Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways

Abstract

Background: c-erbB2, a proto-oncogene coding epidermal growth factor receptor-like receptor, also as a chemosensitivity/prognosis marker for gynecologic cancer, may be involved in initiation of growth of rat primordial follicles. The aim of the present study is to investigate the role and signal pathway of c-erbB2 in onset of rat primordial follicle development.

Methods: The expression of c-erbB2 mRNA and protein in neonatal ovaries cultured 4 and 8 days with/without epidermal growth factor (EGF) were examined by in situ hybridization, RT-PCR and western blot. The function of c-erbB2 in the primordial folliculogenesis was abolished by small interfering RNA transfection. Furthermore, MAPK inhibitor PD98059 and PKC inhibitor calphostin were used to explore the possible signaling pathway of c-erbB2 in primordial folliculogenesis.

Results: The results showed that c-erbB2 mRNA was expressed in ooplasm and the expression of c-erbB2 decreased after transfection with c-erbB2 siRNA. Treatment with EGF at 50 ng/ml significantly increased c-erbB2 expression and primary and secondary follicle formation in ovaries. However, this augmenting effect was remarkably inhibited by c-erbB2 siRNA transfection. Furthermore, folliculogenesis offset was blocked by calphostin (5 x 10(-4) mmol/L) and PD98059 (5 x 10(-2) mmol/L), but both did not down-regulate c-erbB2 expression. In contrast, the expressions of p-ERK and p-PKC were decreased obviously by c-erbB2 siRNA transfection.

Conclusions: c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways, suggesting an important role of c-erbB2 in rat primordial follicle initiation and development.

Figure 1

In situ hybridization detection of c-erbB₂ mRNA in the ovaries. (A), Control (ovary of 2-day-old rat); (B), Ovary of 2-day-old rat cultured for 4 days; (C), Ovary of 2-day-old rat cultured for 8 days; (D), Ovary of 2-day-old rat cultured for 4 days with 50 ng/ml EGF; (E), Ovary of 2-day-old rat cultured for 8 days with 50 ng/ml EGF; (F), Negative control. Scale bar: 2.5 × 10^-2 mm.

Figure 2

RT-PCR detection of c-erbB₂ mRNA in the ovaries. (A), The expressions of c-erbB₂ mRNA (322 bp) by RT-PCR; (B), Semiquantitative analysis of the RT-PCR result; (1), Control (ovary of 2-day-old rat); (2), Ovary of 2-day-old rat cultured for 4 days; (3), Ovary of 2-day-old rat cultured for 8 days; (4), Ovary of 2-day-old rat cultured for 4 days with 50 ng/ml EGF; (5), Ovary of 2-day-old rat cultured for 8 days with 50 ng/ml EGF. Data are presented as means ± SEM (n = 3). *, P < 0.05; **, P < 0.01 vs control group; Δ, P < 0.05 vs group 3.

Figure 3

Western blotting detection of PCNA protein in the ovaries. (A), The expressions of. PCNA protein by western blot; (B), Semiquantitative analysis of the western-blot result. (1), Control (ovary of 2-day-old rat); (2), Ovary of 2-day-old rat cultured for 4 days; (3), Ovary of 2-day-old rat cultured for 8 days; (4), Ovary of 2-day-old rat cultured for 4 days with 50 ng/ml EGF; (5), Ovary of 2-day-old rat cultured for 8 days with 50 ng/ml EGF. Data are presented as means ± SEM (n = 3). **, P < 0.01 vs control group; Δ, P < 0.05 vs group 3.

Figure 4

Effect of c-erbB₂ siRNA on c-erbB₂ mRNA expression. (A), Expression of c-erbB₂ mRNA (322 bp) measured by RT-PCR in cultured ovaries after c-erbB₂ siRNA transfection. (B), Semiquantitative analysis of the RT-PCR results. Control is the group without transfection; Negetive control is the group transfected with negative control siRNA. Data are presented as means ± SEM (n = 3). **, P < 0.01.

Figure 5

Effect of c-erbB₂ siRNA on ErbB₂ protein expression. (A), Expression of c-erbB₂ protein in cultured ovaries after siRNA transfection by Western blot. (B), Semiquantitative analysis of the western-blot result. Control is the group without transfection; Negetive control is the group transfected with negative control siRNA. Data are presented as means ± SEM (n = 3). **, P < 0.01 vs control group.

Figure 6

Effect of c-erbB₂ siRNA on the initiation of primordial follicle growth. (A), Ovaries were cultured for 8 days with treatments: A, control; B, 0.1 mmol/L siRNA targeted c-erbB₂; C, 50 ng/ml EGF; D, 0.1 mmol/L siRNA+50 ng/ml EGF. (B), The number of follicles was counted in serial cross-sections. Percentage of the number of each category over the total number was plotted. Data are presented as means ± SEM (n = 5). Bars with different superscripts are statistically different (P < 0.01).

Figure 7

Effect of c-erbB₂ siRNA on the expressions of ErbB₂, p-ERK and p-PKC protein. (A), The expressions of ErbB₂, p-ERK and p-PKC protein in the ovaries by western blot; (B), Semiquantitative analysis of the western-blot result. Control is the group without transfection; Negetive control is the group transfected with negative control siRNA. Data are presented as means ± SEM (n = 3). **, P < 0.01 vs control group; ΔΔs, P < 0.01 vs negative control.

Figure 8

Effect of MAPK inhibitor PD98059 and PKC inhibitor calphostin on the expression of c-erbB₂ mRNA. (A), The expression of c-erbB₂ mRNA (322 bp) in the ovaries by RT-PCR; (B), Semiquantitative analysis of the RT-PCR results. Data are presented as means ± SEM (n = 3).

Figure 9

Effect of c-erbB₂ siRNA, PD98059 and calphostin on the initiation of primordial follicle growth. (A), Ovaries were cultured for 8 days with treatments: A, control; B, 0.1 mmol/L siRNA; C, 5 × 10^-2 mmol/L PD98059; D, 5 × 10^-4 mmol/L calphostin. (B), The number of follicles was counted in serial cross-sections. Percentage of the number of each category over the total number was plotted. Data are presented as means ± SEM (n = 5) **, P < 0.01 vs control.

Figure 10

EGF signal transduction figure.