT, and R.R. WATSON, Proc. Sot. Exp. Biol. Med. 218 76-82 (1998). 20. S.P. PERSENGIEV, and S. KYURKCHIEV, Int. J. Biochem. 25 441-444 (1993). 21. A.V. SHAJI, S.K. KULKARNI, and J.N. AGREWALA, Clin. Exp. Immunol. 111 181-185 (1998). 22. R. JONES, C.R. BROWN, K.I.GLOS. VON, and M.G. PARKER, Biochem. J. 188 667-676 (1980). 23. A. MENENDEZ-PELAEZ, and R.J. REITER. J. Pineal Res. 15 59-69 (1993). 24. S.Y.W. SHIU. N. NG, and S. F. PANG, J. Pineal Res. 20 198-204 (1996). 25. S.M. REPPERT, and D.R. WEAVER, Cell 83 1059-1062 (1995). 26. S.Y.W. SHIU, and S.F. PANG, Biological Signals Receptors 7 244-248 (1998). 27. G. D’ANGELO, H. LEE, and R.I. WEINER, J. Cell Biochem. 67 353-366 (1997). 28. W.J. OH, CO. JOE, and K.H. CHOI, Biochem. Mol. Biol. Int. 39 991-999 (1996). 29. T. ONISHI, and K. HRUSHA, Endocrinology 138 1995-2004 (1997). 30. G. BENITEZ-KING, and F. ANTON-TAY, Hormone Res. 21 154-l 59 (1996). 3 1. F. ANTON-TAY, G. RAMIREZ, I. MARTINEZ, and G. BENITEZ-KING, Neurochem. Res. 23 601-606 (1998). 32. G.W. BOOZ. D.E. DOSTAL, H.A. SINGER, and K.M. BAKER, Am. J. Physiol. 267 Cl 3081318 (1994). 33. D. POZO, R.J. REITER, J.R. CALVO, and J.M. GUERRERO, J. Cell Biochem. 65 430-442 (1997). 34. A. ZETTERBERG, and 0. LARSSON, Proc. Nalt. Acad. Sci. USA. 82 5365-5369 (1985). 35. A.B. PARDEE, Science 246 603-608 (1989). 36. S.F. PANG, F. TANG, and P.L. TANG, J Exp. Zool. 229 41-47 (1984). 37. V. SERRE, and B. ROBAIRE, Biol. Reprod. 58 497-5 13 (1998).
PIISOO24-3205(99)00337-9
ELSEVIER
MELATONIN-INDUCED
STIMULATION OF RAT CORPUS EPIDIDYMAL CELL PROLIFERATION
EPITHELIAL
Li Li’, Joseph T.Y. Wong 2, Shiu F. Pang’ and Stephen Y.W. Shiu’ ‘Department of Physiology, The University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong; and *Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
(Receivedin final form May 5, 1999) Summary Stimulation of rat epididymal epithelial cell proliferation by melatonin was demonstrated by thymidine incorporation and flow cytometric analyses. The stimulatory effect of melatonin was dependent on the hormone concentration and the duration of cell exposure to the hormone. Maximal stimulation of [3H]thymidine incorporation into epididymal epithelial cells by melatonin was observed at lx 1V9 M So-dihydrotestosterone in medium, while lower or higher concentrations of androgen attenuated the stimulatory effect of melatonin. Interestingly, a nuclear melatonin receptor agonist (1-[3-allyl-4-0x0thiazolidine-2-ylidene]-4-methyl-thiosemi-carbazone, CGP 52608) induced opposite effect on epithelial cell proliferation to that produced by melatonin. Our data suggest that melatonin-induced stimulation of rat epididymal epithelial cell proliferation is not likely to be mediated by nuclear receptor. Furthermore, sequential changes of cell cycle distribution with melatonin treatment also supports a stimulatory action of melatonin on epididymal epithelial cell proliferation. Key n%fs: melatonin, Sadihydrotestosterone, CGP 52608, cell proliferation, ~11 cycle distribution
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone synthesized and secreted predominantly by the pineal gland in the dark (1). Recent research has provided considerable evidence that this evolutionarily conserved signaling biomolecule subserves multiple functions in mammals, including the regulation of circadian rhythms and reproductive responses (1, 2). Intriguingly, both receptor and non-receptor mediated mechanisms have been proposed for the actions of melatonin (3). To date, specific G protein-coupled (MELBA/ mti, MEL& MT2, Mel,, and ML2/ MTj) and nuclear melatonin receptors (RZRo) have been reported (4, 5, 6) and the tissue distribution of some of these receptors have been mapped using pharmacological, immunological and molecular biological methods (7, 8, 9). Functionally, it has been shown that the G protein-coupled MELBA and MELis receptor subtypes provide the molecular basis for two
Correspondence to: Dr. SYW Shiu, Department of Physiology, The University Shu Fan Building, 5 Sassoon Road, Hong Kong, China
