Melatonin anterior pituitary of the female Wistar rat

FOLIAHISTOCHEMICAETCYTOBIOLOGICAVol. 48, No. 2, 2010pp. 278-283

Melatonin modulates the effects of diethylstilbestrol(DES) on the anteriorpituitary of the female Wistarrat

Weijiang Zhao1,2, Zhongfang Shi2, Fang Yuan2, Guilin Li2, Yilin Sun2,Yazhuo Zhang2, Zhongcheng Wang2

1Neuroscience Center, Shantou University Medical College, Shantou, Guangdong Province 515041, China2Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, China

Abstract:We studied the anti-tumorigenic effect of melatonin in diethylstilbestrol (DES)-treated anterior pituitaries in rats.Twenty-one female Wistar rats were randomly allocated into three groups: vehicle control rats, DES-treated rats, and DES-treated rats co-administrated with melatonin beginning at week 13. At the end of 16 weeks, rats were weighed and decapi-tated for morphological studies, including an H+E staining-based score evaluation in regard to cell proliferation, angiogen-esis, immunostaining for VEGF, MMP-9, and AQP-1, and electron microscopy. Compared with vehicle, long-term treatmentof DES significantly reduced rat body weight and increased H+E score, both of which were counteracted by melatonin.Administration of melatonin also reduced the expression of VEGF and MMP-9, although no changes were detected in AQP-1 expression. In rats cotreated with melatonin, the RER loosened and accumulated more secretion granules. We thus con-cluded that melatonin can modulate the effects of DES on the rat anterior pituitary by downregulating expression of VEGFand MMP-9 and suppressing the release of secretion granules, suggesting a therapeutic potential in estrogen-induced pitu-itary malfunctions.

Key words:melatonin, vascular endothelial growth factor, VEGF, aquaporin-1, AQP-1, MMP-9, ultrastructure

Introduction

It has been reported that estrogen exposure is one ofthe factors leading to the development of prolactino-ma, a tumor of the pituitary [1]. Experimental estrogenadministration can induce prolactinoma in mice andrats, which is characterized by cellular hyperprolifera-tion and angiogenesis, thus providing a means forinvestigating the mechanisms of pituitary tumorigene-sis and effective therapeutic methods against it [2-4].Melatonin, a neuroendocrine hormone derived from5-hydroxytryptamine (5-HT) and initially synthesizedin the pineal body, is widely distributed in a variety oforgans and tissues. Wu et al. [5] demonstrated strongexpression of melatonin receptors (MT) in several cere-bral nuclei and weak expression of the receptor MT1,through which melatonin might exert its physiologicalfunctions and pharmaceutical effects, in both the ante-rior pituitary and the posterior pituitary glands. In addi-tion, melatonin can protect neurons, enhance immune

Correspondence:F. Yuan and G. Li, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, China;

e-mail: florayuan@, liguilin40@

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Folia Histochem Cytobiol. 2010:48(2): 278(278-283) 10.2478/v10042-010-0023-1

function, delay senescence, and regulate cell division[6,7]. The antiproliferative effects of melatonin werealso observed in multiple carcinoma cell lines of humanand mouse, as well as in diethylstilbestrol (DES)-induced prolactinoma [8]. Recent investigations showthat melatonin suppresses the expression of estrogenreceptor (ER), inhibits ER binding to the estrogenresponse element (ERE), reduces mutations in theestrogen enhancer, and induces apoptosis of prolactin-oma cells, leading to reductions in tumor volume [9-11]. However, the molecular mechanisms of melatonintreatment remain unclear.

Angiogenesis is a typical characteristic of pituitarytumors and is a pivotal event in tumor invasion[12,13]. The roles of VEGF, MMP-1, and AQP-1 havebeen established in angiogenesis, suggesting roles intumor behavior [14-16]. Lissoni et al. [17] evaluatedthe effect of melatonin on senior cancer patients, dis-covering that control of tumor growth by melatoninwas related to reduced serum VEGF concentrations,which suggested that melatonin functions as a naturalanti-angiogenesis agent to inhibit tumor growth. How-ever, we are not aware of any prior reports on the effectof DES on angiogenesis in pituitary tumors.

Melatonin modulates DES effects on the pituitary

melatonin on DES-induced expression of VEGF,In the present study, we investigated the effect ofMMP-9, and AQP-1 and its possible effect on theultrastructure of the anterior pituitary. Compared withrats without melatonin treatment, rats concurrentlyreceiving melatonin and DES demonstrated signifi-cantly reduced expression of VEGF and MMQ-9,accompanied by diminished ultrastructural deteriora-tion of the pituitary tissue. Our findings suggest bene-ficial effects of melatonin in estrogen-related pituitarymalfunction.

