Alam Md Hasanur, Lee Jibak, Miyano Takashi
Laboratory of Developmental Biotechnology, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan.
J Reprod Dev. 2018 Oct 12;64(5):423-431. doi: 10.1262/jrd.2018-078. Epub 2018 Jul 21.
The role of oocytes in follicular antrum formation is not well understood. We examined the effect of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the formation of antrum-like structures by cultured bovine oocyte-granulosa cell complexes (OGCs). OGCs containing growing oocytes (105‒115 µm in diameter) were collected from early antral follicles (1.2‒1.8 mm) and used to prepare oocytectomized complexes (OXCs) and granulosa cell complexes (GCs). The mRNAs of GDF9 and BMP15 were expressed in the oocytes, but not in the granulosa cells. The complexes were cultured for five days with or without GDF9 and BMP15 either alone or in combination. The OGCs maintained their complex integrity and developed antrum-like structure, whereas OXCs and GCs neither maintained their integrity nor developed any antrum-like structure without growth factors. GDF9 or BMP15 alone increased the integrity of these complexes and induced antrum-like structures in OXCs and GCs. Moreover, the combination of GDF9 and BMP15 was more potent for both phenomena in all types of complexes. In OXCs and GCs cultured without GDF9 and BMP15 or with BMP15 alone, outgrowing granulosa cells differentiated into fibroblast-like cells. The combination of GDF9 and BMP15 suppressed the appearance of fibroblast-like cells in OXCs and GCs during incubation. Instead, the granulosa cells appeared rhomboid and pebble-like in shape, similar to those in OGCs cultured without supplementation of GDF9 and BMP15. These results suggest that oocytes maintain complex integrity by preventing granulosa cell differentiation and participate in follicular antrum formation via GDF9 and BMP15.
卵母细胞在卵泡腔形成中的作用尚未完全明确。我们研究了卵母细胞源性生长因子，即生长分化因子9（GDF9）和骨形态发生蛋白15（BMP15），对培养的牛卵母细胞-颗粒细胞复合体（OGCs）形成类似卵泡腔结构的影响。从早期卵泡（直径1.2 - 1.8 mm）中收集含有生长中卵母细胞（直径105 - 115 µm）的OGCs，用于制备去卵母细胞复合体（OXCs）和颗粒细胞复合体（GCs）。GDF9和BMP15的mRNA在卵母细胞中表达，但在颗粒细胞中不表达。将这些复合体在有或无GDF9和BMP15单独或联合存在的情况下培养5天。OGCs保持其复合体完整性并发育出类似卵泡腔的结构，而OXCs和GCs在没有生长因子的情况下既不能保持其完整性，也不会发育出任何类似卵泡腔的结构。单独的GDF9或BMP15增加了这些复合体的完整性，并在OXCs和GCs中诱导出类似卵泡腔的结构。此外，GDF9和BMP15的联合对所有类型复合体中的这两种现象都更有效。在没有GDF9和BMP15或仅含BMP15培养的OXCs和GCs中，生长出的颗粒细胞分化为成纤维细胞样细胞。GDF9和BMP15的联合在培养过程中抑制了OXCs和GCs中成纤维细胞样细胞的出现。相反，颗粒细胞呈现菱形和卵石样形态，类似于未添加GDF9和BMP15培养的OGCs中的颗粒细胞。这些结果表明，卵母细胞通过防止颗粒细胞分化来维持复合体完整性，并通过GDF9和BMP15参与卵泡腔形成。
