Objective

In vitro maturation (IVM) is an assisted reproductive technology used in the generation of embryos in the laboratory. In mammalian ovarian folliculogenesis, it has been identified that growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are essential proteins that form part of the transforming growth factor-β (TGF-β) superfamily; and their signalling pathways play a significant role in IVM. In a 2015 study, these two proteins were identified to form a heterodimer called cumulin (GDF9/BMP15). This paper will aim at investigating if the pro-region of GDF9 forms part of cumulin. Specific objectives are: purifying a form of the heterodimer, FLAG-cumulin, binding an anti-FLAG tag antibody to detect the FLAG-tagged pro-region of GDF9, and developing a transcriptional reporter assay for use in assessing the bioactivity of FLAG-cumulin.

The heterodimer FLAG-tagged GDF9 / 8-histidine tagged BMP15 was purified using immobilised metal affinity chromatography (IMAC). Western blotting and silver staining techniques were then used to characterise the purified protein in preparation for further studies. Anti-FLAG tag antibody was bound to the FLAG-tagged GDF9 to detect the presence of GDF9 pro-region. The activity of FLAG-cumulin was then studied using a transcriptional reporter assay, which consisted of CAGA luciferase/BMP response element (BRE) luciferase. FLAG-cumulin was successfully purified (this is shown from the results of IMAC purification shown on western blots silver-gel staining experiments). It was also characterized successfully. Anti-FLAG-tagged antibody was able to detect the presence of the FLAG-tagged pro-region of GDF9 (suggesting that the pro-region of GDF9 forms part of FLAG-cumulin). However, the transcriptional reporter assay did not show significant bioactivity in the presence of FLAG-cumulin. The discovery that GDF9 pro-region forms part of FLAG-cumulin contributes relevant scientific information to the current database on the heterodimer. This will facilitate its use in future researches on how to improve female fertility using IVM.