“Changes in the proliferation index of rats with polycystic ovarian disease”.

FULLANA L; SALVETTI NR; BARAVALLE C; MIRA GA; ORTEGA HH

Buenos Aires

Congreso; ICLAS Regional Meeting - Advances in the Care and Use of Laboratory Animals; 2004

Introduction To investigate the influence of the changes produced by the presence of ovarian follicular cyst in the cellular proliferation of the female reproductive tract, we analyze the expression of PCNA in the uterus of rat with PCOD induced by exposition to continuous light.  Materials and Methods All the procedures were carried out according to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Wistar rats were provided by the Center for Experimental Biology and Laboratory Animals Sciences, Faculty of Veterinary Sciences, UNL. Before the experiment, the animals were kept with a controlled cycle of light-darkness (lights on between the 6:00 and the 20:00), and to a temperature of 20-24º C with free access to water and commercial food. Twenty female virgin puber rats were used. They were 8 weeks old with a weight of 160 g (+/- 20). Ten of these animals were placed in the mentioned conditions except for the cycle of light that was extended to 24 Hs (continuous light), where they remained during 15 weeks (Treated Group). A parallel group of ten animals, under normal environmental conditions, were used as controls and sacrificed in proestrous (Control Group). After being anesthetized with CO2, the animals were sacrificed and the uterus were dissected and fixed in 10% buffered formalin. Then, the samples were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. Serial sections of 5 µm in thickness were obtained. On the sections was carried out the immunohistochemical technique to detect PCNA (proliferation cell nuclear antigen, clon PC-10, Novocastra). In all cases, was achieved antigen retrieval in microwaves. Immunostaining was made by streptavidin-peroxidase method; it was used 3.3-diaminobenzidine as chromogen.  Finally, the slides were washed in distilled water and counterstained with Mayer’s haematoxylin, dehydrated and mounted. To verify immunoreactions specificity, adjacent control sections were subjected to the same immunohistochemical method replacing primary antibodies by non immune serum (negative control). Sections of intestine were used as positive controls (Woods and Ellis, 1994).  Images were digitized by a CCD colour video camera (Sony, Montvale, NJ, USA) mounted on top of a conventional light microscope (Olympus BH-2) using a objective magnification of x40.Taking as pattern the intensity of positive nuclei of the controls, the percentage of positive cells was quantified on the total of cells (% of positivity) in the epithelium and stroma cells of the uterus in both groups. The data were analyzed using t-Student test. Results All the cells presented immunostaining, as much in the epithelium as in the subepithelial stroma. In stromal cells, the positivity index was significant different (P<0,05) between groups, being 40,16 % (+/- 12,33) in control group (Fig 1) and 27,96% (+/- 10,53) in treated group (Fig 2). In the epithelial cells no differences were observed (proliferation index = 47,09 % vs. 49,59 %). The results are summarized in the Fig 3. Discussion Maliqueo et al. (2003), studying endometria of women with PCOD, postulated that the endocrine and metabolic disorders underlying this syndrome probably modify the hormonal milieu of the endometrial tissue. The changes of the microenvironment could be related with alterations in the expression of proteins that regulate cell survival, which might be potentially associated with the disruption of the endometrial cell cycle.  In our work, we detect an increase in the proliferation index in stromal cells of PCOD animals with regard to control animals without differences in the proliferation in the uterine epithelium. Estrogen stimulates uterine and vaginal epithelial proliferation in vivo. Also play a critical role in other aspects of uterine and vaginal growth and adult function, and it is obligatory for normal epithelial morphogenesis, cytodifferentiation, and secretory activity in these organs. This hormone exerts its action through estrogen receptor (ER). ERα (the most important in the uterus) is expressed in both epithelial and stromal cells of adult uterus and vagina, and possibly the mitogenic and other important action of the estrogens on uterine epithelium could be mediated through stromal ER. Apparently the mitogenic activity of estrogen on the epithelium can be induced only when epithelial and stromal cells express ERα. The action mechanism would be: estrogen bind to ERα in uterine mesenchymal / stromal cells and trigger the production of paracrine factors, which then act on epithelial uterine cells to stimulate mitogenesis (Cooke et al. 1998). In this model of PCOD, constant levels of estrogen and/or gonadotrophins could be altering the expression of ERα in the distinct cells of the uterus, and this way could be altering the cellular proliferation index in the different uterine tissues.