Identification Of Different Commercial FSH Glycoforms And Regulatory Effects On Follicular Development

Follicle-Stimulating Hormone(FSH)is a key endocrine regulator during follicle development,and is currently the most commonly used gonadotropin in livestock superovulation and human assisted reproduction.FSH is a glycoprotein hormone,and glycosylation modifications determine its biological activity.Our previous studies have shown that compared to the fully-glycosylated form,hypo-glycosylated FSH can promote the maturation of more follicles in the body and improve the health index of follicles.However,due to the limitations of in vivo models,the specific regulatory roles and differences of FSH glycoforms in different stages of follicular development are not clear.Therefore,some commercial FSH was screened for glycoform identification,and used an in vitro follicle culture system to investigate the effects of different commercial FSH products on the development of preantral follicles and small antral follicles from the perspective of glycoform modification.The main research results are as follows:1.Identification of Different Commercial FSH GlycoformsRecombinant human follicle-stimulating hormone is fully-glycosylated,and pituitary follicle-stimulating hormone is a mixture of fully-glycosylated and hypo-glycosylated,which this study calls mixed-glycosylated2.Study on the Expression Pattern of FSHRThe FSHR expression level of Type 3 and Type 4 follicles was relatively low,and the expression of FSHR in Type 5b follicles was significantly increased.Consequently,type 5b follicles were selected as the pre-antral follicle model to study the effects of FSH glycosylation patterns.3.Effects of FSH Glycosylation Patterns on Pre-antral Follicle Development(1)Different glycosylation patterns of FSH can induce follicle development in vitro:Compared to the control group,both fully-glycosylated and mixed-glycosylated treatments significantly increased follicle diameter growth,growth rate,and antrum formation rate.(2)Mixed-glycosylated FSH significantly promotes the transition from pre-antral to antral follicles: Compared to the fully-glycosylated group,the mixed-glycosylated treatment group exhibited a faster follicle growth rate,with a 110% increase in follicle diameter growth and a 41% increase in antrum formation rate.Further construction of the mixed-glycosylated rescue model revealed no significant differences in diameter growth(121.28 ± 19.10 μm vs 130.53 ± 12.50 μm)and antrum formation rate(73.75 ± 4.73% vs89.38 ± 3.98%)between the rescue group and the mixed-glycosylated group,but they were significantly higher than the fully-glycosylated treatment group.Conversely,using mixedglycosylated FSH initially and then switching to fully-glycosylated FSH resulted in a significant decrease in the antrum formation rate compared to the mixed-glycosylated treatment group(66.25 ± 3.75% vs 89.38 ± 3.98%).Calcein-AM/PI fluorescence labelling showed that the proportion of dead cell area in the mixed-glycosylated treatment group was significantly lower than that in the control group and the fully-glycosylated treatment group.This result showed that mixed-glycosylated FSH enhanced antral follicle conversion rates and improved follicle vitality.(3)Mixed-glycosylated FSH significantly promotes estradiol secretion: The concentration of estradiol in all groups in 4d was significantly higher than that in 2 d,and the concentration of estradiol in the 2 d mixed-glycosylated FSH group was 46.27 times that of the fully-glycosylated group(4245.00 ± 569.70 pg/m L vs 91.73±58.35 pg/m L),the concentration of estradiol in the 4 d mixed-glycosylated FSH group was 4.88 times that of the fully-glycosylated group(6427.00 ± 685.70 pg/m L vs 1318.00 ± 143.90 pg/m L).(4)Mixed-glycosylated FSH significantly stimulates follicle development-related signaling pathways and gene expression: Upon examining the downstream signaling pathways of FSH and the expression of follicle development-related genes,the results showed that,compared to fully-glycosylated FSH,mixed-glycosylated FSH can significantly activate the PKA and PI3K/AKT signaling pathways.Additionally,mixedglycosylated FSH promoted cell proliferation and inhibited cell apoptosis.Compared to the fully-glycosylated treatment group and control group follicles,the expression levels of 3β-HSD and CYP19A1 in the follicles after mixed-glycosylated FSH treatment were significantly upregulated(p < 0.01),and the expression level of AMH was significantly downregulated(p < 0.01).3.Effects of FSH Glycosylation Patterns on the Development of Small Antral Follicles(1)Mixed-glycosylated FSH significantly promotes the growth of small antral follicles: In the culture system,both fully-glycosylated FSH and mixed-glycosylated FSH were added.Compared to the fully-glycosylated group,the mixed-glycosylated treatment group showed greater vitality and faster growth,with an increase of 155% in follicle diameter growth.(2)Mixed-glycosylated FSH significantly promotes steroid hormone secretion and related gene expression: The concentration of estradiol in the mixed-glycosylated FSH group was 2.09 times that of the fully-glycosylated group(3630.00 ± 140.40 pg/m L vs1734.00 ± 25.84 pg/m L),and the concentration of progesterone was 2.83 times that of the fully-glycosylated group(17.00 ± 2.73 pg/m L vs 6.00 ± 0.41 pg/m L).Compared with the control group and the fully-glycosylated group,the expression of 3β-HSD,17β-HSD,and CYP19A1 in the mixed-glycosylated FSH group was significantly upregulated.The results of this study showed that there were differences in the glycoform of different FSH commercial preparations,and different glycoforms could promote the development of pre-antral follicles and small antral follicles,but compared to fullyglycosylated FSH,the mixed-glycosylated FSH exhibits higher biological activity in promoting the conversion efficiency from preantral follicles to antral follicles,and enhancing the growth rate of small antral follicles,hence having better effects.This research elucidates the glycoform differences present in commercial FSH preparations and the differences in their effects on follicular development,providing new insights for further development of efficient commercial FSH preparations and establishing new methods for regulating animal reproduction.