RESUMO
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.
Assuntos
Proliferação de Células , Genes erbB-2/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Folículo Ovariano/fisiologia , Proteína Quinase C/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Genes erbB-2/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
<p><b>OBJECTIVE</b>To study the expression and possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles.</p><p><b>METHODS</b>Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth culture system. In-situ hybridization, RT-PCR and immunohistochemistry were performed to assess the expressions of c-erbB2 mRNA and protein during the initiation growth of primordial follicles and after the effect of EGF. Western blot was used to observe the PCNA, p-ERK1/2 contents and correlation analysis was used to study the correlation relationship between contents of p-ERK1/2 and expressions of c-erbB2 mRNA at the same time of the primordial follicles growth.</p><p><b>RESULTS</b>PCNA protein levels appeared to be more intense during the initiation growth of primordial follicles, EGF could promote the proliferation and differentiation of the primordial follicles. c-erbB2 mRNA existed in the oocytes endochylema and ErbB2 existed in the oocytes membrane, the expressions of c-erbB2 mRNA and ErbB2 appeared to be more intense when primordial follicles were cultured for 8 d than cultured for 0 d in the Waymouth culture system and were further increased with 50 ng/ml EGF for 4 d and 8 d. The same results were observed by RT-PCR, too. p-ERK1/2 protein levels were consistent with the changes of c-erbB2 mRNA and protein. Furthermore, Spearman rank correlation analysis showed there was a significant positive correlation relationship between the changes of p-ERK1/2 and the changes of c-erbB2 mRNA during the primordial follicles growth and after the effect of EGF (rs = 0.900, P < 0.05).</p><p><b>CONCLUSION</b>It was suggested that proto-oncogene c-erbB2 may be play an important role during the initiation growth of primordial follicles with EGF, and it is indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.</p>
Assuntos
Animais , Feminino , Ratos , Animais Recém-Nascidos , Fator de Crescimento Epidérmico , Farmacologia , MAP Quinases Reguladas por Sinal Extracelular , Metabolismo , Técnicas de Cultura de Órgãos , Folículo Ovariano , Ovário , RNA Mensageiro , Genética , Metabolismo , Ratos Sprague-Dawley , Receptor ErbB-2 , Genética , Metabolismo , Transdução de SinaisRESUMO
Little is known about the factors that control the initiation of growth of primordial follicles. The objective of the present study was to investigate the effect of c-erbB₂ on the onset of primordial follicle development, and whether c-erbB₂ mediates the effect of epidermal growth factor (EGF) in this process. We synthesized three pairs of siRNAs targeting the c-erbB₂ mRNA and transferred them into the newborn rat ovary cultured in vitro with Metafectene. After siRNAs transfection, the efficiency of siRNAs was tested by examining c-erbB₂ mRNA and protein levels. The level of c-erbB₂ mRNA was reduced by 49.6%, 46.7% and 82.6% respectively after transfecting siRNA1, siRNA2 and siRNA3, and the level of ErbB₂ protein also reduced remarkably after siRNA3 transfection. c-erbB₂ siRNA3 significantly inhibited the primordial follicle initiation and development; EGF augmented primordial follicles formation, but the effect was abolished by c-erbB₂ siRNA3. All of these results suggest that c-erbB₂ plays an important role in primordial follicle development and folliculogenesis initiation, and mediates the effect of EGF on primordial follicle development.
