Figure 2.

Active ISR marker detection in immature follicles of the mouse and human ovary. (A) Phosphoserine 51-eukaryotic Initiation Factor 2 subunit alpha (P-eIF2$\alpha$) is detectable via immunofluorescence (purple color is positive signal) in histological preparations of the mouse ovary. PFs are shown in A (arrowheads), primary follicles in (B) (arrowheads), and a small pre-antral follicle is shown in (C). Note that in all cases, both the oocyte and granulosa cells (GCs) exhibit signal; intense punctate staining is visible in some growing oocytes (example in C). A control image where the first antibody to was omitted is shown in (D) (asterisk indicates autofluorescent background) and is the adjacent tissue section to the image shown in C. Three panels are provided in (E) that show wild-type (WT) levels of P-eIF2$\alpha$ for direct comparison with galactose-1-phosphate uridylyltransferase knockout mouse (GalTKO) ovaries in F, images collected using identical settings. (G) and (H) show representative staining for P-eIF2$\alpha$ in immature human follicles from separate patient biopsies (DNA/DAPI, P-eIF2$\alpha$ channels and merged image labeled). (I) is a merged image of DAPI and background staining when first antibody to P-eIF2$\alpha$ is omitted, identical settings as H and I. Panels (J) and (K) show colorimetric staining of mouse ovaries for two DNA damage marks, P-T21 replication protein A2 (Rpa2) and P-S345 checkpoint kinase 1 (Chk1), respectively. Insets show electronically magnified areas that include PFs. Panel (L) is a no 1st antibody control. Scale bars for Panels A–D = 100, G–I = 20 and J–L = 75 $\mathrm{\mu }$m, respectively.