Changes in the subcellular and tissue location of estrogen and progesterone receptors in rat uterus after long-term treatment with analogs of gonadoliberin

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ginekologia

Changes in the subcellular and tissue location of estrogen and progesterone receptors in rat uterus after long-term treatment with analogs of gonadoliberin

Zmiany w subkomórkowej i tkankowej lokalizacji receptorów

estrogenowych i receptora progesteronowego w macicy szczurów poddanych długoterminowemu działaniu analogów gonadoliberyny

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1 Students Scientific Society, Medical University of Silesia, Katowice, Poland

2 Department of Cytophysiology, Medical University of Silesia, Katowice, Poland

3 Department of Histology, Medical University of Silesia, Katowice, Poland

Abstract

Objectives: Certain therapies with the use of analogs of gonadotropin-releasing hormone (GnRH, gonadoliberin) aim at achieving the effect of desensitization of the pituitary gland that causes inhibition of the hypothalamic- pituitary-gonadal axis. The resulting hormonal changes may influence the location and expression of estrogen and progesterone receptors, as well as their endogenous functions.

The aim: The aim of the study was to investigate whether long-term administration of low doses of dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist) affected subcellular and tissue-specific location of ER_ and ER`

estrogen receptors and progesterone receptor (PR) in rat uterus, as well as explore the extent to which the changes were reversible.

Material and methods: Analogs were administered to SPD adult females in the course of 3 months, at a dose of 6 μg/kg b.w. Afterwards, the ovaries and the uterus were resected – in the course of 4 weeks after treatment completion. Tissue paraffin-embedded samples were stained with hematoxyline-eosin for morphological studies or incubated with specific antibodies for the immunohistochemical studies (ABC method).

Results: GnRH analogs induced desensitization, resulting in specific and relatively persistent histological changes in the ovaries and the uterus. Strong nuclear reaction for ER_ in the lining and the glandular epithelial cells in dalarelin- treated rats, and lack of expression changes in cetrorelix-treated rats, were observed in the uterus. Epithelial ER_

expressions were accompanied by diminished ER` and elevated PR expression, as well as diminished ER_ and ER` expression, and unchanged PR expression in the stromal and muscle cells, in both dalarelin- and cetrorelix- treated rats. The majority of the changes were reversible after treatment discontinuation.

Otrzymano: 29.09.2013

Zaakceptowano do druku: 30.11.2013 Corresponding author:

Piotr Czekaj

Department of Cytophysiology, Medical University of Silesia;

Medyków 18 Str., 40-752 Katowice, Poland;

tel/fax. 32 2088374; 322526574; e-mail: pcz@sum.edu.pl

Abbreviations: PR, progesterone receptor; ER, estrogen receptor;

GnRH, gonadoliberin

Skróty: PR, receptor progesteronowy; ER, receptor estrogenowy;

GnRH, gonadoliberyna

Introduction

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Conclusions: Long-term exposure to low doses of GnRH analogs causes morphological changes in the uterine tissues, accompanied by reversible changes of the ER_, ER` and PR expression, possibly influencing tissue sensitivity. These changes indicate that agonist and antagonist regulate ER_ expression by means of different mechanisms. A functional interaction between the receptors, depending on ER` expression, direct influence of analogs on the local hormonal axes, and dose-dependent effects, cannot be excluded. After discontinuation of the analog treatment, the time needed for stabilization of ER and PR expression is shorter than the period of time required to restore histological structure of the uterus.

Key words: HVWURJHQUHFHSWRUĮ/ HVWURJHQ UHFHSWRU ȕ / progesterone receptor /

/ GnRH agonist / GnRH antagonist / dalarelin / cetrorelix / long-term treatment / uterus /

Streszczenie

Wstęp: W terapiach hormonalnych z  użyciem analogów GnRH, dąży się do uzyskania efektu desensytyzacji przysadki mózgowej powodującego zahamowanie osi podwzgórze-przysadka-jajnik. Wywołane zmiany hormonalne mogą wpływać na lokalizację i  ekspresję receptorów estrogenowych i  receptora progesteronowego w  macicy, istotnie modyfikując ich endogenne funkcje.

Cel: Celem pracy było zbadanie, czy długoterminowe podawanie szczurom niskiej dawki agonisty receptora GnRH - dalareliny i antagonisty receptora GnRH – cetroreliksu, wpływa na subkomórkową i tkankową lokalizację receptorów estrogenowych ER_ i ER` oraz receptora progesteronowego (PR) w macicy, a także, w jakim stopniu te zmiany są odwracalne.

Materiał i  Metody: Badania wykonano na dojrzałych samicach szczurów Spraque-Dawley. Oba analogi były podawane w  ciągu trzech miesięcy, w  dawce 6 μg/kg m.c. Po tym okresie czasu oraz w  ciągu miesiąca od zakończenia iniekcji, pobierano jajniki oraz macicę. Do badań morfologicznych, skrawki parafinowe barwiono hematoksyliną i eozyną. Do badań immunohistochemicznych, skrawki inkubowano z odpowiednimi przeciwciałami poliklonalnymi. Wizualizacji kompleksów antygen-przeciwciało dokonano za pomocą metody ABC.

