ABSTRACT
<p><b>OBJECTIVE</b>To explore the role of c-src on the initiation of primordial follicles.</p><p><b>METHODS</b>2-days-old female SD rats' ovaries were cultured in Waymouth culture system and were used HE staining and immunohistochemy to observe the number of follicles after 0, 4, 8 days cultured. Use chemically synthesized small interference RNA (siRNA) transfected into ovarian tissue in cultured for RNA interference, and use HE staining and RT-PCR to detect the best siRNA and packaging it by lentiviruses to test the interference effect.</p><p><b>RESULTS</b>With the increase of culturing days, the nummber of the primordial follicles in ovarian gradually reduced. We packed the best siRNA by lentiviruses to doing RNA interference and found comparing with the blank control group and blank vector group, c-src mRNA of the best interference group were significantly decreased. The total number of primordial follicles was relatively greater and the development of primordial folliculars was inhibited.</p><p><b>CONCLUSION</b>c-src plays an important role in primordial follicle development and folliculogenesis initiation.</p>
Subject(s)
Animals , Female , Rats , Animals, Newborn , Base Sequence , Culture Techniques , Molecular Sequence Data , Ovarian Follicle , Metabolism , Ovary , Metabolism , Proto-Oncogene Proteins pp60(c-src) , Genetics , Physiology , RNA Interference , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Rats, Sprague-Dawley , TransfectionABSTRACT
Our previous studies showed that the proto-oncogene c-erbB₂ played an important role in primordial follicles growth. The present study was conducted to investigate the role of MAPK and PKC signaling pathways in the primordial follicle onset in neonatal rats, and the relationship between c-erbB₂ and MAPK/PKC signaling pathways. Ovaries collected from 2-day-old Sprague-Dawley rats were cultured in the Waymouth culture system in vitro. Ovaries were transfected with c-erbB₂ siRNA, or treated with PD98059 (50 mumol/L) or Calphostin (0.5 mumol/L) in the culture medium. RT-PCR was performed to measure the expression of c-erbB₂ mRNA, and Western blot analysis was performed to measure the expression of ErbB₂, MAPK and PKC protein after the neonatal rat ovaries were cultured for 8 d. The quantities of every-stage follicles of ovaries cultured for 8 d were obtained in histological section stained with hematoxylin eosin. The results showed that c-erbB₂ siRNA reduced the levels of c-erbB₂ mRNA (P<0.01) and the levels of ErbB₂, MAPK and PKC protein (P<0.01) significantly. But the levels of c-erbB₂ mRNA and ErbB₂ protein exhibited no change (P>0.05) in the ovaries cultured with PD98059 or Calphostin. After the ovaries were transfected with c-erbB₂ siRNA or cultured with PD98059 or Calphostin for 8 d, the quantities of primary follicles and second follicles were lower than those in the control group (P<0.05 or P<0.01), but the quantity of the primordial follicles was higher than that in the control group (P<0.01). These results suggest that proto-oncogene c-erbB₂ promotes the initiation of primordial follicle growth through the MAPK and PKC signal transduction, and c-erbB₂ is possibly the upstream of PKC and MAPK signaling pathway in the regulation of primordial follicle onset.