Objective: to determine the immunoexpression of cytochrome P-450 (CYP19), an enzyme associated with the conversion of androstenedione to estradiol in the ovaries of pinealectomized female rats after melatonin treatment. Methods: Thirty female rats (Rattus norvegicus albinus), adult, virgin from Universidade de São Paulo-Escola Paulista de Medicina (UNIFESP/EPM) biotery were separated in three groups of 10 animals each: GI-Sham (falsely pinealectomized) received vehicle; GIIpinealectomized who received vehicle; GIII-pinealectomized with melatonin reposition (10μg/night, each animal) for 60 consecutive days (Fig. 1 and 2). After this period, animals were anesthetized and ovaries were collected, fixed in 10% buffered formaldehyde and processed for paraffin embedding. From the paraffin blocks, 5μm thick sections were collected to silanized slides and submitted to immunohistochemistry for CYP19 detection. Images were obtained using a light microscope (Axiolab Standard 2.0-Carl Zeiss) attached to a high definition camera (AxioCam MRC-Carl Zeiss) and by the image analyzing image (AxioVision Rel. 4.8.2-Carl Zeiss). Reaction expression was analyzed and quantified according with the color intensity with the aid of the Image J Pro Plus, having photographed 5 fields each slide, with the 40× objective. Obtained data was submitted to statistical analysis using ANOVA test complemented by the Tukey-Kramer test (p<0,05). Results: Histological sections underwent immunostaining CPY19 showed that the expression of CPY19 was higher in pinealectomized (GII = 84.43 ± 5.90 *) compared to Sham groups (GI = 64.71 ± 4.06) and pinealectomized treated with melatonin (GIII = 58.35 ± 8.90) (* p <0.05)(Fig. 3) Conclusions: Our results showed that melatonin affects ovarian steroidogenesis in female rats. [2]

Objective: To analyze the expression of genes related to steroidogenesis in ovary of pinealectomized rats. Methods: 32 adult female virgin rats, with regular estrous cycle, were pinealectomized (PNE) and equally divided into two groups, as follows: GIcontrol group (PNE that received vehicle solution) and GII (PNE treated with melatonin, 10µg/night, per animal), during 60 consecutive days. After treatment, the animals were euthanized by overdose of ketamine and xilazine, some ovaries were collected, kept in liquid nitrogen and stored at-80°C for posterior expression analyses by cDNA microarray (Kit GeneChip® Rat Genome 230 2.0 Array, Affymetrix) of genes related to ovarian functions. The microarray assay was carried out in triplicate for each group. Data were normalized and subjected to the GeneChip® Operating Software and later confirmed by the DNA-Chip Analyzer (dChip) software of secundar analyses. Gene expressions were considered significantly different when they were 1.5x over or low expressed, when compared to the control group. Some samples were appropriately kept for posterior RT-PCR analyses and others were processed for paraffin embedding. Results: The GII showed one hundred one overexpressed genes and seventy-two low expressed genes compared to GI. The genes related to ovarian steroidogenesis were statistically significant overexpressed (Inibin beta-A, Folistatin and Abl-Interactor1) and low expressed (Prostaglandin D2 synthase, LIM Homeobox 9 and Glutathione S-transferase Mu 1), in the GII. Among the overexpressed genes, the Inibin beta-A showed the higher expression. Based on these data, we later confirmed the overexpression of Inibin beta-A by RT-PCR (GII > GI, p<0.01) and by immunohistochemistry, which showed higher immunoreactivity in GII (74.43±2.89) in the follicular and interstitial cells, as well as in the inner teca cells, compared to GI (54.32±4.32). Conclusion: Our data show that melatonin may influence ovarian function by the overexpression and down-regulation of steroidogenesis related genes in ovary of PNE rats.