Defining the role of notch family members in the luteinization of murine granulosa cells in vitro

Abstract

OBJECTIVE: Spontaneous luteinization of murine granulosa cells occurs in culture, and is characterized by increased production of progesterone, changes in cellular morphology, and biochemical markers. The Notch signaling pathway has been implicated in the processes of cellular proliferation and differentiation. Our goal was to investigate the significance of Notch signaling in the process of granulosa cell (GC) differentiation/luteinization.DESIGN: Analysis of protein expression and progesterone levels in murine GC cultures after inhibition of Notch protein activation or specific knockdown of Notch2.MATERIALS AND METHODS: Groups of C57BL/6 mice (6-9 weeks of age/6-14 per group) were injected (SQ) with Pregnant Mare Serum Gonadotropin (PMSG) to stimulate follicular development. Granulosa cells were obtained from the ovaries 40-44 hours later by follicular puncture. Cells were cultured in 12-well dishes, and the media and cell lysates were collected at 24 hour intervals for 96 hours. For the inhibition experiments, DAPT (γ-secretase inhibitor which prevents the activation of Notch proteins), was added to the media after 24 hours of culture. For the Notch2 knockdown experiments, siRNA transfection was performed after 24 hours of culture. Media and cell lysates were collected at 72 hours of culture. Media samples were tested for progesterone content using ELISA. Cell lysates were tested for expression of Notch2 protein by Western blot.RESULTS: Notch2 protein expression increased in concert with progesterone production in culture. When DAPT was used to inhibit the activation of Notch proteins, the resulting 20% decrease in Notch2 led to an 80% decrease in progesterone levels. Specific knock-down of Notch2 expression resulted in approximately 30% decrease in the protein concentration, and yielded a consistent 30-50% reduction in progesterone levels.CONCLUSIONS: The inhibition of progesterone production by DAPT indirectly suggests that members of the Notch family are involved in the differentiation/luteinization of granulosa cells. The siRNA knockdown experiments identify Notch2 as one of the potential specific players. OBJECTIVE: Spontaneous luteinization of murine granulosa cells occurs in culture, and is characterized by increased production of progesterone, changes in cellular morphology, and biochemical markers. The Notch signaling pathway has been implicated in the processes of cellular proliferation and differentiation. Our goal was to investigate the significance of Notch signaling in the process of granulosa cell (GC) differentiation/luteinization. DESIGN: Analysis of protein expression and progesterone levels in murine GC cultures after inhibition of Notch protein activation or specific knockdown of Notch2. MATERIALS AND METHODS: Groups of C57BL/6 mice (6-9 weeks of age/6-14 per group) were injected (SQ) with Pregnant Mare Serum Gonadotropin (PMSG) to stimulate follicular development. Granulosa cells were obtained from the ovaries 40-44 hours later by follicular puncture. Cells were cultured in 12-well dishes, and the media and cell lysates were collected at 24 hour intervals for 96 hours. For the inhibition experiments, DAPT (γ-secretase inhibitor which prevents the activation of Notch proteins), was added to the media after 24 hours of culture. For the Notch2 knockdown experiments, siRNA transfection was performed after 24 hours of culture. Media and cell lysates were collected at 72 hours of culture. Media samples were tested for progesterone content using ELISA. Cell lysates were tested for expression of Notch2 protein by Western blot. RESULTS: Notch2 protein expression increased in concert with progesterone production in culture. When DAPT was used to inhibit the activation of Notch proteins, the resulting 20% decrease in Notch2 led to an 80% decrease in progesterone levels. Specific knock-down of Notch2 expression resulted in approximately 30% decrease in the protein concentration, and yielded a consistent 30-50% reduction in progesterone levels. CONCLUSIONS: The inhibition of progesterone production by DAPT indirectly suggests that members of the Notch family are involved in the differentiation/luteinization of granulosa cells. The siRNA knockdown experiments identify Notch2 as one of the potential specific players.