Inhibition of the increased 17ß-estradiol-induced mast cell number by melatonin in the testis of the frog Rana esculenta, in vivo and in vitro
Dipartimento di Medicina Sperimentale-Sezione di Fisiologia Umana e Funzioni Biologiche Integrate "F. Bottazzi", Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, via Costantinopoli 16, 80138 Napoli, Italy
* Author for correspondence (e-mail: sergio.minucci{at}unina2.it)
Accepted 7 November 2003
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Summary |
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Key words: melatonin, 17ß-estradiol, mast cell, testis, frog, Rana esculenta
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Introduction |
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It is well established that MCF7 cells, a cell line derived from a human
mammary adenocarcinoma (Hill and Blask,
1988), have high levels of estrogen receptors and that estradiol
stimulates their growth, while melatonin, a pineal hormone that plays a key
role in a variety of important physiological responses including reproduction
(Reiter, 1991
;
Morgan et al., 1994
), inhibits
their growth. In addition, melatonin acts as an antiestrogen
(Molis et al., 1994
) since it
inhibits the expression of estrogen-regulated genes
(Molis et al., 1995
),
potentiates the sensitivity of MCF7 to tamoxifen
(Wilson et al., 1992
) and
modulates the transcription of estrogen receptor in this cell line.
Recently, we have demonstrated that melatonin has a direct inhibitory
effect on the basal and estradiol-stimulated mitotic activity of primary
spermatogonia in the testis of the frog Rana esculenta
(d'Istria et al., 2003). As
melatonin interferes with the activation of the estrogen receptors and
destabilizes the binding of the estradiol-estrogen receptor complex to the
estrogen-responsive element (Rato et al.,
1999
), we attempt in the present paper to correlate the effects of
the administration of melatonin alone or in combination with estradiol to mast
cell number in the frog Rana esculenta testis.
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Materials and methods |
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Experiment 1: in vivo (24 h and 48 h)
Adult males (N=30), collected in March (10 h:14 h L:D), were
injected with either vehicle alone [100 µl of Krebs Ringer bicarbonate
buffer (KRB), pH 7.4] or with 1 µg of melatonin (dissolved in 100 µl
KRB). Twenty-four hours after the treatment, each group of animals
(N=15) was further divided into three experimental groups: (1) five
animals were used as a control; five animals were injected with 2 µg of
estradiol (E2; dissolved in 100 µl KRB) 24 h later and (3) five
animals received two injections of E2 (2 µg per 100 µl KRB)
24 h and 48 h later. The injections were given into the dorsal lymph sac
during the scotophase.
Experiment 2: in vitro
Adult males (N=10), collected in October (12 h:12 h L:D), were
killed during the scotophase and the excised testes were cut in half.
Half-testes of five animals were incubated in tubes containing either KRB
alone or KRB + E2 (10-6 mol l-1) for 0 h, 6 h
or 12 h. Half-testes of the remaining five animals were incubated in KRB +
melatonin (10-6 mol l-1) alone or in KRB + melatonin
(10-6 mol l-1) + E2 (10-6 mol
l-1) for the same time periods. All the tubes were placed at room
temperature (20°C) in a shaking water bath.
Experiment 3: in vivo (8 days)
Adult males (N=32), collected in October (12 h:12 h L:D), were
divided as follows: five animals were immediately sacrificed as an initial
control; nine frogs were injected with vehicle alone (KRB) and nine frogs were
injected with melatonin (1 µg per 100 µl KRB; Day 0). All animals were
injected with E2 (2 µg per 100 µl KRB) 24 h before being
sacrificed and were decapitated 48 h, 72 h or 8 days after the injection of
melatonin or KRB solution. An additional nine frogs that had been injected
with KRB alone were used as controls at each time point (3 animals per time
point). The injections were given into the dorsal lymph sac during the
scotophase.
Experiment 4: in vitro (dose-response)
Adult males (N=8), collected in October (12 h:12 h L:D), were
killed during the scotophase and their testes were excised. Testes of three
animals were used as an initial control and were then incubated in KRB alone;
the testes of the remaining five frogs were cut in half. Half-testes of each
animal were incubated for 6 h in one of the following: (1) KRB + E2
(10-6 mol l-1), (2) KRB + melatonin (10-6 mol
l-1) + E2 (10-6 mol l-1), (3) KRB
+ melatonin (10-9 mol l-1) + E2
(10-6 mol l-1) or (4) KRB + melatonin (10-12
mol l-1) + E2 (10-6 mol l-1). All
the incubations were carried out at room temperature (20°C) in a shaking
water bath.
