Bioactivity of recombinant hFSH glycosylation variants in primary cultures of porcine granulosa cells

Abstract

Previous studies have reported hypo-glycosylated FSH and fully-glycosylated FSH to be naturally occurring in humans, and these glycoforms exist in changing ratios over a woman's lifespan. The precise cellular and molecular effects of recombinant human FSH (hFSH) glycoforms, FSH21 and FSH24, have not been documented in primary granulosa cells. Herein, biological responses to FSH21 and FSH24 were compared in primary porcine granulosa cells. Hypo-glycosylated hFSH21 was significantly more effective than fully-glycosylated hFSH24 at stimulating cAMP accumulation and protein kinase A (PKA) activity, leading to the higher phosphorylation of CREB and β-Catenin. Compared to fully-glycosylated hFSH24, hypo-glycosylated hFSH21 also induced greater levels of transcripts for HSD3B, STAR and INHA, and higher progesterone production. Our results demonstrate that hypo-glycosylated hFSH21 exerts more robust activation of intracellular signals associated with steroidogenesis than fully-glycosylated hFSH24 in primary porcine granulosa cells, and furthers our understanding of the differing bioactivities of FSH glycoforms in the ovary.

Keywords: Cell signaling; Follicle; Ovary; Protein kinase A; Steroidogenesis; β-Catenin.

Figure 1. (single column). Effect of FSH glycoform preparations on cAMP accumulation and PKA activation in porcine granulosa cells.

Granulosa cells were treated with increasing concentrations of hFSH21 or hFSH24 for 30 minutes. A, cAMP levels in the medium were measured by a cAMP chemiluminescent immunoassay kit as described in the Methods. B, representative Western blot result of phosphorylation of PKA-substrates after hFSH glycoform treatment. Actin was used as an internal control. C, fold increases in the phospho-PKA-substrates/Actin ratio. Results are presented as means ± SEM; n = 3 separate experiments. #P < 0.05 and ##P < 0.01 compared with respective control; *P < 0.05 and **P < 0.01 compared with the other glycoform at the same concentration.

Figure 2. (Single column). Effect of FSH glycoform preparations on CREB phosphorylation in porcine granulosa cells.

Granulosa cells were treated for 30 minutes with increasing concentrations of hFSH21 or hFSH24. Cell lysates were prepared and Western blot analysis was performed using phospho-Ser133-CREB (P-CREB) antibody, and β-actin antibodies. A, Fold increases in the phospho-CREB/Actin ratio; results are presented as means ± SEM; n = 3 separate experiments. #P < 0.05 and ##P < 0.01 compared with respective control; *P < 0.05 and **P < 0.01 compared with the other glycoform at the same concentration. B, representative Western blot analysis of CREB phosphorylation after hFSH glycoform treatment. Actin was used as an internal control.

Figure 3. (Single column). Effects of hFSH glycoform preparations on the number and intensity of nuclear phospho-CREB staining in porcine granulosa cells.

Granulosa cells were treated for 30 minutes with increasing concentrations of hFSH21 or hFSH24. A, the number of phospho-CREB-positive nuclei expressed as a percent of the total cells counted in each experiment. B, the mean fluorescence intensity of phospho-CREB in cell nuclei. *P < 0.05 and **P < 0.01 compared with the other glycoform at the same concentration.

Figure 4. (Single column). Effects of hFSH glycoform preparations at stimulating CRE-mediated transcriptional activity.

Granulosa cells were infected with an adenovirus expressing a CRE luciferase reporter (CRE-Luc) or pRL-Luc as an internal control. After 24 hours, granulosa cells were treated for 6 hours with either hFSH21or hFSH24 ranging from 0–100 ng/mL. Relative CRE-luciferase activity (rlu) was determined as described in the Methods. Bars represent means± SEM; n = 4 separate experiments. #P < 0.05 and ##P < 0.01 compared with control; *P < 0.05 and **P < 0.01 compared with the other glycoform at the same concentration.

Figure 5. (Single column). Effect of FSH glycoform preparations on β-catenin phosphorylation in porcine granulosa cells.

Cells were treated with increasing concentrations of hFSH21 or hFSH24 for 30 min. Cell lysates were prepared for Western blot analysis using phospho-Ser552 and phospho-Ser675-β-catenin (P-β-catenin) antibodies, and actin antibodies. A, fold changes in the phospho-β-catenin Ser552/Actin ratio; B, fold changes in the phospho-β-catenin Ser675/Actin ratio; C, representative Western blot analysis of phosphorylation of β-catenin after FSH glycoform treatment. Actin was used as an internal control. Data are expressed as means ± SEM; n = 3 separate experiments. #P < 0.05 and ##P < 0.01 compared with respective control; *P < 0.05 and **P < 0.01 compared with the other glycoform at the same concentration.

Figure 6. (1.5 column). Relative expression levels of steroidogenic-related genes after hFSH glycoform preparation treatment in porcine granulosa cells.

Granulosa cells were treated with hFSH21 or hFSH24 for 48 h. Real-time, quantitative PCR was performed. The results were evaluated as the relative ratio of the expression level of each mRNA to that of GAPDH. Data are expressed as means ± SEM; #P < 0.05 and ##P < 0.01 compared with control; *P < 0.05 and **P < 0.01 between the indicated experimental condition.

Figure 7. (Single column). Relative expression levels of steroidogenic-related genes and differentiation-related genes after forskolin treatment in porcine granulosa cells.

Granulosa cells were treated with 10 μM forskolin for 48 h. Real-time, quantitative PCR was performed. The results are expressed as the relative ratio of the expression level of each mRNA to that of GAPDH. Data are expressed as means ± SEM, n = 3 or 4 separate experiments; *P < 0.05 and **P < 0.01 compared with respective control.

Figure 8. (1.5 column). Relative expression levels of differentiation-related genes after hFSH glycoform treatment in porcine granulosa cells.

Granulosa cells were treated with hFSH21 or hFSH24 for 48 h. Real-time, quantitative PCR was performed. The expression level of each mRNA was normalized to that of GAPDH. Data are expressed as means ± SEM, n = 3 or 4 separate experiments; #P < 0.05 and ##P < 0.01 compared with control; *P < 0.05 and **P < 0.01 between the indicated experimental condition.

Figure 9. (Single column). Progesterone and estradiol synthesis in porcine granulosa cells.

Granulosa cells were treated with increasing concentrations of hFSH21 or hFSH24 for 48 h. A, Cell lysates were prepared and Western blot analysis of HSD3B1 protein was performed, with actin as an internal control. B and C, progesterone and estradiol secretion in the culture medium, respectively. D, Cell viability assay (MTT). Data are expressed as means ± SEM; n = 3 separate experiments. #P < 0.05 and ##P < 0.01 compared with control; *P < 0.05 and **P < 0.01 compared with the other glycoform at the same concentration.