Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice

Abstract

The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn7 and Asn24 residues that are co-translationally attached early during subunit biosynthesis. Differences in the number of N-glycans (none, one or two) on the human FSHβ subunit contribute to macroheterogeneity in the FSH heterodimer. The resulting FSH glycoforms are termed hypo-glycosylated (FSH21/18, missing either an Asn24 or Asn7 N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH24, possessing of both Asn7 and Asn24 N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH21/18 and FSH24) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH21/18 exhibits faster FSH- receptor binding kinetics and is much more active than FSH24 in every assay tested to date. However, the in vivo bioactivity of the hypo-glycosylated FSH glycoform has never been tested. Here, we evaluated the in vivo bioactivities of FSH glycoforms in Fshb null mice using a pharmacological rescue approach. In Fshb null female mice, both hypo- and fully-glycosylated FSH elicited an ovarian weight gain response by 48 h and induced ovarian genes in a dose- and time-dependent manner. Quantification by real time qPCR assays indicated that hypo-glycosylated FSH21/18 was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH24. Western blot analyses followed by densitometry of key signaling components downstream of the FSH-receptor confirmed that the hypo-glycosylated FSH21/18 elicited a response similar to that by fully-glycosylated FSH24 in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH21/18 was more active than the fully-glycosylated FSH24 in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH21/18 glycoform elicits bioactivity in vivo similar to the fully-glycosylated FSH. Our studies may have clinical implications particularly in formulating FSH-based ovarian follicle induction protocols using a combination of different human FSH glycoforms.

Keywords: FSH-responsive genes; N-glycosylation; Ovary; Pharmacological rescue; Testis.

Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Figures

Fig. 1

A summary of N-linked sugar chains attached at the Asn residues in human FSH subunits and the FSH glycoform nomenclature (A). Western blot analysis (B) using a mouse anti-human FSH monoclonal antibody (RFSH-20) indicates the molecular size of purified recombinant GH3-derived FSH21/18 and FSH24 glycoforms (Lanes 1 and 2). Purified human pituitary FSH reference preparation (AFP 7298) was used as positive control (Lane 3). An independent blot with FSH samples loaded in the same order as in panel B, was probed with an anti-alpha 15-2 monoclonal antibody (C).

Fig. 2

Ovarian gene expression analyzed by Taqman real time qPCR assays in Fshb null female mice injected with recombinant FSH glycoforms at 1μg dose. Gene expression was assayed between 2h – 24h at different time points. For each time point, triplicate cDNA samples from ovaries from 4-5 mice were used. * P < 0.05 vs. PBS-injected control group. The genes analyzed are listed in the Supplementary Material Table - 1.

Fig. 3

Ovarian gene expression analyzed by Taqman real time qPCR assays in Fshb null female mice injected with recombinant FSH glycoforms at 1μg dose. Gene expression was assayed at different time points between 0.5h – 2h as indicated. For each time point, triplicate cDNA samples from ovaries from 4-5 mice were used. * P < 0.05 vs. PBS-injected control group and ** P < 0.05 vs. the other glycoform at the same time point. The genes analyzed are listed in the Supplementary Material Table - 1.

Fig. 4

Ovarian gene expression analyzed by Taqman real time qPCR assays in Fshb null female mice injected with recombinant FSH glycoforms at 1μg dose. Expression of 11 known FSH-responsive marker genes was analyzed 0.5h, 1h and 2h after the FSH glycoform injection. Ovarian gene expression was separately compared for each time point. For each marker expression assay, triplicate cDNA samples from ovaries from 4-5 mice were used. * P < 0.05 vs. PBS-injected control group and ** P < 0.05 vs. the other glycoform. The genes analyzed are listed in the Supplementary Material Table - 1.