Histology and ultrastructure
For histological studies, the testes of each animal were fixed in Bouin's
fluid, dehydrated in a graded ethanol series and cleared in xylene. Serial
paraffin sections (5 µm) were stained with hematoxylin-eosin and 0.2%
Toluidine Blue in Walpole buffer at pH 4.2
(Gabe, 1968) for the
recognition and evaluation of mast cell number (MCN).
For the electron microscopic study, small pieces (1 mm3) of
testis were fixed for 2 h at 4°C in Karnovsky's fluid
(Karnovsky, 1968) in 0.1 mol
l-1 phosphate buffer at pH 7.4 and post-fixed in 1% osmium
tetroxide in the same buffer at 4°C. Samples were dehydrated and embedded
in TAAB 812 (epoxy resin-araldite 812; TAAB Lab., Berkshire, UK). Ultrathin
sections were counterstained with 4% uranyl acetate followed by 1% lead
citrate and observed with a Philips 301 transmission electron microscope.
Numerical and statistical analysis
For each experiment, three randomly chosen sections from each
animal/experimental group were viewed under a light microscope at a
magnification of 400x. The mast cell number (MCN) within interstitial
tissue and the tubule numbers of the testes were counted to give a value of
MCN per 100 tubules per animal. For each experiment, the groups were compared
by one-way analysis of variance (ANOVA) followed by Duncan's test (at
P<0.05 and P<0.01) for multi-group comparisons.
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Results |
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At the ultrastructural level, mast cells filled with many mature and
non-homogeneous granules can be seen in the testis of E2-injected
animals at 24 h. The granules contain regularly arranged lamellae, which form
parallel straight or curved structures. The nuclei are irregular and contain
large masses of heterochromatin, particularly near the nuclear envelope. The
endoplasmic reticulum and the Golgi apparatus were not visible in the
cytoplasm due to the abundance of secretory granules
(Minucci et al., 1997).
Experiment 1: in vivo (24 h and 48 h)
Testes of March frogs injected with E2 showed a significant
increase (P<0.01) in MCN either at 24 h or 48 h compared with the
untreated control testes (Fig.
1). No significant differences were found in the testes of animals
injected with melatonin alone and melatonin plus E2 at either 24 h
or 48 h compared with those of untreated control testes
(Fig. 1).
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Experiment 2: in vitro
Testes of October frogs incubated for 6 h or 12 h in KRB containing
E2 alone showed a significant increase in MCN compared with
untreated control testes (Fig.
2; P<0.01). No differences were found between the MCN
of the testes incubated in melatonin alone or in melatonin plus E2
and the untreated control group either at 6 h or 12 h
(Fig. 2).
|
Experiment 3: in vivo (8 days)
Testes of frogs treated with E2 during October showed a
significant increase (P<0.01) in MCN compared with the untreated
initial control testes at all time points
(Fig. 3). In addition, testes
of animals sacrificed at 48 h showed a higher MCN compared with those observed
in the testes of animals sacrificed at 72 h (P<0.05) and on day 8
(P<0.01).
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Testes of animals injected on day 0 with melatonin and at 24 h before the sacrifice with E2 showed a significant increase in MCN at all time points. The increase was more pronounced at 72 h and on day 8 (Fig. 3; P<0.01) compared with the value observed in the testes of untreated animals.
Interestingly, in the testes of frogs injected on day 0 with melatonin and then with E2 at 48 h and 72 h (24 h before the sacrifice), MCN is lower than that observed in the testes of frogs injected with E2 alone at the same time points (Fig. 3; P<0.01). No differences in MCN were observed in the testes of frogs injected on day 0 with melatonin and then with E2 and sacrificed on day 8, as compared with MCN observed in the testes of frogs injected with E2 alone at the same time point (Fig. 3).
