Morfofunkcionalne osobine reproduktivnog sistema nezrelih i acikličnih ženki pacova nakon tretmana fitoestrogenima iz soje

Abstract

Fitoestrogeni iz soje, pre svega genistein i daidzein, su veoma zastupljeni u životu savremenog čoveka, kako kroz ishranu, tako i kroz pomoćna lekovita sredstva. Zbog svog selektivno modulatorskog, tj. tkivno specifičnog estrogenskog delovanja, fitoestrogeni poslednjih godina postaju atraktivno polje intenzivnih studija. Period gašenja reproduktivne funkicije kod žena, tj. menopauza, vrlo često je praćen tegobama i poremećajima koje su posledica smanjenja koncentracije endogenog estradiola, odnosno funkcije ovarijuma. Stoga je jedna od mogućih terapijskih aplikacija genisteina i daidzeina upravo sprečavanje i ublažavanje ovih tegoba, a kao alternativa supstitucionoj hormonskoj terapiji. Međutim, njihovo delovanje na organe ženskog reproduktivnog sistema, naročito u periodu menopauze, još uvek nije u potpunosti istraženo. Cilj ove studije obuhvata rasvetljavanje dejstava fitoestrogena na reproduktivni sistem nezrelih i acikličnih ženki pacova. Od posebnog interesa je jasno definisanje potencijala fitoestrogena u poboljšanju funkcije reproduktivnog sistema u animalnom modelu menopauze, kao i poređenje efekata njihove aplikacije sa onima koje ima aplikacija terapijske doze estradiol dipropionata, koji se koristi kao lek izbora u prevenciji i terapiji menopauzalnih simptoma. Za ispitivanje estrogenske aktivnosti fitoestrogena (genisteina i daidzena) korišćen je uterotrofni esej po protokolu Organizacije za ekonomsku saradnju i razvoj (OECD). Ženkama pacova starosti 19 dana aplikovani su fitoestrogeni (genistein i daidzein), tokom tri dana u tri doze (visokoj, srednjoj i niskoj). U okviru protokola predviđene su i kontrolne grupe - pozitivne kontrolne grupe, koje su primale 17α-etinil estradiol (u dve doze), negativne kontrolne grupe koje su primale rastvarače, kao i intaktna kontrolna grupa. Nakon uterotrofnog eseja, utvrđeno je koja doza fitoestrogena (genisteina i daidzeina) ispoljava minimalan estrogenski efekat. Ta doza je, tokom četiri nedelje, aplikovana acikličnim ženkama pacova starosti 12 meseci, koje predstavljaju eksperimentalni model menopauze. Na ovaj način utvrđeno je da li ispitivani fitoestrogeni (genistein i daidzein) imaju efekat na strukturu i funkciju organa reproduktivnog sistema, hipofize, ovarijuma, uterusa i vagine. Dodatno su efekti fitoestrogena poređeni sa efektima koje u organima reproduktivnog sistema izaziva terapijska doza estradiol dipropionata. Promene u strukturi i funkciji organa reproduktivnog sistema analizirane su histološkim, imunofluorescentnim, savremenim stereološkim, morfometrijskim i molekularno biološkim metodama. Imunofluorescentno obeležavanje specifičnih antigena, omogućilo je vizuelizaciju gonadotropnih i laktotropnih ćelija hipofize, i izračunavanje relativnog intenziteta fluorescentnog signala. Istim metodama, detektovano je i prisustvo laktoferina, kao i steroidnih receptora u uterusu nezrelih i acikličnih ženki pacova. Metodama savremene stereologije, uz pomoć newCast stereološkog softverskog sistema, određeni su volumeni organa, volumenske gustine osnovnih gradivnih faza organa, kao i broj i volumen pojedinačnih granuloza luteinskih ćelija u žutim telima. Izračunavanje visine endometrijalnog epitela, broja endometrijalnih žlezda, broja ovarijalnih folikula u različitim stadijumima folikulogeneze i atrezije kao i broja, kako pojedinačnih klasa, tako i ukupnog broja žutih tela, omogućila je primena morfometrijskih metoda. Molekularno biološka metoda, reakcija lančanog umnožavanja u realnom vremenu “Real-time” PCR, korišćena je za određivanje relativne ekspresije gena za estrogenski receptor alfa (ERα), estrogenski receptor beta (ERβ) i progesteronski receptor (PR) u uterusu acikličnih ženki pacova. Dobijeni rezultati su statistički obrađeni. Uterotrofnim esejem pokazano je da genistein u dozama od 35 i 50 mg/kg telesne mase na dan ispoljava estrogensko delovanje, koje se ogleda u gravimetričkom porastu uterusa, te možemo reći da deluje kao agonista estradiola, dok daidzein ne izaziva ove efekte. Evaluacijom visoke, srednje i niske doze ustanovljeno je da genistein aplikovan u visokoj i srednjoj dozi, ima ulogu estrogenskog agoniste u pogledu svih ispitivanih karakteristika u uterusu, hipofizi