of Hong Kong, Li
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distinct separable effects of melatonin on the physiology of suprachiasmatic nucleus (SCN) in mice. and that MELlu receptor can possibly serve as a back-up for MEL,* receptor in mediating melatonin-induced phase shifts in mouse SCN (10). This highlights the importance of MEL,* receptor of the SCN in regulating the circadian and reproductive responses in seasonal mammal (11). The expression of specific melatonin receptors in tissues outside the central nervous system adds further weight to the long-believed direct regulatory action of the pineal neurohormone on peripheral tissue or cell biology (1). Indeed. it has recently been found that nuclear melatonin receptors may be responsible for repressing 5-lipoxygenase gene expression in human B lymphocytes (12). while enhancing cytokine production by human Thl cells and monocytes (13). Previous studies by our laboratories have demonstrated the expression of high affinity 2[‘2SI]iodomelatonin binding sites, satisfying the pharmacokinetic properties of a specific receptor, in the corpus epididymis of rats (14, 15). These high affinity binding sites in the rat epididymis have been identified by cDNA cloning to be G protein-coupled MEL!* and MELia receptors (7, 16). In situ hybridization studies have demonstrated that both MEL,* and MELia melatonin receptor mRNAs are expressed by epithelial cells of rat corpus epididymis (7). Although these high affinity melatonin receptors, whose activities are regulated by androgens, are negatively coupled to adenylyl cyclase via pertussis toxin-sensitive Gi proteins (7) the biological functions of these Gi-coupled melatonin receptors in epididymal epithelial cells are yet undefined. Given that the androgen can regulate the expression of these membrane receptors (14) and enhance the inhibition of cyclic AMP by melatonin (7), investigations were conducted to examine if melatonin can reciprocally modulate androgen-mediated cellular processes, such as cell proliferation, in the epididymis of male reproductive tissues. Materials and Methods Materials Chemicals for cell culture were purchased from GIBCO BRL chemical Co. (Grand Island, N.Y.). Melatonin and 5a-dihydrotestosterone (SIX-DHT) were supplied by Sigma Chemical Co. (St. Louis, MO). [3H]Thymidine (5 Ci/mmol) was purchased from Amersham International Co. (Aylesbury, UK) l-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-Methyl-thiosemi-c~b~one (CGP 52608) a nuclear melatonin receptor agonist (6) was kindly provided by Dr. I. Wiesenberg (Novartis Pharma Inc., Basel, Switzerland). Male Sprague-Dawley (SD) rats (6-week-old) were obtained from the Laboratory Animal Unit of The University of Hong Kong, and kept under a 12 h light, 12 h dark cycle (light on from 0300-1500 h) for 1 week. Light was provided by ceilingmounted fluorescent lamps with an intensity of about 150-200 lux at the top of the cage. Temperature was kept constant at 23 + 2 “C. Standard food and water were provided ad libitum. The epididymides of animals were collected after sacrifice at mid-light (0900 h). Rat corpus epididymal epithelial cell culture Primary culture of rat corpus epididymal epithelial cells has been previously reported (7). Briefly, rat corpus epididymides were cut into small pieces and placed in sterile Hank’s balanced salt solution (HBSS) containing 0.25% trypsin. Tissues were incubated for 30 min at 32 “C and separated by centrifugation at 800 g for 5 min. The pellet was resuspended in Eagle’s minimum essential medium (EMEM), containing collagenase IA (1 mg/ml) for 2 hr at 32 “C. Cells were separated by centrifugation at 800 g for 10 min. The pellet was then resuspended in EMEM containing nonessential amino acids, sodium pyruvate (1 x lo” M), glutamine (4x 10” M), 5adihydrotestosterone (lx lo-” M), 10% fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 pg/ml). The cell suspension was maintained at 32 “C in a humidified atmosphere of 5% CO2/95% air for 8 hr. During this period, non-epithelial cells such as fibroblasts and smooth muscle cells attached rapidly to the plastic surface of the flask, whereas
