182 IMMUNOHISTOCHEMISTRY LOCALIZATION OF GROWTH DIFFERENTIATION FACTOR 9 (GDF-9) AND BONE MORPHOGENETIC PROTEIN 15 (BMP-15) IN CANINE FOLLICLES THROUGHOUT ESTRUS CYCLE

Abstract

Among different developmental proteins, growth differentiation factor-9 (GDF-9) and bone morphogenic protein 15 (BMP-15), members of the transforming growth factor β (TGFb) superfamily, have been described in many species as key mediators for folliculogenesis. The aim of this study was to analyse in canines the presence and localization of GDF-9 and BMP-15 proteins in follicle cells and oocytes during follicle development. To determine the immunolocalization of GDF-9 and BMP-15, sections of 4% paraformaldehyde in PBS ovaries obtained from 15 bitches at different stage of oestrous cycle (proestrus-oestrus n = 5; diestrus n = 5; anestrus n = 5) were dehydrated (xylene–ethanol solutions) and embedded into paraffin blocks. Individual paraffin sections (5 µm) were inactivated with methanol/H₂O₂. Sections were blocked at 37°C in the peroxidase reagent and incubated with rabbit anti-human GDF-9 (1 : 200) or goat anti-human BMP-15 (1 : 150) antibodies overnight at 4°C and with secondary antibodies goat anti-rabbit IgG and donkey anti-goat IgG, both HRP conjugated. Previously negative control sections received rabbit serum. Sections were stained with 1 mg mL^–1 3,3-diaminobenzidine (DAB) and counterstained with haematoxylin. All sections were examined at magnification × 200 using an IX71 inverted microscope (Olympus, Center Valley, PA, USA), with an IX2-RFA lamp and a ProgRes CapturePro camera (Jenoptik AG, Jena, Germany). The same exposure time was applied to all samples and digital photos were taken and subsequently analysed. The intensity of immunostaining in both oocyte and follicular cells of each follicle was evaluated with Image J version 1.45s software (National Institutes of Health, Bethesda, MD, USA). The images were transformed to grey scale and the corrected total cell mark was calculated by subtracting the mean staining of background reading to the integrated density of each sample. The results showed that samples of all slices contained primordial, primary, secondary and pre-antral follicles as well as different sizes of antral follicles showed positive GDF-9 and BMP-15 immunoreactions in the oocyte cytoplasm and in the follicles cells. No such labelling was found in the negative controls. Follicle sizes differed in GDF-9 and BMP-15 immunoreactions according to oestrous cycle. The pattern of GDF-9 immunostaining in proestrus, oestrus, and diestrus was more intense within preantral rather than antral follicles/oocytes, whereas BMP-15 immunoreaction was prominent mainly in proestrus stage increasing to antral stage. These preliminary results indicated temporal pattern of GDF-9 and BMP-15 in canine ovarian follicles which might play important roles in follicular/oocyte development and ovulation during oestrous cycle.

Research was funded by FONDECYT Grant 1140658.