Materials and methods

Animals and materials.Animal manipulation was approved bythe Animal Use Committee of Beijing Neurosurgical Institute. Atotal of 21 female Wistar rats, 3 weeks old, and weighing 70-80 gwere used. All animals were housed with free access to tap waterand standard pellet food. They were kept at a controlled tempera-ture (24±1°C) and humidity (55±5%), and a 12-hour day-nightcycle (10 a.m.-10 p.m.) was maintained. Rats were randomlydivided into 3 groups of 7, with each group receiving a differenttreatment regimen: the vehicle control group, in which rats wereintraperitoneally administrated with sunflower seed oil (1 mL/kg,twice a week) for 16 weeks; the DES group, which received DES(5 mg/kg, twice a week) for 16 weeks; and the DES + melatoningroup, in which DES was administered for 16 weeks at the samedose as in the DES group, with melatonin (1 mg/day) co-adminis-tered at 5 p.m. each day from weeks 13 to 16. Rats in both vehicleand DES groups received the same dose of vehicle in place ofmelatonin, while the DES + melatonin group received melatonin.

DES and melatonin were purchased from Sigma Chemical Co.(St Louis, MO, USA). Anti-VEGF antibodies were purchased fromBoster Biotech (Wuhan, China). MMP-9 and prolactin (PRL) anti-bodies were purchased from Dako (Denmark). AQP-1 antibodywas purchased from Chemicon (Temecula, CA, USA). The DESinjection was prepared by dissolving DES powder in sunflowerseed oil to obtain a final concentration of 5 mg/mL.

Morphological score of anteriorpituitary gland.After 16weeks, rodents were anesthetized with chloroaldehyde, 10%hydration (300 mg/kg) and perfused intracardially with a 40g/Lsolution of paraformaldehyde in PBS (0.01M, pH 7.4). Tissueswere then embedded in paraffin, sectioned at 4 µm for H+E stain-ing, and observed immunohistochemically. An evaluation scoresystem based on H+E staining was employed to compare thechanges in the anterior pituitary gland between each group.Immunohistochemistry.After deparaffinization, sections wererehydrated through a graded series of ethanol to phosphate buffer.For antigen retrieval, the sections were incubated in 3% Hclear endogenous peroxidase and were then saturated with 10%2O2tonormal goat serum for 10 minutes. Afterwards, sections were indi-vidually incubated with anti-VEGF, -MMP-9, or -AQP-1 antibody(1:50 dilution) at 4°C overnight. The antigen-antibody complexeswere visualized using the avidin-biotin-peroxidase complex (ABC)method. Counterstaining was done with Meyer's hematoxylin.Table 1 depicts the H+E scoring system used.

Transmission electron microscopy.randomly selected for transmission electron microscopic investiga-One rat in each group wastion. For electron microscopy, pituitary tissues were fixed in 2%glutaraldehyde in 0.1 M PBS (pH 7.4) and then subjected to post-fixation in 2% osmium tetroxide. Tissues were embedded inAraldite, and the ultra-thin sections were examined under a PhilipsEM208 electron microscope.

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Table 1.Score based on H+E staining.

Ethical issues.The study was monitored by the Bioethical Com-mittee of Beijing Tiantan Hospital, Capital Medical University,China. The number of the license for animal use is SYXK (Beijing)2008-005.

Statistical analysis.Results were expressed as mean ± standarddeviation (SD), and one-way ANOVAanalysis was used to com-pare differences in body weight and H+E score, with post-hoctesting by Bonferroni test. Frequency distribution was applied totest the normal distribution of data in each group using Skew-ness method. P-values of less than 0.05 were considered signifi-cant.

Results

Physical and histological changes inducedby DES and melatonin

Fur loss and weight loss are the initial superficialeffects of intraperitoneal injection of DES in pared with continuously DES-treated rats, mela-tonin treatment appeared to reduce fur loss. The bodyweights of rats in the vehicle control group, the DESgroup, and the DES + melatonin group were282.9±35.6 g, 239.9±20.7 g, and 248.7±17.4 g, respec-tively. The frequency of values in each group was dis-tributed in a normal mode. Compared with controls,continuous treatment of DES without melatonin sig-nificantly reduced body weight, which was mitigatedby coadministration of melatonin, although no signifi-cance was observed.

