Oral Presentation ESA-SRB Conference 2015

BMP15 and GDF9 increase intracellular cyclic AMP concentration and junctional protein mRNA expression in granulosa cells (#130)

Michael J Bertoldo 1 , Zhi K Sia 1 , David Agapiou 1 , Sonia Bustamente 2 , Dulama Richani 1 , Craig A Harrison 3 , Robert G Gilchrist 1
  1. Oocyte Biology Research Unit, School of Women’s and Children’s Health, UNSW, Sydney, NSW, Australia
  2. Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, UNSW, Sydney, NSW, Australia
  3. Hudson Institute, Clayton, Victoria, Australia

Adherens junctions, tight junctions and gap junctions are at the nexus of intercellular communication within the ovarian follicle. Adherens and tight junctions establish and maintain the follicle’s 3D architecture while gap junctions mediate somatic cell-somatic cell and somatic cell-oocyte bidirectional communication. The oocyte-secreted factors (OSFs) growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been implicated in the regulation of somatic cell-oocyte gap-junctional communication. The regulation of adherens and tight junctions and their role in oocyte and follicle development are poorly understood. This study aimed to assess the role of BMP15 and GDF9 in regulating intracellular cyclic AMP (cAMP), and to determine if OSFs control mRNA expression of gap junction, adherens junction and tight junction genes. We used the human granulosa cell line COV434,mass-spectrometry and RT-qPCR to obtain our readouts. Firstly, cells were treated with hBMP15(100ng/ml), mGDF9 (100ng/ml) and BMP15 + GDF9 (50ng/ml each) for 1h. Compared with controls, BMP15 and BMP15 + GDF9 tended to increase intracellular cAMP (1.4 ± 0.1, 1.7 ± 0.09 and 2.2 ± 0.3 pmol/mg protein, respectively; P=0.06), while there was no effect of GDF9 alone. A 24h time course experiment with GDF9 (25ng/ml) or dbcAMP (250µM) did not demonstrate an effect on mRNA expression. Conversely BMP15 (25ng/ml) increased mRNA expression of genes encoding vimentin, connexin43, zona occludens-1 and claudin at 6, 9 and 24h of culture. Vimentin encoding mRNA expression increased in a dose-responsive manner to increasing concentrations of BMP15 (range tested 0-100ng/ml; P<0.01). Vimentin, connexin43 and zona occludens-1 encoding mRNA increased with 6.25 and 100ng/ml of GDF9 at 24h (P<0.05). These results suggest that OSFs regulate granulosa cell mRNA expression of cytoskeletal vimentin and the mRNA of proteins specific to gap junctions (connexin43) and tight junctions (zona occludens-1 and claudin). Cyclic AMP may regulate intercellular communication via post-transcriptional mechanisms.