Wyniki: Analogi GnRH wykazały działanie desensytyzacyjne skutkujące charakterystycznymi i stosunkowo trwałymi zmianami histologicznymi w  jajnikach i  w  macicy. W  macicy zaobserwowano pojawienie się silnego jądrowego odczynu ER_ w komórkach nabłonka wyścielającego i gruczołowego u szczurów traktowanych dalareliną oraz brak zmian tej ekspresji w komórkach szczurów, którym podawano cetroreliks. Zmianom nabłonkowym towarzyszyły:

obniżona ekspresja ER` oraz podwyższona ekspresja PR, a także obniżona ekspresja ER_ i ER` oraz niezmieniona ekspresja PR w komórkach zrębowych endometrium i mięśniowych myometrium zarówno szczurów traktowanych dalareliną, jak i cetroreliksem. Większość tych zmian była odwracalna zaraz po zaprzestaniu iniekcji.

Wnioski: W  tkankach macicy, długoterminowe podawanie niskiej dawki analogów GnRH wywołuje zmiany morfologiczne, którym towarzyszą odwracalne zmiany ekspresji receptorów ER_, ER` i PR mogące wpływać na wrażliwość tkanek. Zmiany te wskazują, że agonista i antagonista regulują ekspresję ER_ poprzez różne mechanizmy.

Nie można wykluczyć interakcji funkcjonalnej pomiędzy receptorami, zależnej od ekspresji ER`, bezpośredniego oddziaływania analogów na lokalne osie hormonalne w  macicy oraz efektów zależnych od wielkości dawki. Po odstawieniu analogów, czas powrotu ekspresji ER i PR do wartości kontrolnych jest krótszy niż czas potrzebny do przywrócenia struktury histologicznej macicy.

Słowa kluczowe: receptor estrogenoZ\ Į / receptor estrogenoZ\ ȕ /

/ receptor progesteronowy / agonista receptora GnRH / antagonista receptora GnRH /

/ dalarelina / cetroreliks / d]iaáanie dáugoterminowe / macica /

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Results

Ovarian and uterine tissue morphology

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Figure 1. ER_ immunolocalization in the uterus of rats administered placebo for one month (IK), dalarelin for one (ID) and three months (IIID) and cetrorelix for two months (IIC). Abbreviations: e – lining epithelium; g – glandular epithelium, s – endometrial stroma, mi – muscularis interna, me – muscularis externa, a – arteries, v – veins.

Microscope magnification: 200x.

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Discussion

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Figure 2. ER` immunolocalization in the uterus of rats administered placebo for one (IK) and two months (IIK), dalarelin for one (ID) and three months (IIID) and cetrorelix for three months (IIIC). Abbreviations: e – lining epithelium; g – glandular epithelium, s – endometrial stroma, mi – muscularis interna, me – muscularis externa, a – arteries, v – veins.

Microscope magnification: upper pictures: 400x; IIK and lower IIIC: 200x; IIID and lower IK: 100x.

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Figure 3. PR immunolocalization in the uterus of rats administered placebo for one month (IK), dalarelin for two months (IID) and cetrorelix for two (IIC) and three months (IIIC).

Abbreviations: e – lining epithelium; g – glandular epithelium, s – endometrial stroma, mi – muscularis interna, me – muscularis externa, v – veins.

Microscope magnification: upper and lower pictures: 100x; other pictures: 400x.

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Figure 4. ER_, ER` and PR immunolocalization in the uterus of rats after one (III+1), two (III+2) and four (III+4) weeks following the three month of dalarelin (D) and cetrorelix (C) treatment duration. Microscope magnification is also shown.

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Oświadczenie autorów

1. Kamil Sołtysik – opracowanie koncepcji i założeń badań, zebranie i  opracowanie materiału, wykonanie i  interpretacja wyników badań laboratoryjnych, przygotowanie manuskryptu i  piśmiennictwa, korekta i  aktualizacja literatury, współautor tekstu pracy, ostateczna weryfikacja i akceptacja manuskryptu.

2. Piotr Czekaj – opracowanie koncepcji i  założeń badań, analiza i interpretacja wyników, współautor protokołu, przygotowanie manuskryptu i piśmiennictwa, korekta i akceptacja ostatecznego kształtu manuskryptu, uzyskanie funduszy na realizację badań laboratoryjnych - autor zgłaszający i odpowiedzialny za manuskrypt.

3. Aleksandra Suszka-Świtek – opracowanie koncepcji i  założeń pracy, współautor protokołu, zebranie materiału, korekta i  aktualizacja literatury, korekta i akceptacja ostatecznego kształtu manuskryptu.

4. Karolina Kociszewska – zebranie materiału, wykonanie badań laboratoryjnych, opracowanie wyników badań, przygotowanie manuskryptu i  piśmiennictwa, korekta i  aktualizacja literatury, korekta i  akceptacja ostatecznego kształtu manuskryptu.

5. Danuta Plewka – korekta i  aktualizacja literatury, wykonanie badań immunohistochemicznych, opracowanie i  interpretacja wyników badań, ostateczna weryfikacja i akceptacja manuskryptu.

Źródło finansowania: Praca finansowana ze środków Śląskiego Uniwersytetu Medycznego w Katowicach przeznaczonych na badania własne – umowa nr: KNW-2-026/10.

Konflikt interesów: Autorzy nie zgłaszają konfliktu interesów oraz nie otrzymali żadnego wynagrodzenia związanego z powstawaniem pracy.

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