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.

Follicle-Stimulating Hormone Glycosylation Variants Distinctly Modulate Pre-antral Follicle Growth and Survival

“…As FSH24 increases with age, with potential to promote follicle atresia, it may in part contribute to the accelerated follicle depletion observed with aging ( 51 , 52 ). Furthermore, the differences in FSH target gene expression (steroidogenic enzymes) are in concordance with previous in vitro studies in cultured human GCs ( 53 ) and in vivo studies ( 41 , 43 ).…”

“…Previous studies investigating the effects of partially glycosylated FSH21/18 and fully glycosylated FSH24 glycoform preparations have predominately utilized acute measurements in heterologous cell lines expressing FSHR, isolated GCs, and in vivo and ex vivo ovaries ( 43 , 41 ) This study provides important next steps in understanding the effects of FSH glycoforms on longer-term pre-antral follicle cultures, analyzing the effects of FSH glycoforms when follicles become sensitive to FSH. Moreover, this study presents the first ratiometric analysis of FSH21/18:FSH24, mimicking the reported aging-dependent changes and showing titratable effectiveness of FSH21/18 in promoting follicle growth and survival with increasing in FSH24.…”

“…Importantly, 2 of the main signaling pathways activated by FSH via PKA are the PI3K and ERK pathways ( 28 ). Moreover, the canonical PKA-dependent pathway and MAPK pathways are both required for full FSH biological activity, while the PI3K pathway plays a pivotal role in FSH-induced activity including gene transcription, proliferation, and protein translation ( 41 ). Herein, we highlight, using pharmacological inhibition of these pathways, the loss of FSH21/18 induced proliferation in the presence wortmannin (PI3K inhibitor) and U0126 (MAPK inhibitor).…”

Differential FSH Glycosylation Modulates FSHR Oligomerization and Subsequent cAMP Signaling

“…Functional analysis has shown that this difference in glycosylation pattern results in modulation of binding to the FSHR (14) and the amplitude of the canonical FSHR signaling pathway, the Gas/cAMP/PKA signaling, with FSH21/18 displaying faster binding kinetics and more potent activation of Gas signaling (15)(16)(17). Recent studies suggest that FSH glycoforms may display distinct signaling profiles, or signal bias (18) in activation of AKT (19,20), b-arrestin (17) and calcium signaling (17,21). Important functional differences have also been observed, with differences in ovarian and testicular gene expression in mice injected with either FSH21/18 or FSH24 (19).…”

Follicle-Stimulating Hormone Induces Lipid Droplets via Gαi/o and β-Arrestin in an Endometrial Cancer Cell Line

“…This complexity in signal profiles offers the ability of GPCRs such as FSHR to exhibit bias in their signaling, where one, or more, pathways, may be preferentially activated over others. Such bias has been well documented and may be both ligand and cell/ system specific (30)(31)(32)(33)(34). The pluri-dimensional signaling capacity of FSHR offers a mechanism underlying the multiple and dynamic functions of FSH/FSHR in target cells including proliferation, steroidogenesis, apoptosis and differentiation (31,(35)(36)(37).…”