Effects of α-enolase Gene Silencing on Reproductive-related Hormone Receptor Expression and Steroid Hormone Synthesis of Primary Granulosa Cells from Goose F1 Follicles

Abstract

Introduction: Enolases are enzymes in the glycolytic pathway, which catalyse the reversible conversion of D-2-phosphoglycerate into phosphoenol pyruvate in the second half of the pathway. In this research, the effects of α-enolase (ENO1) on steroid reproductive-related hormone receptor expression and on hormone synthesis of primary granulosa cells from goose F1 follicles were studied.

Material and methods: Primary granulosa cells from the F1 follicles of eight healthy 8-month-old Zi geese were separated and cultured. An ENO1 interference expression vector was designed, constructed and transfected into primary cultured granulosa cells. The mRNA expression levels of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LHR), oestrogen receptor α (ER α), oestrogen receptor β (ER β), growth hormone receptor (GHR) and insulin-like growth factor binding protein-1 (IGFBP-1) in the cells were evaluated as were the secretion levels of oestradiol, activin, progesterone, testosterone, inhibin and follistatin in cell supernatant.

Results: α-enolase gene silencing reduced the expression of FSHR, LHR, ERα, ERβ, GHR, and IGFBP-1 mRNA, potentiated the secretion of oestrogen, progesterone, testosterone, and follistatin of granulosa cells, and hampered the production of activin and inhibin.

Conclusion: ENO1 can regulate the reactivity of granulosa cells to reproductive hormones and regulate cell growth and development by adjusting their hormone secretion and reproductive hormone receptor expression. The study provided a better understanding of the functional action of ENO1 in the processes of goose ovary development and egg laying.

Keywords: Zi goose; follicular granulosa cell; hormone; hormone receptor; α-enolase.

Fig. 1

Transfection of shRNA-ENO1 in granulosa cells and detection of ENO1 expression. A – transfection of shRNA-ENO1 in granulosa cells (100 ×) including granulosa cells under ordinary light and fluorescence. B – ENO1 mRNA expression by qRT-PCR in granulosa cells transfected with shRNA-ENO1 after 48 h. ENO1 mRNA expression by qRT-PCR in granulosa cells was measured by qRT-PCR. C – ENO1 expression by Western blot in granulosa cells transfected with shRNA-ENO1 after 48 h. The significance of difference for the ENO1 expression level among the different groups was determined by Student’s t-test. ** P < 0.01, and * P < 0.05 when compared with the control sample

Fig. 2

Quantitative RT-PCR–detected expression of FSHR, LHR, ERα, ERβ, GHR, and IGFBP-1 mRNA in the granulosa transfected with shRNA-ENO1 after 48 h detected by qRT-PCR. A – FSHR, B – LHR, C – GHR, D – IGFBP-1, E – ERα, F – ERβ. The significance of difference for the hormone receptor levels were determined by Student’s t-test. ** p < 0.01, and * p < 0.05 when compared with the control sample

Fig. 3

ELISA-detected concentration of six hormones in the supernatant of granulosa transfected with shRNA-ENO1 after 48 h. A – oestradiol. B – activin. C – progesterone. D – testosterone. E – inhibin. F – follistatin. The significance of difference for the hormone levels was determined by Student’s t-test. ** P < 0.01, and * P < 0.05 when compared with the control sample