Differential expression of GDF-9 and BMP- 15 duringfollicular development in canine ovaries evaluated by flowcytometry

Abstract

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) playimportant functions in follicular and oocyte development in many species. This study eval-uated the dynamic expression of GDF-9 and BMP-15 in canine follicles cells using flowcytometry analysis. Follicular cells were removed from three sizes of antral follicles (small,medium and large) from ovaries of bitches throughout the estrus cycle. Cells were incu-bated with anti-human GDF-9 polyclonal and anti-mouse BMP-15 monoclonal antibodies.A size and complexity discriminatory gate was used for the cytometryc analysis in theinitial dot plot and, additionally, a CD45 marker for leukocyte and propidium iodide (PI)were used for erythrocyte and debris discrimination. The evidence corroborated the pres-ence of both proteins in canine follicle cells, but these proteins were not expressed equallyduring follicular development. The results analyzed by ANOVA showed that GDF-9 expres-sion decreased (P < 0.05) during follicular growth in anestrus and proestrous/estrous, butincreased in diestrus (P < 0.05). The expression levels of BMP-15 rose (P < 0.05) from small tomedium sizes in anestrous without changing at diestrus. Small antral follicles expressed thehighest values of GDF-9 at anestrus while only BMP-15 showed higher value in small antralfollicles at proestrous-estrus compared to diestrus and anestrus. Both proteins decreasedin proestrous/estrous (P < 0.05) with increasing follicle size, registering the lowest levels inlarge follicles. The flow cytometric assay was able to assess GDF-9 and BMP-15 expressionin canine follicular cells, showing that these proteins were differentially expressed duringfollicular development, possibly related to the special features of canine reproduction.