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epididymal cells remained suspended in the medium. The epithelial cells in the supematant were then cultured at a concentration of 1x10’ cells per ml at 32 “C. The epididymal epithelial cell monolayers became confluent after 3 days of culture. Cells in the second or third passages were used for determination of [3H]thymidine incorporation or cell cycle distribution. [3H]thymidine incorporation assay Cells (2~10~) in the second or third passages were plated in 6-well plastic dishes and maintained in medium containing 10% FBS at 32°C until they were approximately 70-80% confluent. Quantitation of pre-existing melatonin concentration in FBS-supplemented medium used for culturing cells for [3H]thymidine incorporation studies was performed by radioimmunoassay ( 17) and found to be less than 1O-” M. The cells were incubated for 48 hr in medium containing 10% FBS with melatonin (5~10‘~ M to 5x10-’ M), CGP 52608 (5x10_’ M, 1~10~~ M, 2~10~~ M) or an equal volume of vehicle (0.015% DMSO). For experiments involving melatonin pulse-treatment, cells were incubated with melatonin (5~10~~ M to 5x10“ M) for 6 or 12 hr per day for 3 consecutive days. For So-DHT experiments, cells were treated for 48 hr in medium added with 5x lo-” M So-DHT, 1~10~~ M So-DHT, lx 10m8M Sa-DHT or without Sa-DHT, in the presence of melatonin (5~10~~ M to 5x10-’ M) or vehicle (0.015% DMSO). After 48 hr incubation, [3H]thymidine (5 Ci/mmol, 1 @i/well) was added, and incubation was resumed for another 24 hr. Cells were incubated in 1 ml 10% (wt/vol.) trichloroacetic acid (TCA) for 30 min. The supematant was removed and the precipitate was solubilized in 0.42 ml 1N NaOH for 1 hr. After saving 10 pl aliquots in duplicates for later protein determination by Lowry’s method (18) 0.1 ml glacial acetic acid was added to neutralize the remaining solution. The content of each well was then transferred to a vial with 4 ml scintillation cocktail. The amount of radioactivity was determined by scintillation spectrometry (Beckman LS 6500 multi-purpose scintillation counter). Flow cytometric analysis of cell cycIe distribution Rat epididymal epithelial cells (4~10~) were seeded into 25 cm* flasks until they were approximately 70 - 80 % confluent. Cells were then incubated in medium added with melatonin (5~10.~ M) or an equal volume of vehicle (0.015% DMSO). After 8, 16 and 24 hr of incubation, cells were collected by centrifugation (1000 g x 5 min) at 4 “C after treatment with 0.25% trypsin in PBS solution. The cells were washed three times in PBS and fixed two times, for 5 min each, in 95% ethanol buffered with PBS at 4°C. Fixed cells were rehydrated with PBS and treated with RNase (100 ug/ml) for 20 min at 37 “C, prior to staining with propidium iodide (50 ug/ml) for 30 min in the dark. Using red propidium-DNA fluorescence, 20,000 events were acquired with a vantage cytometer for each sample and the percentage of cells in Go/r, S and Gz-M phases of the cell cycle was determined by the Cell-Fit analytical software (Becton-Dickinson, San Jose, CA, USA). Statistics Each set of experiment, repeated three or four times, was conducted with individual sample in triplicate or quadruplicate. Numerical results were expressed as mean f SE and expressed as a percentage of the mean vehicle-treated control level, which was arbitrarily defined as 100%. The data were analyzed with one way ANOVA and Bonferroni’s t test as appropriate. The level of significance was set at P < 0.05. r
Results
Effect of melatonin on r3H/thymidine incorporation into epididymal epithelial cells Melatonin induced an increase of cellular [3H]thymidine incorporation in a concentrationdependent manner (Fig. 1). Significant (P < 0.05) increase in [3H]thymidine incorporation was observed in cells which were incubated, continuously, with 5x 10m8M to 5x IO-’ M melatonin.
