Evaluation of in vivo bioactivities of recombinant hypo- (FSH 21/18 ) and fully- (FSH 24 ) 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 hypoglycosylated 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 48h 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.

FSH for the Treatment of Male Infertility

“…Interestingly, different data emerged from male FSHB null mice, where hFSH 21 /hFSH 18 promoted the increase of testicular weight more efficiently than the fully glycosylated FSH 24 , correlating with testis tubule size and the number of germ cells per tubule. In Sertoli cells of these animals, less glycosylated FSH promoted significantly higher expression levels of target genes and activation of proliferation markers than hFSH 24 [65]. Taken together, these data are suggestive of a relevant physiological impact of FSH glycoforms, even though this impact is likely to be sex-specific.…”

“…However, in vivo studies have only partially confirmed what was found in vitro; female FSHβ-encoding gene (FSHB) null mice injected with different doses of hFSH 21 /hFSH 18 or hFSH 24 had higher estradiol production with less glycosylated forms. These two glycoforms also impact on specific subsets of FSH-responsive genes, but no different activation of PKA, CREB, p38 MAPK, ERK and AKT were found, as well as ovarian weight gain, between less and fully glycosylated FSH [65,66]. These experiments suggest that the biased activity of FSH variants observed in vitro is likely attenuated in vivo.…”

In Vivo and In Vitro Impact of Carbohydrate Variation on Human Follicle-Stimulating Hormone Function

“…with different doses of FSH glycoforms separately and at different times postinjection, ovaries were collected for subsequent selected gene expression analysis by quantitative real-time PCR. In these studies, hypo-glycosylated FSH 21/18 elicited in vivo bioactivity comparable to that of FSH 24 ; however, these analyses also indicated that differences exist between FSH 21/18 and FSH 24 glycoforms in inducing a unique subset of FSH-responsive genes ( 32 ). Second, to assess the upstream signaling pathways which control FSH-induced gene expression, immunofluorescence analysis was performed on ovarian sections obtained from Fshb null female mice injected with FSH 21/18 and FSH 24 glycoforms using p-CREB and p-PKA substrate antibodies.…”

“…This led to the concern that we were studying a physiologically irrelevant glycoform. However, subsequent demonstration of significant biological activity differences between other pituitary and recombinant FSH glycoform preparations eliminated this concern ( 18 , 31 , 32 ).…”

“…Second, to assess the upstream signaling pathways which control FSH-induced gene expression, immunofluorescence analysis was performed on ovarian sections obtained from Fshb null female mice injected with FSH 21/18 and FSH 24 glycoforms using p-CREB and p-PKA substrate antibodies. At three different time points tested (0.5, 1, and 2 h), both glycoforms were equally effective and significantly upregulated p-PKA and p-PKA substrates (nuclear accumulation in granulosa cells) over PBS-injected controls, with maximal induction observed at the 1-h time point ( 32 ).…”