Female reproductive aging is characterized by a rise in follicle-stimulating hormone (FSH) levels during peri-menopause. N-linked glycans are co-translationally attached to the Asn7 and Asn24 residues on the FSHβ subunit. Differences in the number of N-glycans on the FSHβ subunit result in distinct glycoforms: hypo-glycosylated (FSH21/18, glycans absent on either Asn24 or Asn7, respectively) or fully-glycosylated (FSH24, glycans present on both Asn7 and Asn24). The relative abundance of FSH glycoforms changes with advanced reproductive age, shifting from predominantly FSH21/18 in younger women to FSH24 in older women. Previous in vitro studies in granulosa cell lines and in vivo studies using Fshb-null mice showed these glycoforms elicit differential bioactivities. However, the direct effects of FSH glycoforms on the mouse ovarian follicle have not yet been determined. In this study, we isolated secondary follicles from pre-pubertal mice and treated them with 20- or 100 ng/mL purified recombinant FSH glycoforms for 1 h or 18–20 h. Analysis of phosphorylated PKA substrates showed that glycoforms were bioactive in follicles following 1-h treatment, although differential bioactivity was only observed with the 100 ng/mL dose. Treatment of follicles with 100 ng/mL of each glycoform also induced distinct expression patterns of FSH-responsive genes as assessed by qPCR, consistent with differential function. Our results, therefore, indicate that FSH glycoforms are bioactive in isolated murine follicles.
Supplemental Figure 1. Permeabilization by proteinase K treatment of formalin-fixed follicles allows antibody binding. Fixed follicles incubated with 20µg/mL Proteinase K at 37°C showed greater localization of PKA substrates (B) in comparison to untreated control follicles (A). (C) Non-immune IgG control shows that PKA substrate expression was specific. Insets show rhodamine phalloidin (red) and DAPI (blue). N=5 follicles per condition group. Scale bar = 50µm.
Supplemental Figure 2. Expression patterns of genes that did not show a response to glycoform treatment. Gene expression was assayed by Taqman real time qPCR assays following one-hour and 18-20-hour glycoform treatment. Groups of 100 follicles were analyzed in triplicate.
Supplemental Table 1. Alphabetical list of genes quantified by real time qPCR assays