Endocrine Abstracts

Objective: Recently rare heterozogous AMH genetic variants have been identified in women with polycystic ovary syndrome (PCOS) that result in reduced AMH signaling. However, the exact functional mechanism remains unknown. Therefore, we have performed functional analyses to analyze the processing, secretion and signaling of these PCOS-specific AMH rare variants.

Methods: Six PCOS-specific AMH variants containing mutations (V12G, P151S, P270S, P352S, P362S, H506Q) were selected based on previous findings. The variants were introduced in an AMH expression vector containing either a wild type (AMH-RAQR) or optimized cleavage site (AMH-RARR) and co-expressed with the BRE-Luc reporter in the mouse granulosa cell line KK-1. The AMH expression vectors were stably expressed in HEK293 cells for Western Blot analysis and ELISA.

Results: Expression of AMH-P151S and AMH-H506Q decreased AMH signaling by ~90% (P<0.001), depending on the presence of a RAQR or RARR cleavage site. Signaling of the other four variants was comparable to wild-type (wt)-AMH. Coexpression of the variants with wt-AMH at equal amounts confirmed that AMH-P151S and AMH-H506Q significantly inhibited the signaling activity of wt-AMH by ~30% (P<0.001). Transfection of increasing amounts of these two variants resulted in a further inhibition, which was independent of the cleavage site. To explain this dominant negative effect, we next analyzed the impact of AMH cleavage on AMH signaling. Cells were transfected with an AMH construct containing an inactive cleavage site (AMH-RAGA) in combination with exogenous AMH treatment. We observed that exogenous AMH-induced signaling was suppressed by 30% (P<0.01) in the presence of AMH-RAGA. In contrast, exogenous AMH-induced signaling was not affected when AMH-P151S or AMH-H506Q was transfected. Indeed, Western blot analysis showed that AMH-P151S and AMH-H506Q proteins were only detected in the cell lysate but not in the supernatant, even in the presence of RARR cleavage site. In contrast, wt-AMH and the P352S and P362S variant were detected in both the cell lysate and the supernatant. Further supporting these results, confocal image analysis showed that cells expressing AMH-P151S and AMH-H506Q retained significantly higher cellular AMH protein levels with a highly abnormal subcellular localization in the ER compared to the cells expressing wt-AMH and AMH-P352S.

Conclusions: Our results show that the PCOS-specific AMH variants P151S and H506Q disrupt normal processing and secretion of AMH. Our results further suggest that these AMH variants hamper secretion of wt-AMH, explaining the dominant negative effect of these variants on AMH signaling.