Fig 2: Sites of pro-anti-Müllerian hormone (AMH) cleavage and conversion to active AMHN, C. AMH is secreted into follicular fluid predominantly as the uncleaved, inactive proprotein, proAMH, with small amounts of the active form, AMHN, C. ProAMH diffuses through the granulosa layer slowly and will encounter increased prohormone convertase (PCSK) activity in the theca and stromal layers. The newly converted AMHN, C becomes available to bind to receptors on cells/structures found in theca and stroma. Diffusion of AMHN, C will preferentially occur into theca and stroma because diffusion back into the granulosa layer will be impeded by the net negative charge found on the protein.

Fig 3: Anti-Müllerian hormone (AMH) diffusion and proteolytic activation of proAMH in ovarian stroma. (A) Microdialysis probes were inserted adjacent into ovary stroma where dialysis buffer was pumped through the porous probe allowing molecules up to 1000 kDa in molecular weight to enter the dialysate. The dialysate was collected for AMH assay. Needles were placed adjacent to an antral follicle as shown in the hematoxylin and eosin-stained sheep ovary section (Scale bar = 1 mm). AMH concentrations in follicular fluid or adjacent stroma were quantified in sheep and human ovaries. (B) 2D-PAGE separating recombinant AMH protein by size on the vertical dimension and isoelectric focusing (IEF) on the horizontal direction. Western blot was conducted on the dashed area using an antibody against the AMH N-terminal fragment (AMHN). The recombinant AMH preparation contains predominantly proAMH (arrow) and lesser quantities of AMHN. (C) Western blot of recombinant proAMH treated with (+) or without (−) recombinant PCSK3 in artificial extracellular fluid or artificial secretory vesicle fluid. ProAMH appears as a 72 kDa band (arrowhead) and the cleaved AMHN fragment as a 64 kDa band (double arrowhead). (D) PCSK activity in mouse ovary fragments was visualized with a fluorogenic reagent that releases rhodamine 110 when exposed to protease activity (green: rhodamine 110 and blue: Hoescht 33342 DNA-stain, scale bar = 100 µm). A yellow ring denotes the basal lamina that separates the granulosa and theca layers. The upper graph shows the relative fluorescence intensity units (RFU) along the straight-line trace shown in the rhodamine 110 image and the lower graph shows mean (±SE) fluorescence intensity in the antrum, granulosa, theca, and stroma was compared with 1-way ANOVA (F(3,32) = 15.8, P < 0.001) and Tukey’s B post hoc test (regions that share the same letter were not significantly different, n = 9).

Fig 4: The effects of anti-Müllerian hormone (AMH) on primordial and antral follicles. (A) Dose–response curve of recombinant AMH on primordial follicle activation rates in neonatal mouse ovary cultures (blue open diamonds) and AMHR2-transfected P19 reporter cell line (green closed circles). Activation is expressed as the number of remaining primordial follicles as a fraction of all follicles (primordial + developing). (B) Confocal microscope images of antral follicles from adult Amh+/+ or Amh−/− mice fused to Amh+/+ neonatal ovaries in co-culture with immunofluorescence for AMH, DDX4 (oocyte marker), and DAPI (DNA label). (C) Primary follicle expressing AMH in granulosa cells, (D) adult follicle from an Amh+/+ mouse, and (E) adult follicle from an Amh−/− mouse, scale bars = 50 µm. (F) Distances between the antral follicle and all primordial or primary follicles in the antral follicle-neonatal ovary co-culture were divided into quadrants to investigate the primordial follicle activation rate at increasing distances from the antral follicle (2-way ANOVA with Tukey’s B post hoc test. Groups that share letters are not significantly different). (G) Amh+/+ and Amh−/− small antral follicle growth in culture (* indicates P < 0.05, repeated measures ANOVA with Tukey’s B post hoc test) with representative images of follicles at 0 and 96 h in vitro.