Abstract
Anti-Müllerian hormone (AMH) is a gonadal hormone that induces part of the male phenotype. However, it is also present in the blood of both sexes, and is a putative local regulator of adult gonadal function. This suggests that AMH, like other members of the TGFβ superfamily, is a pleiotropic regulator. The TGFβ superfamily ligands share receptors and binding proteins (BPs), leading to context-dependent signaling. AMH is the only ligand with a unique type-2 receptor (AMHR2), but it shares type-1 receptors with other TGFβs. Its ability to interact with other TGFβ superfamily members via common BPs is unknown. This thesis investigated whether TGFβ-superfamily BPs can regulate the activity of AMH.
A (BRE)2-Luc reporter gene assay was established to measure AMH signaling, at physiological levels of AMH. The assay was optimized by varying key parameters, such as the cell type and the type of serum, and by refining the critical technical steps. P19 cells were selected in preference to DU145 and LNCaP cells, as they had superior growth characteristics and as the AMH-reporter assay had a higher signal-to-background ratio. P19 cells express AMH type-1 receptors. However, the expression of endogenous AMHR2 by P19 cells was minimal. Transfection of AMHR2 was therefore essential to produce robust AMH signaling by P19 cells. The receptor binding component of AMH (AMHC) and the physiological form of AMH (AMHN,C) both activated the reporter assay, with physiologically-relevant EC50s.
The influence of fifteen TGFβ-superfamily BPs on AMH signaling was examined,using multiple screens. The influence of each BP on the AMH dose-response curve was examined, along with their effect on an EC50-like concentration of AMH. AMHC and AMHN,C were both examined, enabling the influence of interactions on both the N and C terminal domains of AMH to be examined. The follistatins (FSs) had the greatest effect on AMH signaling, with their influence being maximal when AMH levels were low (adult circulating-like levels). FS288 appeared to be more potent than FS315.
Brorin, decorin and FS-like 4 also caused a statistically significant change in the doseresponse curve. The effect of these BPs were small and were only evident when the concentration of AMHC was low. Little or no effect was observed when the concentration of AMH was close to or above the EC50. Endoglin and chordin-like 2 reduced the bottom (zero AMH) of the dose-response curve, but did not affect the reporter activity produced by AMH. This suggests that endoglin and chordin-like 2 influence the reporter assay through a mechanism that is unrelated to AMH.
The assay was designed to detect AMH BPs, and different types of studies will be needed to identify how FS288, FS315, brorin, decorin and/or FS-like 4 affects the functions of AMH in vivo. The other eight BPs tested appeared to have little or no effect on AMH signaling.
AMH is partially secreted as an inactive precursor (proAMH), and the bioactivity of AMH in vivo may be influenced by when and where proAMH is cleaved to AMHN,C. BPs potentially influences this process, but none of the BPs examined in the Thesis affected the cleavage of proAMH by one of its putative cleavage enzymes, furin.
AMH shares type-1 receptors and the intracellular signaling cascade with bone morphogenetic proteins (BMPs). This raised the possibility that AMH does not always signal as an independent regulator. In some contexts, AMH and BMP may interact to regulate gene expression. Consistent with this, AMH and BMP exhibited redundant or co-operative activation of the reporter, depending on the concentrations of each ligand. This question was largely outside of the scope of this thesis, and further testing of this hypothesis was not undertaken. However, these preliminary observations lay a foundation for the future research directions outlined in Chapter 6.
In summary, this thesis presents the first evidence that certain TGFβ-superfamily BPs may influence AMH signaling. FS288 was the most significant of these, and may be most important when AMH levels are low, as in men and women. In vivo experiments are needed to prove this. BPs such as FS288 may integrate the biological actions of AMH with those of other TGFβ-superfamily ligands.