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OJHAS: Vol. 4, Issue
4: (2005 Oct-Dec) |
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Effect Of Oestradiol On
Mouse Vaginal Epithelium |
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Prakash Shetty
Associate Professor
Latha V Prabhu
Professor
Mangala M Pai
Assistant Professor
Department of Anatomy, Center
for Basic Sciences, Kasturba Medical College, Bejai, Mangalore- 575004,
Karnataka, India. |
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Address For Correspondence |
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Dr. Prakash Shetty
Department of Anatomy, Center
for Basic Sciences,
Kasturba Medical College,
Bejai, Mangalore- 575004,
Karnataka, India.
E-mail:
docprakashshetty@yahoo.co.in |
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Shetty P, Prabhu LV, Pai MM. Effect Of Oestradiol On
Mouse Vaginal Epithelium.
Online J Health Allied Scs.2005;4:1 |
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Submitted: Nov 09,
2005; Revised: Jan 19, 2006; Accepted: Jan 22, 2006; Published:
Mar 31, 2006 |
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| Abstract: |
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Oestradiol
which induces the proliferation of the vaginal epithelium is used in
the oestrogen replacement therapy for menopausal and postmenopausal
disorders. The exact onset of induced cell proliferation and recovery
to the controlled level has not been reported before. Hence this study
was done in ovariectomised mice and the period of cellular response
to ethinyl estradiol, a semisynthetic ester (0.025mg/kg) was recorded.
The vaginal epithelium was studied for histological changes and incidence
of mitotic figures. The onset of proliferation started at 4h after exposure
and reached its maximum at 32h and declined gradually thereafter to
reach the base level at 60h.
Key Words:
Oestradiol, Mitotic
figures, Vaginal epithelium |
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Oestradiol,
a naturally occurring oestrogen or its semisynthetic esters are employed
in the oestrogen replacement therapy for menopausal, postmenopausal
or menstrual disorders.(1) Oestrogens impart their side effects on veterinary
and human health especially on development and morphology of the reproductive
system.(2) Oestrogen treatment induces 2-15 fold increase in the risk
of endometrial cancer formation, with some relation to the doses and
duration employed.(3) Although the role of oestrogens in the breast
carcinogenesis is not clear, there seems to be some positive correlation
between the use of this drug and breast cancer.(4)
Considerable
number of reports is now available on the effects of oestradiol and
its analogues on the vagina. Exposure of oestradiol benzoate causes
anomalous vaginal development and the epithelium rapidly develops into
stratified keratinized type probably due to the direct effect of steroid.(5)
In ovariectomised rats, delta-tetra hydrocannabinol induces hyperplasia
of the uterus and stratification of the vaginal epithelium.(6) Oestradiol
administration to the overiectomised rats and mice results in the proliferation
of the epithelium,(7, 8) even though the duration of proliferative and
recovery phases was not studied. Sanyal et al (9) reported that oestradiol
induces the cell proliferation in the rat vaginal epithelium. However,
the onset of induced cell proliferation and its recovery to the control
level in oestradiol treated vaginal epithelium is unknown and this study
was planned to investigate this phenomenon in mice.
A
total of 50 adult female albino swiss mice weighing 25-30g were chosen
and were maintained under the standard laboratory conditions with access
to food and water ad libitum. Mice were segregated into 16 groups of
3 in each except the control group, which had 9 animals. They were anaesthetized
(Pentobarbitol sodium, 40 mg/kg; Sigma Chemicals), and overiectomised
as per standard procedure. (9) Two weeks after the ovariectomy, they
were given a single dose of 0.025 mg of oestradiol (ethinyl oestradiol
– Duoluton-L; German Remidies, Mumbai), dissolved in arachis oil.