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Maximal stimulation to 181.0 f 5.1% of the control value was recorded when melatonin concentration was 5~10~~ M. Increase in [3H]thymidine incorporation into the cells which received pulse treatment of melatonin was found to depend on the pulse duration. No significant increase in [3H]thymidine incorporation was noted for cells which were pulsed-treated with melatonin for 6 hr per day for 3 consecutive days. For cells treated with melatonin for 12 hr per day consecutively for 3 days, [3H]thymidine incorporation into the cells increased significantly to 115.7 + 4.4% (P < 0.05) and 126.0 k 4.8% (P < 0.01) of control value when the respective concentrations of melatonin were 5x 10e6M and 5x 1O-’ M.
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Fig. 1 Effect of melatonin on [3H]thymidine incorporation into rat epididymal epithelial cells. Cells were incubated in medium containing 1~10~~ M SWDHT with melatonin for 6 hr (open circle), 12 hr (open quadrate) and 24 hr (open triangle) per day for 3 consecutive days. Data shown are the mean XLSE of 3 separate experiments assayed in triplicates or quadruplicates. Results are expressed as a percentage of the mean [3H]thymidine incorporation into vehicle-treated control (100%). *, P < 0.05; **, P < 0.01 vs. control. Effects of melatonin with and without 5 a-DHT on J3H]thymidine incorporation The levels of [3H]thymidine incorporation varied with the concentrations of Sa-DHT in the medium. [3H]Thymidine incorporation levels were 12837 + 1201 dpm per mg protein, 13289 + 1010 dpm per mg protein, 14010 + 454 dpm per mg protein, and 18720 f 742.2 dpm per mg protein (three separate experiments) when the cells were incubated with 0 M, 5x 1O-” M, 1x 1Oe9 M and 1x10-* M Sa-DHT, respectively. without melatonin addition (Fig. 2). In the absence of Sa-DHT, significant increases in cellular [3H]thymidine incorporation to 127.3 + 2.5% (P< 0.05) and 133.0 + 5.5% (P < 0.01) of the control value were recorded when the concentrations of melatonin were 5x 1Om6M and .5x1Om5M respectively. An increase of androgen concentration up to 1x 10m9 M Sa-DHT enhanced the sensitivity and magnitude of cellular [3H]thymidine incorporation to melatonin. The lower concentrations of melatonin that induced a significant (P< 0.05) increase in cellular [3H]thymidine incorporation were 5~10.~ M and 5x10-* M for cells that were, respectively, co-incubated with 5x 1OF” M and 1x 1OW9 M Sa-DHT. In the presence of 5x 1U5 M melatonin, [3H]thymidine incorporation was increased to 145.3 + 6.1% (P< 0.01) and 181 .O + 5.1% (P< 0.01) of the control value when the cells were respectively co-incubated with 5~10-‘~ M and lx low9 M Sa-DHT. Despite a 1.46-fold elevation of basal [3H]thymidine incorporation when Sa-DHT was increased from 0 M to 1x 10m8M, a decrease in the cellular responses to
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melatonin in the presence of l~lO_~ M So-DHT was recorded. While 5~10~~ M melatonin increased [3H]thymidine incorporation into cells to 139.6 k 7.1% (P < 0.01) of the control value, the lowest concentration of melatonin that could induce a significant (P < 0.05) increase in [3H]thymidine incorporation was 5~10~~ M.
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Fig. 2 Effect of melatonin on [3H]thymidine incorporation into rat epididymal epithelial cells incubated with different concentrations of So-dihydrotestosterone (So-DHT). Cells were incubated in medium containing increasing concentrations of melatonin (5~10~~ to 5~10~~) with 5x10-” M So-DHT (closed lozenge), 1x1w9 M So-DHT (open triangle), 1x 10m8M So-DHT (cross), or without So-DHT (open circle) for 3 days. Data shown are the mean f SE of 3 separate experiments assayed in triplicates or quadruplicates. *, P < 0.05; **, P < 0.01 vs. control. Effect of CGP 52608 on r3H]hymidine incorporation In contrast to the stimulatory effect of melatonin, [3H]thymidine incorporation was decreased, concentration-dependently, to 98.6 k 4.5%, 87.6 f 2.9% and 66.5 k 3 .O% of the control value when the cells were incubated with 5x10-’ M, 1x 10m6M and 2~10~~ M CGP 52608, respectively. Significant differences between CGP 52608-treated cells and controls were observed at 1x 10m6M (P < 0.05) and 2~10~~ M (P < 0.01) concentrations of the ligand (Fig. 3). Effect of melatonin on cell cycle distribution Flow cytometric analysis of cellular DNA content was conducted to study the effect of melatonin on epididymal epithelial cell cycle. The experiment was repeated four times and representative DNA profiles with melatonin or vehicle treatment are shown in Fig. 4. Compared with vehicletreated control, treatment of the cells with melatonin for 16 hours induced a significant decrease in Go/G, cells (61.7 Z!Z1.7% versus 46.2 f 3.1%, P < 0.05) and a coordinate increase in Gz+M cells (12.5 * 1.7% versus 25.2 k 2.4%, P < O.OS), while no significant changes in the proportion of cells in Go/Gi, S and Gz+M phases were detected when the cells were exposed to melatonin for 8 and 24 hours (Table 1). In addition, no significant changes in the DNA profiles were observed among control cells harvested at 0, 8, 16 and 24 hr and among melatonin-treated cells at 0, 8 and 24 hr after the start of the experiments.
