Female reproductive ageing is associated with a loss of GDF9 signalling capability by granulosa cells but not by oocytes. (#16)
Female reproductive aging is caused by a continuous loss of follicles/oocytes and a concomitant loss of oocyte quality. Aims: to determine whether; 1) normal granulosa cell (GC) function is maintained in older mice, and 2) oocyte-paracrine signalling to GCs declines with age. GCs and oocytes from young (3 wks) and old (1-2 years) 129/Sv mice were collected 46h post-eCG. Cells were collected for RNA or protein analyses or cultured for 24h. In response to GDF9, 3H-thymidine incorporation (DNA synthesis) was 7-times higher in young vs older GCs (p=0.006), but no different with BMP15 treatment. FSH stimulated GC progesterone production 43-fold vs 5-fold in young and old mice, respectively (p=0.005). FSH-stimulated progesterone production was potently suppressed in young GCs by GDF9, BMP6 and BMP15 (6-, 21-, 27-fold, respectively), and in older GCs by BMP6 and BMP15 but not GDF9. Hence we next examined whether there is an age-dependent decline in GDF9 signalling integrity in older GCs. An age-dependent decline in GC mRNA expression of BMPRII, ALK6 (p<0.05; BMP signalling) and ALK5 (p=0.054; GDF9) was found, but not of ALK4, ALK7 or SMAD2/3. P-SMAD3 activity was higher in young vs old GCs in response to GDF9 (17-fold higher, p=0.005) and following coculture with young oocytes (5.5-fold higher, p<0.05), but BMP15- and BMP6-induced p-SMAD1/5/8 activity was unaffected by age. Collectively, these results suggest an age-dependent decline in GDF9, but not BMP, signalling capability by GCs. We next examined if the oocyte’s capacity for paracrine signalling also declines with age. Young and old oocytes stimulated 3H-thymidine incorporation in GCs to the same extent, regardless of GC donor age. This was supported by GDF9 Western blot analysis where age did not affect the amount or forms (17 and 65 kDa) of GDF9 in denuded oocyte extracts. These results suggest an age-related effect on GDF9 function. A primary defect lies, not in the oocyte’s capacity to signal to GCs, but rather dysfunctional GC responsiveness to oocyte-secreted GDF9, but not to BMPs. As this communication axis is integral to oocyte quality, this defect likely contributes significantly to reproductive ageing.