Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: enhanced transcription and PI3K and MAPK signaling

Abstract

Study question: Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)?

Summary answer: Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling.

What is known already: Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells.

Study design, size, duration: This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways.

Participants/materials, setting, methods: The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways.

Main results and the role of chance: Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK.

Large scale data: RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author.

Limitations, reasons for caution: The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries.

Wider implications of the findings: Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART.

Study funding/competing interest(s): This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.

Keywords: FSH; assisted reproduction; cell signaling; fertility; follicle development; glycosylation; gonadotropin action; granulosa cell; ovary; transcription.

Figure 1.

The effects of hypo- and fully-glycosylated human recombinant FSH (hFSH) on follicular development in vivo. (a) Automated western blot of recombinant hFSH preparations. The primary FSHβ antibody was 7-13.B5 diluted 1:5000. Lanes 1 and 2, 100 ng pituitary FSH AFP7298A; lanes 3 and 4, 100 ng Gonal-f^® (FSH used in clinical ART); lanes 5 and 6, 100 ng hFSH18/21; lanes 7 and 8, 100 ng hFSH24. (b) Total growing follicles, including pre-antral follicles, and antral follicles, 48 h after treatment with hFSH18/21 and hFSH24. (c) Numbers of antral follicles per ovary in control, hFSH18/21 and hFSH24 treatment groups. (d) Number of large antral follicles per ovary in control, hFSH18/21 and hFSH24 treatment groups. (e) Serum estradiol concentrations (pg/ml) in different treatment groups. (f) Serum anti-Müllerian hormone (AMH) concentrations (ng/ml) in different treatment groups. (g–i) Total RNA was extracted for qRT-PCR in Control, hFSH18/21, and hFSH24 treatment groups. (g) Ovarian transcripts for Bax. (h) Ovarian transcripts for Bcl2. (i) Bax versus Bcl2 gene expression ratio. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2.

Apoptosis-related gene and protein expression in mouse ovaries. Mouse ovaries were isolated 48 h after PBS, hFSH18/21 or hFSH24 treatments. Total RNA and protein were extracted for qRT-PCR and western blot analysis. (a) Representative images of ovarian protein expression levels of Bcl2, Bax and Cyclin D detected by western blot. (b) Quantification data showing the Bax versus Bcl2 protein ratio. (c) Ovarian mRNA levels of Ccnd in Control, hFSH18/21 and hFSH24 treatment groups. (d) Quantification data showing the Cyclin D protein expression level. (e) Immunofluorescence staining of the proliferation marker Ki67 in mouse ovaries isolated 48 h after PBS (Ctrl), FSH18/21 and FSH24 treatment. Neg: Primary antibody negative control. (f) Quantification of Ki67 immunosignal intensity (n = 6 ovaries). (g) Immunofluorescence staining showing the apoptosis marker cleaved-Caspase 3 (Clv-Cas 3) in the ovaries. (h) Quantification of Clv-Cas 3 foci number (n = 6–11 ovaries). Scale bar: 200 μm. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 3.

Bioinformatics analysis of early ovarian response genes induced by hypo-glycosylated hFSH18/21 and fully glycosylated hFSH24. Mice were treated for 2 h with Control (PBS), hypo-glycosylated FSH (hFSH18/21) or fully-glycosylated FSH (hFSH24). Ovaries were isolated and processed for RNA-sequencing. (a) Principal component analysis (PCA) of the RNA-sequencing data of ovaries. (b) Upregulated and downregulated genes in response to FSH18/21 versus Control, FSH24 versus Control and FSH18/21 versus FSH24. (c) Venn diagram showing the number of common or differentially expressed ovarian genes between FSH18/21 versus Control, FSH24 versus Control and FSH18/21 versus FSH24. (d) Analysis of top 5 molecular and cellular functions determined by Ingenuity pathway analysis (IPA) for FSH18/21 versus Control treatment (top) and FSH24 versus Control treatment (bottom) groups. Red text indicates the unique pathways in each group. (e) Top 5 molecular and cellular functions determined by IPA between the FSH18/21 versus FSH24 treatment groups. (f) Top 5 canonical pathways (absolute Z score > 3.0, -log P-value < 1.3, exclude Degradation/Utilization/Assimilation and Disease Specific Pathways) between FSH18/21 versus FSH24 treatment. Red color indicates upregulated genes in each pathway; white color indicates no overlap with dataset. (g) Overlay of protein coding DEGs of FSH18/21 versus FSH24 treatment on the FSH pathway using the Path Explorer tool in IPA.

Figure 4.

Differential effects of hFSH18/21 and hFSH24 glycoforms on expression of transcription factors. (a) Heatmap of transcripts for 34 differentially expressed transcription factors. (b–e) Relative expression of groups of transcription factors following treatment with FSH18/21 or FSH24 compared to control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5.

Screening of hypo- and fully-glycosylated human recombinant FSH (hFSH) crosstalk with receptor tyrosine kinase (RTK) signaling and downstream signaling pathways. PND 17 mice (n = 5) were treated i.p. with PBS (Ctrl), or 1 µg of either FSH18/21 or FSH24 for 2 hrs. Ovaries were isolated and total proteins were prepared. (a) Total protein pool from each group (n = 5) was used to perform the RTK assay. Selected image of Mouse RTK array showing the differentially activated EGFR signaling responding to the different FSH glycoform treatments (upper panel). Quantification data showing the relative phosphorylation levels of various EGFR family members (lower panel). (b) Selected image of the RTK array (upper panel) and quantification data (lower panel) showing the differential activation levels of the FGFR family. The immunosignal intensity was normalized to the control spot on each array. Each bar represents the mean immunosignal intensity ± SEM of duplicates of each RTK. (c) Protein extracts from one ovary from each mouse was prepared for Western blot analysis. The image shows the activation of RTKs and downstream pathways. (d) Human granulosa cells were treated with increasing concentrations of either FSH18/21 or FSH24 for 30 min. Western blot image showing the activation of pathways in human primary granulosa cells. Forskolin (FSK, 10 µM) was used as positive control.

Figure 6.

The effect of PI3K and MEK inhibitors on FSH glycoform-dependent regulation of apoptotic and proliferative gene expression in ovarian wedge sections. Ovary sections were pre-treated with either media containing vehicle (Control), 10 μM U0126 (MEK inhibitor) (a–c) or 100 nM Wortmannin (Wort, PI3K inhibitor) (d–f) for 60 min. Following pre-treatment, ovarian sections were treated with media alone (Control), 30 ng/ml FSH18/21 or FSH24. Ovary wedge sections were snap-frozen and analyzed by quantitative RT-PCR. (a and d) Proliferating cell nuclear antigen (Pcna) gene expression is increased in ovary wedge sections treated with FSH18/21. (b and e) Bcl2/Bax ratio of ovary wedge sections, a high Bcl2 to Bax ratio is indicative of follicle health as seen in the FSH18/21 treatment group. (c and f) Apoptosis related expression of Caspase3 transcripts is increased following treatment with Wortmannin and U0126. Ovarian sections treated with FSH18/21 expressed reduced levels of Caspase 3 transcripts compared to sections treated with FSH24. Data was analyzed using one-way ANOVA with Dunnett’s post hoc test. Bars represent means ± SEM for six individual ovarian wedge sections. Bars marked ^a indicate a significant difference from Control (open bar). Comparisons among indicated treatments, *P < 0.05, **P < 0.01, ***P < 0.001.