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CGP 52608 concentration (M) Fig. 3 Effect of CGP 52608 on [‘Hlthymidine incorporation into rat epididymal epithelial cells. Cells were incubated with CGP 52608 (5x 1O-’ M, 1x 1Oe6M and 2x 1Om6M) for 3 days, *. P < 0.05; **, P < 0.01 vs. control. Data shown are the mean k SE of 3 separate experiments assayed in triplicates or quadruplicates. Results are expressed as a percentage of the mean [3H]thymidine incorporation into vehicle-treated control (100%).
Fig. 4 Representative DNA histograms of vehicle- and melatonin-treated rat epididymal epithelial cells. Cells were treated with vehicle (a, b, c, d, respectively) or 5x10-’ M melatonin (A, B, C, D, respectively) for 0, 8, 16 and 24 hrs. The relative fluorescence intensity (channel number) and the cell number per channel are represented by the x- and y-axis of the histogram respectively.
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Table 1. Effect of melatonin on cell cycle distribution corpus epididymal epithelial cells vehicle
ohr
8hr
64.3 + 2.9 22.2 f. 1.5 13.6 + 2.1
64.4 f 3.5 21.6 + 2.0 14.0 f 2.6
%GdG,
65.7 + 3.7 20.2 k 2.7 14.1 + 2.9
61.5 + 4.5 23.0 f 2.5 15.5 f 3.3
61.7 + 1.7 27.8 + 3.3 12.5 k 1.7
46.2 f 3.1* 28.4 * 3.7 25.2 f 2.4*
67.5 k 2.1 19.4 + 1.8 13.1 + 2.5
64.9 + 3.1 20.4 f 1.5 14.5 f 2.3
%Go/G, 16hr %S %G2+M
%Go/G1 24hr %S %G2+M
of rat
melatonin
%G,,/G, %S %G2+M
%S %G2+M
1073
The rat corpus epididymal epithelial cells were treated with 5 x 10m7M melatonin or vehicle for 0, 8, 16,24 hr. The data are expressed as mean f SE (n=4). *, P < 0.05, vs. vehicle. Discussion In the present investigation, epididymal epithelial cell proliferation was found to be stimulated by melatonin in a concentrationand time-dependent manner (Fig. 1). While exposure of the epitbelial cells to 6 hours of melatonin daily induced no effect on cell proliferation, increases in cellular proliferative responses to melatonin were observed when the cells were incubated with high concentrations (5~10~~ M and 5x10-’ M) of the pineal hormone daily for 12 hours. In addition, epididymal epithelial cell proliferation was augmented with daily exposure of the cells to melatonin continuously for 24 hr, as evidenced by a lowering in the effective concentration of melatonin in inducing a stimulatory action and an increase in the magnitude of cellular [3H]thymidine incorporation (Fig. 1). Similar to our findings, proliferation of other cell types, such as the lymphoid cells, has been reported to be stimulated concentration-dependently by supraphysiological concentrations of melatonin (19,20,21). Furthermore, our results suggest that the action of melatonin on cell proliferation is likely to be dependent on the time of exposure to the hormone. Being an anabolic sex steroid, testosterone stimulates cell proliferation of target male reproductive tissues including the epididymal epithelial cells. The cellular proliferative responses to melatonin were found to vary with the concentrations of So-DHT, the active metabolite of testosterone, in the culture medium. The epididymal epithelial cells were most sensitive to the stimulatory action of melatonin in the presence of 1~10~~ M So-DHT, while, lower or higher concentrations of 5~DHT reduced the sensitivity of the cells to melatonin (Fig. 2). Apparently, an optimal level of So.-DHT, possibly 1x 10A9M under in vitro conditions, is needed for maximal stimulatory effect of melatonin on epididymal epithelial cell proliferation. Interestingly, this optimal concentration of Sa-DHT is within the physiological range of androgen levels reported
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for rat blood plasma, which is 40-fold less than the concentration fluid entering the epididymis (22).