Control animals received equal quantity of oil (i.p.) only. These animals
were treated with colchicines (1 mg/kg; Sigma chemicals, i.p.), dissolved
in water just 2h before the sacrifice to arrest mitosis.(9) Each group
of mice was sacrificed at an interval of 4h till 60h. Vagina was dissected
out carefully by suprapubic incision and fixed in 10% formalin.(9) The
tissue was then processed for paraffin embedding and 10 micron sections
were obtained and stained with Haematoxylin and eosin. Sections from
each animal were randomly selected and mitotic figures were counted
in the epithelium under 40X objective of a high resolution binocular
light microscope (Olympus).The mitotic activity was estimated on the
basis of area of each section and its mitotic index was expressed as
the average number of mitotic figures per unit area of the epithelium
(one square mm being considered as the unit area). The data were expressed
as mean + SD and subjected to analysis by the Mann-Whitney ‘U’
test. P< 0.05 was considered as the level of significance.
In
ovariectomised control mice, vaginal epithelium showed decreased activity
of cell division, and the mitotic figures ranged from 5-35. In ovarectomised
and oestradiol treated animals, epithelial proliferation was noted after 4h
following the exposure [Figure 1]
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Figure 1: Microphotograph of
mouse vagina (4 hr group). Slight proliferation of vaginal epithelium
is seen. Arrows point the mitotic figures in the basal layer indicating
an early response of vaginal epithelium to oestradiol. |
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This was the earliest
sample time employed and following that, the incidence of mitotic
figures increased as a function of time up to 32h [Figure 2]
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Figure 2: Microphotograph
of mouse vagina (32 hr group). Maximum mitotic figures are seen in this
group. Arrows indicate the mitotic figures. |
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At this sample time the
number of cells with metaphase figures ranged from 312-476. This was
the time duration required to show the maximum proliferative response
of oestradiol on vaginal epithelium (Table 1).
Table 1: Effect
of oestradiol on mouse vaginal epithelium
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Sample
time |
Mitotic index
(Mean + SD) |
Range |
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0 (9) |
18.03 +
10.15 |
5-35 |
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4 (3) |
68.26 +
16.90* |
42-113 |
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8 (3) |
107.30 +
19.28* |
84-156 |
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12 (3) |
146.23 +
24.75* |
102-220 |
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16 (3) |
119.43 +
44.15* |
147-346 |
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20 (3) |
245.00 +
44.23* |
192-388 |
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24 (3) |
287.63 +
43.20* |
233-419 |
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28 (3) |
330.10 +
38.37* |
280-451 |
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32 (3) |
384.76 +
42.82* |
312-476 |
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36 (3) |
324.36 +
31.92* |
262-422 |
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40 (3) |
211.90 +
26.75* |
159-312 |
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44 (3) |
179.8 +
27.27* |
145-280 |
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48 (3) |
152.43 +
28.11* |
119-252 |
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52 (3) |
124.50 +
24.91* |
89-204 |
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56 (3) |
78.76 +
18.88* |
63-155 |
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60 (3) |
51.93 +
21.01* |
33-93 |
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Numbers in parentheses represent
the sample size.
* P<0.001 versus control
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From 36h onwards up to
60h, the mitotic counts decreased [Figure 3] and the minimum count at
60h was well within the control range.