Fig. 5

Immunolabeling of ovarian sections with anti-phospho-CREB (A, C and E) and anti-phospho-PKA substrate (B, D and F) antibodies indicate activation of FSH-receptor mediated signaling in ovaries of FSH glycoform injected Fshb null mice. Each FSH glycoform was injected at 1 μg dose and ovaries were harvested after 0.5h (A and B), 1h (C and D) and 2h (E and F), formalin-fixed and processed. Merged images are shown: specific antibody staining was visualized in green and the nuclei were stained red. Multiple sections from ovaries of 3 mice per group and per each time point were analyzed. Bottom panels represent enlarged images of follicles present in white squares in upper panels. White bar represents 100 μm.

Fig. 6

Western blot analysis of ovarian extracts probed with various antibodies against activated forms of signaling components downstream of FSH-receptor (A, C and E). Extracts were prepared from Fshb null mice injected with FSH glycoforms after different times (0.5h, 1h and 2h). Western blot analysis was performed on ovarian extracts from 2-3 mice per group and per time point. Expression of β-tubulin was used as an internal control and relative expression of each phospho-protein was plotted on the Y-axis. Densitometry data (B, D and F) represents an average of 3 independent blots. * P < 0.05 vs. PBS injected group and ** P < 0.05 vs. the other glycoform.

Fig. 7

Loss of FSH results in hypoplastic ovaries and both the FSH21/18 and FSH24 glycoforms elicit the ovarian weight gain response in Fshb null mice (A). Ovarian histology shows antrum in sections obtained from control (Ctrl.) (B) and FSH glycoform-injected (D and E) but not PBS-injected (C) Fshb−/− females. Serum estradiol was significantly high in FSH21/18 injected immature Fshb null mice compared to either FSH24 - injected Fshb null females or PBS- injected control mice (F). * P < 0.05 vs. PBS-injected Ctrl, ** P < 0.05 vs. PBS-injected Fshb−/−, n=6 mice per group. Black bar represents 200 μm.

Fig. 8

In vivo bioactivity of FSH glycoforms was evaluated following the protocols depicted in Supplementary Fig. S1. Both FSH21/18 and FSH24 elicited a testis weight gain response in Fshb−/− male mice in a 5-day injection protocol (A). * P < 0.05 vs. 5d PBS group, ** P < 0.05 vs. 10d PBS group, n=7 mice. Hematoxylin-eosin stained testis histology (B) shows increased number of germ cells in sections from FSH21/18 - injected mice compared to those in FSH24 - injected mice resulting in increased tubule size (bar graph in panel B). Quantification of GCNA1, a pan-germ cell marker - positive germ cells by immunofluorescence indicates the same (C). In panel B and C, * P < 0.05 vs. PBS group, ** P < 0.05 vs. FSH24 group, n=3 mice, approximately 100 tubules were counted. White bar represents 100 μm in panel B and 200 μm in panel C. Taqman real time qPCR analysis (D) shows that 4 (Cdo1, Clu Tbx22 and Zic3) out of the 9 FSH-responsive genes were upregulated by FSH21/18 compared to FSH24. * P < 0.05 vs. PBS group, ** P < 0.05 vs. the other glycoform. The genes analyzed are listed in the Supplementary Material Table -1. In panel E, testes sections from postnatal day 5 pups from control (Ctrl.) and experimental groups were immunolabeled with antibodies against SOX9, the Sertoli cell lineage - specific marker (green) and BrdU (red). The merged images are shown (E). White bar represents 200 μm. Quantification (F) shows that loss of FSH results in 45 % reduction in number of proliferating Sertoli cells (calculated as % of Sox9+BrdU+ /Sox9+ cells) in Fshb−/− males. FSH21/18 significantly increases the percentage of proliferating Sertoli cells in Fshb−/− males compared to those in FSH24 - injected Fshb−/− males (B). Approximately 250 tubules from multiple testis sections from 3 mice per group were counted. In panel F, * P < 0. 05, vs. 5d Ctrl. group, ** P < 0.05 vs. 10d Fshb−/− group and a P < 0.05 vs. FSH24-injected group.