Experiment 4: in vitro (dose-response)
Testes of October frogs incubated for 6 h in KRB containing E2
alone showed a significant increase in MCN compared with that observed in the
control testes (P<0.01; Fig.
4), while no significant differences were found in the testes of
animals incubated in E2 plus melatonin at any of the concentrations
used (10-6 mol l-1, 10-9 mol l-1
and 10-12 mol l-1) compared with the values observed in
the untreated control testes (Fig.
4).
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Discussion |
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In amphibians, mast cells of a connective tissue type have been
characterized at the histochemical, biochemical and ultrastructural levels in
different tissues of the frog Rana esculenta
(Chieffi Baccari et al., 1998).
In particular, the ultrastructure of their cytoplasmic granules is unique and
totally unlike any other previously described granules in other animal species
(Chieffi Baccari et al., 1998
).
It was previously shown that, in the testis of Rana esculenta,
17ß-estradiol increases MCN in vivo and in vitro, while
testosterone has no effect (Minucci et
al., 1997
; Di Matteo et al.,
2000
). Accumulation of mast cells after estradiol treatment was
also observed in the testis of the lizard Podarcis s. sicula Raf
(Minucci et al., 1995
) and in
the testicular interstitium of neonatal rats
(Gaytan et al., 1990
).
Estradiol probably acts on the multiplication and/or differentiation of mast
cells through its direct action on the putative precursor of testicular mast
cells and/or by stimulating growth factors
(Minucci et al., 1997
).
In the present study, we confirm that estradiol treatment increases MCN in
the frog testis and, for the first time, we found that melatonin has an
inhibitory effect on estradiol-stimulated MCN in vivo and in
vitro. Several studies have demonstrated the antiproliferative role of
melatonin using an MCF7 cell model to study the anti-estrogenic effect of this
hormone (Hill and Blask, 1988;
Wilson et al., 1992
; Molis et
al., 1994
,
1995
;
Lissoni et al., 1995
).
Estradiol was used in the present study to induce MCN increase in the testis
to verify the effect(s) of melatonin on mast cell accumulation. The results
obtained from the in vivo experiments confirm that administration of
estradiol increases the MCN in the frog testis within both 24 h and 48 h of
treatment and indicates an inhibitory role for melatonin on mast cell
accumulation induced by estradiol. Interestingly, the inhibitory effect almost
disappears at 72 h and is not present on day 8 after melatonin injection,
indicating that it is reversible. In addition, testis pieces incubated with
estradiol for 6 h showed increased MCN, and this effect is counteracted by
melatonin at 10-6 mol l-1 and was confirmed at a
physiological concentration (10-12 mol l-1).
The inhibitory effect exerted by melatonin on mast cell accumulation could
be explained either through a mechanism involving the
hypothalamus-pituitary-gonadal axis or directly via the local action
of indoleamine on the frog gonads. Although it is now obvious that melatonin
prevents the estrogen-induced MCN increase, more data are needed to verify the
mechanism of its action. Bearing in mind that melatonin interferes with the
activation of the estrogen receptor, destabilizing the binding of the
estradiol-estrogen receptor complex to the estrogen-responsive element
(Rato et al., 1999), and that
the expression of estradiol receptors have been demonstrated in mast cells
(Zhao et al., 2002
;
Nicovani and Rudolph, 2002
),
we suggest that melatonin may be involved in mast cell differentiation and/or
proliferation induced by estradiol treatment via estrogen receptors.
An alternative hypothesis of melatonin action on mast cells comes from the
identification of a melatonin receptor transcript in the RBL-2H3 line of rat
mast cells (Chen et al., 1998
).
This result strongly suggests a direct action of melatonin on mast cells
via its own receptor. This is consistent with a recent report
demonstrating a direct inhibitory effect of melatonin on the basal and
estradiol-induced mitotic activity of primary spermatogonia in the frog testis
(d'Istria et al., 2003
); the
results support the hypothesis that melatonin exerts the inhibitory effect
via its local action on the frog gonads
(d'Istria et al., 2003
).
In conclusion, the results of the present study validate the accumulation of mast cells following estradiol treatment and show, for the first time, that melatonin interacts with the proliferation and/or differentiation of mast cells induced by estradiol in the testis of the frog Rana esculenta either in vivo or in vitro.
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Acknowledgments |
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