sues showed no apparent abnormalities or gland cavi-In six out of seven vehicle-treated rats, pituitary tis-ty narrowing, and the pituitary cells were arrangednormally. One rat showed self-developed pituitaryhyperproliferation. By contrast, pituitary tissues in ratsreceiving continuous administration of DES showed adisappearance of the gland cavity, with subsequentoccupation by proliferated cells. Co-administration ofmelatonin reduced structural abnormalities, withincreased gland cavity volume compared with the DES

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Fig. 1.Immunohistochemical stain-ing of female Wistar rat anteriorpituitary gland for expression ofPRL, VEGF, MMP-9, and AQP-1.V-vehicle control rats, DES-diehyl-stilbestrol treated rats, DES+M-diehylstilbestrol treated rats coad-ministrated with melatonin. scalebars=30µm.

group, possibly attributed to the sharp withdrawal ofproliferated cells.

The H+E staining-based evaluation scores in thevehicle, DES, and DES + melatonin groups were1.42±1.13, 4.57±0.98, and 2.00±0.63, respectively,and frequency distribution of values in each groupwere in agreement with normal distribution. Long-term treatment of DES significantly increased the H+Escore, which was reduced by subcutaneous injection ofmelatonin (p<0.01 vs. DES group). In the sole pituitarywith spontaneous tumor-like changes, the tumor-likecells were closely packed with similar cytoplasmic andnuclear volumes, suggesting that they were from thesame origin.

blood vessels. By contrast, administration of DES ledto extensive expression of VEGF in the cytoplasm ofpituitary cells, accompanied by angiogenesis, whichwas reduced by melatonin at a dose of 1 mg/day for 4weeks (Fig. 1).

No apparent expression of MMP-9 was observed invehicle-treated rats, whereas administration of DESproduced diffuse expression of MMP-9. Co-adminis-tration of melatonin abolished MMP-9 expression inthe pituitary (Fig. 1). At week 16, expression of AQP-1 was increased in the endothelial cells of the pituitaryglands of rats receiving DES, which could not bealtered by melatonin administration (Fig. 1).

Effects of melatonin on VEGF, MMP-9,and AQP-1

In the vehicle-treated pituitary tissues, VEGF wasselectively expressed in pituitary cells surrounding

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Changes in ultrastructure

We previously reported in the DES-treated pituitary,electron microscopic investigation revealed well-developed rough endoplasmic reticulum (RER) andfaulty exocytosis in some cells, indicating the devel-

Melatonin modulates DES effects on the pituitary

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Fig. 2.Electron microscopy on pitu-itary tissues. Accumulated secretiongranules for extrusion into the bloodvessels were observed in the DEStreated rat pituitary gland (A),whereas in the DES treated rat pitu-itary co-administrated with mela-tonin, most of these granules wereseen in the loosened structure of therough endoplasmic reticulum (B).

opment of prolactinoma [16]. Here, we also observedthe accumulation of secretion granules approachingthe blood vessels in the DES treated rat pituitary gland(Fig. 2A). In pituitary tissues of rats co-treated withmelatonin, many premature, abnormally enlarged,medium-density hormone containing granules wereobserved in loosened endoplasmic reticulum, with anaverage diameter of 300 nm (Fig. 2B), suggestingmelatonin may function by inhibiting the granuleextrusion.

Discussion

Prolactinoma is a multi-factorial disease. Althoughdopamine agonists, including bromocriptine,quinagolide, and lisuride, have proved to be effective inmost patients suffering from prolactinoma, there are stillmany patients who show no response to these drugs orwho develop resistance to them [18]. In this investiga-tion, rats were subjected to DES administration with orwithout concurrent melatonin treatment, after whichimmunohistochemistry and electron microscopy wereundertaken to investigate the effects of melatonin on theexpression of VEGF, MMP-9, and AQP-1, as well as itseffect on the ultrastructure of pituitary tissues. Ourresults demonstrate the ability of melatonin to counter-act DES-induced upregulation of VEGF and MMP-9expression and its ability to improve the ultrastructureof DES-treated pituitary tissue.

Although investigations have demonstrated that thepineal body influences PRLrelease, little is knownabout the effects of the pineal body on hyperprolactine-mic diseases, and pineal body activity is usually exclud-ed from clinical investigations of pituitary tissues. Liss-noi et al. [19] investigated the effects of melatonininjection on serum PRLin 19 patients with hyperpro-lactinemia. They found that intramuscular injection ofmelatonin led to the reduction of serum PRLin 3 out of8 patients with congenital hyperprolacthemia. Subse-quent investigation [17] showed that the serum mela-©Polish Histochemical et Cytochemical Society

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tonin level in these patients was significantly lower thanin normal controls. Additionally, patients withacromegaly and pituitary tumors showed no response interms of serum growth hormone or prolactin to acuteadministration of melatonin. Thus, the effects of long-term melatonin treatment need further elucidation.

