Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation

Abstract

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-␤ ␤ (TGF␤ ␤) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocytesecreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGF␤ ␤1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGF␤ ␤ superfamily receptor ectodomains (ECD) to neutralise oocyte-or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGF␤ ␤1 and activin-A bioactivity, demonstrating its specificity. The TGF␤ ␤R-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte-or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dosedependent manner. Consistent with these findings, oocytes, GDF9 and TGF␤ ␤1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGF␤ ␤ superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cellsignalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.

Growth and differentiation factor 9 promotes oocyte growth at the primary but not the early secondary stage in three-dimensional follicle culture

“…This effect is consistent with multiple previous studies in which GDF9 has been shown to stimulate the proliferation of isolated granulosa cells in vitro [26][27][28]. However, no significant further growth of the granulosa cell compartment is seen after 24 h, whereas the oocyte in the primary follicles continues to grow through day 3.…”

“…In addition, neither of these studies evaluated the effects of GDF9 on the granulosa cell and oocyte compartments independently. While it has been shown that GDF9 can promote growth of isolated granulosa cells in culture [26][27][28], studies in GDF9 null mice suggest that it acts to inhibit, rather than promote, growth of the oocyte [29]. The effect of GDF9 alone on the oocyte and granulosa cells during the earliest stages of preantral follicle growth, therefore, remains unclear.…”

Cumulus cell contact during oocyte maturation in mice regulates meiotic spindle positioning and enhances developmental competence

“…As an obvious substrate for cell interactions, it is somewhat surprising that so little is known about TZP remodeling especially with respect to the acquisition of developmental competence after IVM. TZPs are ubiquitous amongst mammals [1][2][3] and have been widely implicated in paracrine signaling within the COC [12,17,19,20,45]. However, the fate and function of TZPs during oocyte maturation under in vivo or in vitro conditions has evaded definition for signaling mechanisms other than those of paracrine or junctional varieties [15,22,29,32].…”

Inferring biological mechanisms from spatial analysis: Prediction of a local inhibitor in the ovary

“…First, growing follicles could start to produce an activator at the primary stage. One candidate is the oocyte-specific Growth Differentiation Factor-9 (Gdf-9), which is expressed from the primary stage onwards in mouse (24), and is known to stimulate granulosa cell mitosis (25,26). Other possible candidates are the bone morphogenetic proteins BMP-4 and -7, which are first detected at the primary and secondary stages respectively (18) and stimulate initiation of follicle growth (27,28).…”