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of androgens
in the testicular
In the literature, G protein-coupled membrane receptors, nuclear receptors and cytosolic and nuclear sites have been reported to mediate the diverse cellular actions of melatonin (5, 6,23). Of the three G protein-coupled melatonin receptor subtypes (MELBA, MELia and Meli,) which have been cloned in vertebrates (5,24), two (MEL,* and MELia) of them have, so far, been reported in mammals (25, 26). While it is hitherto unknown whether nuclear melatonin receptors are expressed in the epididymis, expression of MELi* and MELta receptor mRNAs have recently been demonstrated in rat epididymal epithelial cells (7). In our study, epididymal epithelial cell proliferation was inhibited by micromolar CGP 52608, a nuclear melatonin receptor agonist (13, 6). Since the action of CGP 52608 on epididymal epithelial cell proliferation is opposite to that of melatonin, the observed stimulatory effect of melatonin on epithelial cell proliferation in the rat epididymis is less likely to be mediated by nuclear melatonin receptor. In light of many reports that have linked the CAMP signaling pathways to regulation of cell proliferation (27, 28, 29) and our previous observation that Gi-coupled MEL iA and MELin receptors are expressed in rat epididymal epithelial cells (7), G protein-coupled membrane receptors are possible candidate molecules for mediating the observed action of melatonin on cell proliferation. Apart from membrane receptors, melatonin has also been shown to interact with calmodulin (30) as well as protein kinase C (31), which are known to be important intracellular biomodulators involved in cell proliferation control (32). Melatonin has been demonstrated to inhibit rat cerebellar nitric oxide synthase and cyclic GMP production via complex formation with calmodulin (33). Besides binding to calmodulin, melatonin was reported to activate Ca2+-dependent protein kinase C activity in vitro (3 1). Apparently, G protein-coupled MELBA and MELta membrane receptors, as well as cytosolic proteins like calmodulin and protein kinase C are possible cellular targets, which are involved in the transduction of the stimulatory signal of melatonin on epididymal epithelial cell proliferation in rats. Further clues to the possible intracellular mechanisms of melatonin action on epididymal epithelial cell proliferation were provided by sequential flow cytometric analysis of cell cycle distribution with melatonin treatment for 0, 8, 16, and 24 hr (Fig. 4 and Table 1). Similar DNA profiles of the epididymal epithelial cell population treated with vehicle over 24 hr reflect a dynamic steady state of the control cells, with relatively constant proportion of the cells in different phases of the cell cycle at the 8 hourly time points sampled. Compared with the vehicletreated controls, no significant changes in the proportion of cells in different cell cycle phases were detected up to 8 hr of incubation with melatonin, whereas, a significant increase in G2+M and decrease in Go/G, cells were detected with continuous treatment of melatonin for 16 hr. It is apparent from the data that it takes on the order of 8 to 16 hr for epididymal epithelial cells to become stimulated by melatonin to progress from Go/G, to G2+M. The time span observed is consistent with that reported for growth factor-induced shift of fibroblasts from Go to Gi phases, which takes about 8 hr (34) and that for fibroblasts to transit from Ga to S phases, which takes at least 12 hr (35). It is also noteworthy that further incubation of the epididymal epithelial cells with melatonin up to 24 hr resulted in a drop of Gz+M and rise of GdGi cells back to the fractions observed after 8 hr of melatonin treatment, suggesting that the cycling time of epididymal epithelial cells in the presence of 5x 1O-’ M melatonin may be on the order of 16 hr. Interestingly, the estimated cycling time of epididymal epithelial cells is similar to that reported for exponentially proliferating 3T3 fibroblast cells (34). Our flow cytometric results, therefore, support a stimulatory action of melatonin on epididymal epithelial cell proliferation by increasing the number of proliferating cells, possibly through activation of quiescent Go cells to re-enter the Gi phase of the cell cycle. The flow cytometric analysis can not distinguish between cells in Go phase and cells in Gi phase, that is why the proportion of melatonin-treated cells in Go/G1 phase
