Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: Enhanced transcription and PI3K and MAPK signaling
Hua, Guohua George, Jitu W. Clark, Kendra L. Jonas, Kim C. Johnson, Gillian P. Southekal, Siddesh Guda, Chittibabu Hou, Xiaoying Blum, Haley R. Eudy, James
Hua, Guohua
George, Jitu W.
Clark, Kendra L.
Jonas, Kim C.
Johnson, Gillian P.
Southekal, Siddesh
Guda, Chittibabu
Hou, Xiaoying
Blum, Haley R.
Eudy, James
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Issue Date
2021-06
Type
Article
Genre
Keywords
Fertility,FSH,Gonadotropin action,Ovary,Follicle development,Assisted reproduction,Transcription,Granulosa cell,Cell signaling,Glycosylation
Subjects (LCSH)
Citation
Hua, G., George, J. W., Clark, K. L., Jonas, K. C., Johnson, G. P., Southekal, S., . . . Davis, J. S. (2021). Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: Enhanced transcription and PI3K and MAPK signaling. Human Reproduction (Oxford, England), 36(7), 1891-1906. doi:10.1093/humrep/deab135
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.
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Publisher
Oxford University Press
Journal
Book Title
Series
Human Reproduction;Vol. 36, Iss. 7
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PubMed ID
DOI
ISSN
0268-1161
1460-2350
1460-2350