Morfofunkcionalne osobine reproduktivnog sistema nezrelih i acikličnih ženki pacova nakon tretmana fitoestrogenima iz soje
Morphofunctional features of reproductive system of immature and middle-aged female rats after treatments with soy phytoestrogens
Abstract:
Soy phytoestrogens, primarily genistein and daidzein, are widely used through diet
and as dietary supplements. Since they may act as “natural” selective modulators of
estrogen receptor, scientific interest for phytoestrogens is constantly increasing. Cessation
of a women’s reproductive function, i.e. menopause, is often accompanied with disorders
that are consequence of reduced ovarian function and low estrogen concentration.
Therefore, one of the possible therapeutic application of genistein and daidzein is
prevention and alleviation of menopausal symptoms, as an alternative to hormone
replacement therapy.
The aim of this study is to examine the effects of phytoestrogens on the
reproductive system of immature and middle-aged female rats. Of particular interest is to
define the potential of phytoestrogens for improvement of reproductive system function in
an animal model of menopause, and to compare these effects with the effects of
therapeutic doses of estradiol dipropionate, commonly used in prevention and treatment of
menopausal symptoms.
Uterotrophic assay was used in order to test estrogenic activity of genistein and
daidzein, according to the protocol of the Organization for Economic Cooperation and
Development (OECD). Immature female rats received phytoestrogens (genistein and
daidzein), in three doses (high, medium and low) for three consecutive days, from 19th till
21st day of life. According to the protocol, control groups were formed - the positive
control groups that received 17α - ethynyl estradiol (in two doses), negative control groups
that received solvents, and the intact control group.
After uterotrophyc assay, minimal dose of genistein and daidzein that exerts
estrogenic effect was determined. This dose was administered to 12 months old, middleaged
female rats, for four weeks. After this period it was analyzed whether genistein and
daidzein exerted beneficial effects on the structure and function of the pituitary, ovary,
uterus and vagina. These effects were then compared with the effects of the therapeutic
dose of estradiol dipropionate.
Changes in the structure and function of the organs of reproductive system of
immature and middle-aged female rats were analyzed by histological, immunofluorescence,
design-based stereological, morphometric and molecular biological methods.
Immunofluorescent labeling of specific antigens allowed the visualization of the pituitary
lactotropic and gonadotropic cells, and the calculation of the relative intensity of
fluorescent signal, i.e. concentration of antigen within the cell. In the same manner, the
presence of lactoferrin and steroid receptors in the uterus of immature and middle-aged
female rats was analysed. Unbiased, design-based stereological methods were used for
determination of volume of reproductive organs, volume density of their basic
compartments, as well as volume, number and volume density of granulosa lutein cells of
the corpora lutea. The endometrial epithelium height, endometrial gland number, number of
ovarian follicles at different stages of folliculogenesis and atresia, as well as the number of
individual classes and the total number of corpora lutea were performed using morphometric
methods. "Real-time" PCR was used to determine the relative gene expression of estrogen
receptor alpha (ERα), estrogen receptor beta (ERβ) and progesterone receptor (PR) in the
uterus of middle-aged female rats. All results were statistically analyzed.
Uterotrophic assay showed that genistein, at doses of 35 and 50 mg/kg b.w.,
increased gravimetric uterine growth and acted as an estrogen agonist, while daidzein, in
any dose used, did not cause these effects. It has been shown that genistein, in medium and
high dose, acted as an estrogen agonist in terms of all tested parameters in uterus, pituitary
gland, and vagina. However, the same doses in the ovaries caused antagonistic effects. By
including additional analyses, the sensitivity of uterotrophic assay was increased. This
resulted in an observation that daidzein also exerts estrogenic activity in all tested doses.
The estrogenic activity of daidzein was reflected in the increase of endometrial gland
number and volume density, and stimulation of pituitary gonadotropic and lactotropic cells.
The minimal effective dose, i.e. the dose that causes the estrogenic effect in the organs of
female reproductive system, is the dose of 35 mg/kg b.w. per day of genistein and daidzein.
Chronic estradiol treatment of middle-aged female rats caused an inhibition of
gonadotropic cells, which was reflected not only in the reduction of fluorescent signal
intensity, but also in reduction of the cell size. At the same time, estradiol stimulated
lactotrops, by increasing their density and the relative intensity of the immunolabeled
prolactin in cells.
In contrast to estradiol treatment, genistein and daidzein did not cause changes in
the relative intensity of the fluorescent signal within the gonadotropic or lactotropic cells.
However, changes in cell morphology were observed after treatment with both
phytoestrogens. Gonadotropic cells were larger in size, while lactotropic cells were smaller
comparing to the controls. All types of examined pituitary cells were changed in shape with
unevenly stained cytoplasm, that is, immunolabeled parts of cytoplasm were separated by
unstained regions, which gave cells the appearance of vacuolization.
Chronic treatment with therapeutic doses of estradiol, in the ovaries of acyclic
females, generally had a negative effect. In the group of small follicles, estradiol caused a
significant increase of atretic, primordial, primary and preantral follicles. Similar effect was
observed in the group of antral follicles. Estradiol treatment caused an increase of volume
and number of corpora lutea, which resulted in the ovary volume increase. In contrast to
estradiol, chronic application of genistein and daidzein caused a significant increase in the
number of follicles in initial stage of folliculogenesis (primordial and primary follicles),
without affecting total number of atretic primordial follicles, but significantly reducing the
number of primary atretic follicles. Treatments with phytoestrogens did not affect total
number of preantral and antral follicles, while they caused a decrease of atretic antral
follicle number. Genistein treatment did not change the number of individual classes of
corpora lutea, or their total number. Unlike genistein, daidzein increased the number of
mature corpora lutea, which resulted in an increase of their total number, and ovary volume.
However, due to the preservation of healthy follicles, both treatments caused an increase of
follicular parenchyma volume.
Estradiol treatment increased the absolute and the relative uterine weights. This is
result of increased volume density of endometrium, endometrial epithelium and
endometrial glands. Estradiol caused decrease of ERα mRNA and increase of PR mRNA
expression, while ERβ mRNA expression was not changed. Genistein caused an increase
of uterine weight and endometrial volume density, decrease of ERα mRNA expression, and
increase of PR and ERβ mRNA expression. In cotrast to genistein treatment, daidzein did
not change uterine weight, or morphometric and stereological characteristics of uterus.
Expression of ERα and PR mRNA was not affected, while expression of ERβ mRNA was
increased.
In vagina, application of estradiol caused hypertrophy of the epithelium, followed
by desquamation of epithelial cells. After treatment with phytoestrogens, changes in general
histological appearance of the vaginal epithelium were not observed. However, detailed
light microscopic analysis revealed less numerous pyknotic nuclei, and more numerous
mitotic cells.
Comparing to estradiol, genistein and daidzein, exhibited numerous beneficial
effects on the reproductive system of middle-aged females, especially in the ovary. Bearing
in mind that menopause, among other things, is caused by reduced ovarian function, the
results of this study are significant and applicable, given that the preservation of ovarian
function can significantly improve the quality of life of women. Therefore, it can be
concluded that genistein and daidzein could be a successful alternative to estrogen
replacement hormone therapy.
Keywords:
Female reproductive system; Phytoestrogens; Estradiol; Uterotrophic essay; MenopauseSource:
University of Belgrade, Faculty of Biology, 2013, 1-157Funding / projects:
- The effects of select plant extracts, phytoestrogens, steroid and peptide hormones on the rat neuroendocrine system (RS-MESTD-Basic Research (BR or ON)-173009)
URI
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http://nardus.mpn.gov.rs/123456789/2118
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