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Figure 3: Microphotograph of
mouse vagina (52 hr group). Mitotic figures decreased considerably and
superficial cornified layer almost started desquamating into the vaginal
lumen. Arrows indicate the mitotic figures. A- Basal layer, B- Intermediate
layer, C- Cornified layer
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Ovariectomy
results in depletion of endogenous oestrogens, which affects the normal
female sexual functions. Oestrogens, either natural or synthetic are
mutagenic and carcinogenic in almost all species tested.(10) Developmental oestrogenic exposure to oestrogenic chemicals induces morphological,
functional and behavioral anomalies associated with reproduction.(11)
In mice, neonatal DES treatment induces persistent proliferation and keratinization of vaginal epithelium even in the absence of the ovaries
resulting in cancerous lesions later in life.(12) However, the occurrence
of such irreversible vaginal changes is blocked by concurrent Vit A
treatment.(13) On the other hand, oestrogen exposure affects the normal
embryogenesis in either sex, and fertility parameters in experimental
animals and humans.(2)
In this present
study, single exposure of oestradiol enhanced the mitosis in the vaginal
epithelium and this phenomenon is known to occur in the animals also.(14) However, there are no studies reporting the duration through which
this mitotic activity continues following the exposure. This study has
evaluated the onset of cell proliferation just at 4h following the exposure,
which increased through time up to 32h. The recovery phase also takes
almost same time in the mouse vaginal epithelium. This indicates that
the proliferative response of mitogen oestradiol persists as a prolonged
effect when given as a single treatment. In previous studies, 50 microgram
of 17 beta-oestradiol on day 17 of foetal life induced ovary-independent
proliferation and cornification of vaginal epithelium on postnatal day
3 in mouse.(15) Mitogenic activity of oestradiol is time dependent,
and the cell division is accelerated in ovariectomised-oestradiol treated
mice. This property of oestradiol is reversible by 60h after the exposure.
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Sharpe RM, Skakkebaek
NE. Are oestrogens involved in falling sperm counts and disorders of
the male reproductive tract? Lancet 1993;341:1392-1395.
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Henderson BE. The
cancer question: an overview of recent epidemiologic and retrospective
data. Am J Obstet Gynecol 1989;161:1859-1864.
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Dupont WD, Page
DL. Menopausal oestrogen replacement therapy and breast cancer. Arch
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Hisano N. effects
on hamster vaginal development of single dose of testosterone on oestradiol
given neonatally. Acta Anat (Basel) 1997;97(4):361-370.
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Solomon J, Cocchia
MA, DiMartino R. Effect of delta-tetrahydrocannabinol on uterine and
vaginal cytology of ovariectomised rats. Science 1977;195(5281):875-877.
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Ivasenko IN. Effect
of simultaneous administration of oestradiol and progesterone on proliferative
pool of the uterine and vaginal epithelium in castrated rats. Arkh Anat
Gistol Embriol 1978;74(40: 79-84.
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Wong LM, Bern HA,
Jones LA, Mills KT. Effect of later treatment with estrogen on reproductive
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Sanyal AK, Singh
S, Singh SP. Response of vaginal epithelium and quantitative estimation
of its cellular proliferation in overiectomised rats after a single
administration of oestradiol. Acta Anat 1974;87:82-92.
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Edelman DA. Diethylstillbesterol
exposure and the risk of clear cell cervical and vaginal adenocarcinoma.
Int J Fertil 1989;34(4):251-255.
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Forsberg JG. Treatment
with different antiestrogens in the neonatal period and effects in the
cervicovaginal epithelium and ovaries of adult mice: a comparison to
oestrogen-induced changes. Biol Reprod 1985;32(2):427-441.
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Markey CM, Wadia
PR, Rubin BS, Sonnenschein C, Soto AM. Long term effects of fotal exposure
to low doses of the xenoesterogen bisphenol-A in the female mouse genital
tract. Biol Reprod 2005;72(6):1344-1351.
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Masui F, Matsuda
M, Mori T. Vitamin A prevents the irreversible proliferation of vaginal
epithelium induced by neonatal injection of keratinocyte growth factor
in mice. Cell Tissue Res. 2003;311(2):251-258.
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Tansey
G, Hughs CL Jr, Cline JM, Krummer A, Walmer DK, Schmoltzer S. Effects of dietary
soybean estrogens on the reproductive tract in female rats. Proc oc Exp Biol
Med 1998;217(3):340-344.
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Kimura T, Kawashima S, Nishizuka Y. effects of prenatal treatment with estrogen on mitotic activity of vaginal anlage cells in mice.
Endocrinol
Jpn 1980;27(6):739-745.
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