Fernandez Tomas, Palomino Jaime, Parraguez Victor H, Peralta Oscar A, De los Reyes Monica
Laboratory of Animal Reproduction, Faculty of Veterinary Sciences, University of Chile, Casilla 2 Correo 15, Santiago, Chile.
Laboratory of Animal Physiology, Faculty of Veterinary Sciences, University of Chile, Casilla 2 Correo 15, Santiago, Chile.
Anim Reprod Sci. 2016 Apr;167:59-67. doi: 10.1016/j.anireprosci.2016.02.005. Epub 2016 Feb 3.
Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) play important functions in follicular and oocyte development in many species. This study evaluated the dynamic expression of GDF-9 and BMP-15 in canine follicles cells using flow cytometry 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 incubated 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 the initial dot plot and, additionally, a CD45 marker for leukocyte and propidium iodide (PI) were used for erythrocyte and debris discrimination. The evidence corroborated the presence of both proteins in canine follicle cells, but these proteins were not expressed equally during follicular development. The results analyzed by ANOVA showed that GDF-9 expression decreased (P<0.05) during follicular growth in anestrus and proestrous/estrous, but increased in diestrus (P<0.05). The expression levels of BMP-15 rose (P<0.05) from small to medium sizes in anestrous without changing at diestrus. Small antral follicles expressed the highest values of GDF-9 at anestrus while only BMP-15 showed higher value in small antral follicles at proestrous-estrus compared to diestrus and anestrus. Both proteins decreased in proestrous/estrous (P<0.05) with increasing follicle size, registering the lowest levels in large follicles. The flow cytometric assay was able to assess GDF-9 and BMP-15 expression in canine follicular cells, showing that these proteins were differentially expressed during follicular development, possibly related to the special features of canine reproduction.
生长分化因子9（GDF - 9）和骨形态发生蛋白15（BMP - 15）在许多物种的卵泡和卵母细胞发育中发挥着重要作用。本研究采用流式细胞术分析评估了犬卵泡细胞中GDF - 9和BMP - 15的动态表达。在整个发情周期中，从母犬卵巢的三种大小的窦状卵泡（小、中、大）中取出卵泡细胞。细胞与抗人GDF - 9多克隆抗体和抗小鼠BMP - 15单克隆抗体孵育。在初始点图中使用大小和复杂性鉴别门进行流式细胞术分析，此外，使用白细胞的CD45标记物和碘化丙啶（PI）来鉴别红细胞和碎片。证据证实了这两种蛋白在犬卵泡细胞中的存在，但这些蛋白在卵泡发育过程中的表达并不相同。方差分析结果表明，在乏情期和发情前期/发情期，卵泡生长过程中GDF - 9表达降低（P<0.05），但在间情期升高（P<0.05）。在乏情期，BMP - 15的表达水平从小卵泡到中卵泡升高（P<0.05），在间情期没有变化。小窦状卵泡在乏情期表达GDF - 9的最高值，而与间情期和乏情期相比，只有BMP - 15在发情前期 - 发情期的小窦状卵泡中表达更高。在发情前期/发情期，随着卵泡大小增加，两种蛋白均降低（P<0.05），在大卵泡中达到最低水平。流式细胞术检测能够评估犬卵泡细胞中GDF - 9和BMP - 15的表达，表明这些蛋白在卵泡发育过程中差异表达，可能与犬繁殖的特殊特征有关。