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at 24 hr has no significant different from that of the vehicle-treated cells. It means although the proportion of the GI phase cells treated with melatonin may be increase, the proportion of the cells in G1 plus Go phase treated with melatonin may no change than it treated with vehicle at 24 hr. Given that control of switches in and out of GI is believed to be the main determinant of postembryonic cell proliferation rate in vivo (39, future investigations are warranted to examine whether melatonin, like the growth factors, may serve as a regulator of the molecular cell cycle machinery. In summary, our data showed that melatonin stimulates the proliferation of rat epididymal epithelial cells via mechanisms that probably do not involve the nuclear receptor. It is possible that the observed action of melatonin on cell proliferation may be transduced via membrane receptors, whose activities have been shown to be regulated by androgens (7, 14). Taking into account that aging leads to decrease in pineal melatonin secretion (36) as well as reduction in the number of principal and basal epididymal epithelial cells in rats (37), it is tempting to postulate that the observed stimulatory action of melatonin on cell proliferation may be important for maintaining the normal architectural organization of the functional epithelium in the rat corpus epididymis by replacing effete principal and basal cells in vivo. Acknowledgments This work was supported by CRCG research grant #10200288/18626/21400/301/01, Elaine GCF Tso Memorial Fund and Neuroendocrinology Research Fund of the University of Hong Kong. The authors acknowledge Dr. I. Wiesenberg (Novartis Pharma Inc.) for the gift of CGP 52608, Mr. F. T.W. Wong, Mr. Y.T. Wong and Miss Kimmy Tsang for their technical assistance. Ms. L. Li is a recipient of a postgraduate studentship of the University of Hong Kong and the data presented are derived from part of her PhD thesis work. References 1. S.F. PANG, P.P.N. LEE, Y.S. CHAN, and E.A. AYRE, Melatonin: Biosynthesis, Physiological effects and clinical applications, H.S. Yu and R.J. Reiter (Eds), 129-153, CRC Press, Boca Raton, (1993). 2. R.J. REITER, Endocr. Rev. 12 151-180 (1991). 3. R.J. REITER, Eur. J. Endocrinol. 134 412-420 (1996). 4. M.L. DUBOCOVICH, Trends Pharmacol. Sci. 16 50-56, 1995. 5. S.M. REPPERT, D.R. WEAVER, and C. GODSON, Trends Pharmacol. Sci. 17 loo-102 (1996). 6. I. WIESENBERG, M. MISSBACH, J.P. KAHLEN, M. SCHRADER, and C. CARLBERG, Nucleic Acids Res. 23 327-333 (1995). 7. L. Li, J.N. XU, Y.H. WONG, J.T.Y. WONG, SF. PANG, and S.Y.W. SHIU, J. Pineal Res. 25 219-228 (1998). 8. S.M. REPPERT, C. GODSON, C.D. MAHLE, D.R. WEAVER, S.A. SLAUGENHAUPT, and J.F. GUSELLA, Proc. Natl. Acad. Sci. U. S. A. 92 8734-8738 (1995). 9. Y. SONG, C.W. CHAN, G.M. BROWN, S.F. PANG, and M. SILVERMAN, FASEB J. 1193100 (1997). 10. C. LIU, D.R. WEAVER, X. JIN, L.P. SHEARMAN, R.L. PIESCHL, V.K. GRIBKOFF, and S.M. REPPERT, Neuron 19 91-102 (1997). 11. D.R. WEAVER, C. LIU, and S.M. REPPERT, Mol. Endocrinol. 10 1478-1487 (1996). 12. D. STEINHILBER, M. BRUNGS, 0. WERZ, I. WIESENBERG, C. DANIELSSON, J.P. KAHLEN, S. NAYERI, M. SCHRADER, and C. CARLBERG, J. Biol. Chem. 270 70377040 (1995). 13. S. GARCIA-MAURINO, M.G. GONZALEZ-HABA, J.R. CALVO, M. RAFII-EL-IDRISSI, V. SANCHEZ-MARGALET, R. GOBERNA, and J.M. GUERRERO, J. Immunol. 159 574-
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