170 GENE SILENCING OF BMPRII IN BOVINE GRANULOSA CELLS

Abstract

Granulosa cells (GC) are important constituents of the follicular environment for oocyte competence acquisition. However, their functionality depends on oocyte-derived factors, such as GDF9 and BMP15, which act through BMPRII receptor signalling. Gene silencing using lipofection has been used as an important tool to investigate the role of cell genes and proteins. The aim of this study was to establish the ideal conditions for lipofection in bovine GC and starting from this protocol to establish a methodology for silencing of the BMPRII gene by RNA interference, and to use this strategy to study the functions of BMPRII in GDF9 signalling. GC were obtained from slaughterhouse ovaries by aspiration of follicles (2 to 6 mm) and subsequently cultured in DMEM medium at 38.5°C and 5% CO₂ in air. All data analyzes were performed by GraphPad Prism^® version 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using one-way ANOVA followed by Tukey's test. For optimizing the conditions for lipofection, the GC were treated with different amounts of lipofection agents Lipofectamine^® RNAiMAX (Invitrogen, Carlsbad, CA, USA; 1, 2, and 3 µL) or Lipofectamine^TM 2000 (Invitrogen; 1, 2, and 3 µL) and the transfection indicators Siglo^® (GE Healthcare, Waukesha, WI, USA; 30 to 100 nM) or FUGW transgenic plasmid (100 to 900 nM) during 24 and 48 h. The highest efficiency of lipofection was observed at 24 h of culture with 2 μL of Lipofectamine™ 2000 + 100 nM of Siglo^®. Based on these conditions, different concentrations of siBMPRII (100 to 500 pM) were tested during 24 h of culture and subsequently, different incubation times (0, 6, 12, 18, and 24 h) with the best siRNA concentration in order to establish optimum conditions for gene silencing. GC were evaluated for the relative abundance of mRNA for BMPRII using PPIA and β-actin as endogenous controls by real-time PCR. All concentrations provided similar and highly significant transcript reduction in comparison to control (P < 0.001), so the lowest of them was adopted (100 pM). For different incubation times with 100 pM siBMPRII also a similar decrease was also seen, which was more significant at 24 h (P < 0.01). The best concentration (100 pM) and incubation time (24 h) with the siRNA were analysed by Western blotting, which confirmed the BMPRII reduction also at protein level (P < 0.05). For functional evaluation, GC submitted or no to gene silencing were incubated with or without GDF9 (100 ng) and assessed for expression of genes controlled by GDF9 through its BMPRII receptor (LHR, INHA, and INHBA), besides the CYP17 gene, a marker of potential theca cells contamination. LHR expression was reduced in silenced groups (P < 0.05) and highly suppressed by GDF9 in non-silenced groups (P < 0.001), while INHA and INHBA expression remained constant in the different groups (P > 0.05), suggesting that the control of these genes in bovine does not behave as reported in other species. The lack of amplification CYP17 indicates the nonexistence of the contamination. In conclusion, this study allowed the establishment of an efficient methodology for the silencing of BMPRII by lipofection in bovine GC, which may be used as a tool for the functional study of BMPRII and potentially for other genes of interest.

Research was supported by FAPESP and CNPq.