i vagini nezrelih ženki pacova. Međutim, iste doze su u ovarijumima uslovile antagonističko delovanje. Zahvaljući uključivanju dodatnih analiza, osetljivost uterotrofne probe je povećana i ustanovljeno je da i daidzein poseduje estrogensko delovanje u svim ispitivanim dozama, koje se ogleda u povećanju broja i volumenske gustine endometrijalnih žlezda i stimulaciji gonadotropnih i laktotropnih ćelija hipofize. Minimalne efektivne doze, tj. doze koje izazivaju estrogenski efekat u organima ženskog reproduktivnog sistema, su doze od 35 mg/kg telesne mase na dan genisteina i daidzeina. U animalnom modelu menopauze, tretman estradiolom je izazvao inhibiciju gonadotropnih ćelija, koja se ogleda, ne samo u smanjenju intenziteta fluorescentnog signala, već i u smanjenju veličine ćelija. Istovremeno, estradiol je delovao stimulatorno na laktotropne ćelije, povećanjem njihove zastupljenosti, kao i povećanjem relativnog intenziteta fluorescentnog signala immunoobeleženog prolaktina. Nakon tretmana genisteinom i daidzeinom, nasuprot estradioloskom tretmanu, izostaju promene u relativnom intenzitetu fluorescentnog signala unutar gonadotropnih i laktotropnih ćelija. Međutim, fitoestrogeni su izazvali promene u morfologiji ćelija. Gonadotropne ćelije su veće, dok su laktotropne ćelije nešto manje u poređenju sa kontrolnom grupom. Međutim, fitoestrogeni su u navedenim ćelijskim populacijema uslovili promenu oblika, dok se u citoplazmi uočavaju neobojeni delovi koji joj daju izgled vakuoliziranosti. Hroničan tretman terapijskim dozama estradiola, u ovarijumima acikličnih ženki, generalno je pokazao negativan uticaj. U grupi malih folikula izazvao je značajno povećanje atretičnih, primordijalnih, primarnih i preantralnih folikula. Slična slika je uočena i u grupi antralnih folikula. Tretman estradiolom, uslovio je povećanje broja i volumena žutih tela, koje za posledicu ima povećanje volumena ovarijuma. Međutim, hronična aplikacija kako genisteina, tako i daidzeina, uslovila je višestruko povećanje broja folikula inicijalne faze folikulogeneze (primordijalnih i primarnih). Sa druge strane, genistein i daidzein nisu uticali na promenu apsolutnog broja atretičnih primordijalnih folikula, dok su značajno smanjili atreziju primarnih folikula. Tretmani fitoestrogenima nisu uticali na promenu sume preantralnih i antralnih folikula, dok su izazvali smanjenje apsolutnog broja atretičnih antralnih folikula. Tretman genisteinom, nije uticao ni na promenu pojedinačnih klasa žutih tela, kao ni na promenu njihovog ukupnog broja. Za razliku od genisteina, daidzein je povećao brojnost zrelih žutih tela, što je uzrokovalo povećanje njihovog ukupnog broja, usled čega je došlo i do povećanja apsolutnog volumena ovarijuma. Međutim, usled očuvanja zdravih folikula, oba tretmana su uzrokovala povećanje volumena folikularnog parenhima. Tretman estradiolom izazvao je povećanje apsolutne i relativne mase uterusa, koje je posledica povećanja volumensih gustina endometrijuma, endometrijalnog epitela i endometrijalnih žlezda. Ekspresija ERα je smanjena, PR povećana, dok promene u eksperiji ERβ izostaju. Nakon tretmana genisteinom, uočena je povećana masa uterusa, kao i povećana volumeska gustina endometrijuma. Genistein je smanjio ekspresiju ERα, i povećao ekspresiju PR i ERβ. Za razliku od genisteina, daidzein nije izmenio masu uterusa, njegove morfometrijske i stereološke karakteristike, kao ni ekspresiju ERα i PR, dok je uslovio povećanje ERβ. Aplikacija estradiola izazvala je hipertrofiju vaginalnog epitela, praćenu deskvamacijom ćelija. Nakon tretmana fitoestrogenima, promena opšte histološke slike vaginalnog epitela nije zapažena u poređenju sa kontrolnim presecima. Međutim detaljnom analizom uočava se odsustvo piknotičnih jedara, dok se mogu zapaziti ćelije u mitozi. Genistein i daidzein, u poređenju sa estradiolom, ispoljavaju niz pozitivnih efekata na reproduktivni sistem acikličnih ženki, a posebno na nivou ovarijuma. Imajući u vidu da menopauza, između ostalog, nastaje usled smanjene funkcije ovarijuma, rezultati ove studije imaju veliki značaj i primenu, s obzirom na to da očuvanje funkcije ovarijuma značajno utiče na kvalitet života žena. Stoga se može reći da genistein i daidzein mogu biti uspešna alternativa supstitucionoj hormonskoj terapiji.

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.