Xuet al. [9,20] used controllable pumps containingestradiol to induce the development of prolactinoma inrats, which received melatonin treatment from 7 daysbefore implantation to 90 days after implantation. Theresults demonstrated that melatonin could effectivelyabolish the growth of prolactinoma by inhibiting PRLand ER expression and could partially prevent thebinding of ER to ERE.

Melatonin, at an appropriate dose, can also reducemutations in the PRLgene enhancer, preventing over-expression of PRLand resulting in the decreasedgrowth of prolactinomas [10]. Further investigationsdemonstrated that melatonin can increase the activityof caspase-3, increasing the expression of Bcl-2 andfacilitating decreases in mitochondrial membranepotential, which promote apoptosis and reduce tumormass [11,21]. Notably, melatonin treatment was initi-ated before DES induction in previous studies. In con-trast to this preventive mode of treatment, we adminis-tered melatonin after 12 weeks of DES pre-treatment.Co-administration of melatonin with DES couldpromote the re-emergence of the adenoid cavity, espe-cially in the areas approaching the margin of the ante-rior pituitary gland. This may be caused by the rapidretreat of proliferative cells, allowing greater bloodflow and thus leaving a larger cavity. It can be specu-lated that further increases in blood flow might rupturethe vessels. Therefore, an effective dose of melatoninwill need to be determined to optimize vascular stabil-ity during melatonin treatment.

Electron microscopy is a sensitive and reliablemethod for observing the earliest ultrastructuralchanges related to the development and treatment of apituitary tumor. Using immune electron microscopy,

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Osamura well-developed, double-layered, concentric roughet al. [22] found that PRLwas located in theendoplasmic reticulum. Bromocriptine caused thecytoplasm to shrink and unsecreted PRLaggregate, with the abrogation of exocytosis. Eljarmakgranules toet alistration reduced the volumes of the cell, the rough. [23] found that continuous bromocriptine admin-endoplasmic reticulum, and the Golgi complex. Theultrastructural improvements conferred by melatoninprovided strong evidence for its efficacy as a thera-peutic. Melatonin has been shown to reduce tumor sizeand serum PRLexocytosis or disruption of RER.

concentration without the misplacedfor 4 weeks caused the accumulation of large PRLIn the present study, administration of melatoninsecretory granules in loosened RER, suggesting mela-tonin may inhibit the exocytosis of PRL-containinggranules and reduce the expression of PRLnegative feedback. The structural improvements to thethroughconcentric RER support this.

product of the pineal body and a natural tumorThe anti-cancer activity of melatonin, primarily ainhibitor, has been related to its immune-regulatory,anti-proliferation, and anti-oxidant effects. Angiogene-sis is a key step in the development and metastasis ofmost tumors, including pituitary tumors. However,compelling data concerning melatonin in angiogenesisare not yet available. Soybir melatonin could function in angiogenesis and woundet al. [24] found thatrepair, which was related to the regulatory effects ofmelatonin on monocytes, cytokines, and fibroblastomacells. Cui it the growth of cultured umbilical vein endothelialet al. [25] found that melatonin could inhib-cells, inducing apoptosis by regulating the cell size,which involved p53 and Bax/Bcl expression changes.EGF and VEGF are involved in angiogenesis, stimu-lating cellular proliferation, promoting vasculariza-tion, and sustaining the integrity of vessels, meaningthese proteins play an important role in tumor metasta-sis [26-28]. The present study demonstrated DES-induced upregulation of VEGF and MMP-9, confirm-ing a role for estrogen in angiogenesis in the rat ante-rior pituitary. Melatonin has been reported to down-regulate MMP-9 expression and activity in severalpathological states in rodents, such as reperfusion-induced hemorrhage following transient focal cerebralischemia and spinal cord injury [29-31]. The inhibito-ry effects of melatonin may be associated with reducedexpression of TNF-inhibitor of metalloproteinase (TIMP)-1 [30,32]. αand elevated expression of tissueMMP-9, accompanied by an improvement in H+E score,Melatonin reduced the expression of both VEGF andsuggesting these molecules are targets of melatonin inthe treatment of estrogen-induced pituitary malfunction.Further study is needed to determine why AQP-1 wasupregulated by DES and not suppressed by melatonin.

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Folia Histochem Cytobiol. 2010:48(2): 282(278-283) 10.2478/v10042-010-0023-1

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Submitted: 10 February, 2010

Accepted after reviews: 25 April, 2010

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