PMC

The results indicated that the LH concentrations in the group were not affected by inhibin gene.

The results indicated that the FSH concentration 48-transfection was higher in pshRNA-2 group than those of pshRNA-negative and PBS groups respectively.

The results of cell cycle examination demonstrated that the number of cells in S phase was significantly decreased in pShRNA2 group [p<0.05, compared with pShRNA-negative and PBS groups], indicating S phase arrest.

Three Inhα RNAi recombinant plasmids were transfected in anterior pituitary cells named as pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative, respectively for 24 h, 48 h or 72 h, the best efficient of which was selected for further investigation.

A: Inhibin α-subunit expression in the mouse anterior pituitary gland using immunohistochemical methods. Immunohistochemical analysis of inhibin α subunit expression in the anterior pituitary gland by inhibin monoclonal antibody [A1] and PBS [A2], magnification is ×40. B: Inhibin α subunit expression in the cultured mouse anterior pituitary cells using indirect immunofluorescence. Indirect immunofluorescence analysis of inhibin α subunit expression in the anterior pituitary cells by inhibin monoclonal antibody [B1] and PBS [B2], magnification is ×40.

Transcription levels of INH α gene in the anterior pituitary cells transfected with pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative respectively. The statistical differences were tested using one-way ANOVA. Asterisk [*] means P<0.05, two asterisks means P<0.01. B: INHα subunit expression were detected by western blotting in the anterior pituitary cells after 48 h transfected with pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative respectively.

The levels of INHβB, FSHβB, LHβB, GnRHR and β-glycan were quantified by real-time PCR and western-blotting among pshRNAi-2, pshRNAi-negative and PBS groups, respectively. A: The mRNA levels of INHβB, FSHβB, LHβB, GnRHR and β-glycan were examined by q-PCR. Compared with pshRNA-negative and PBS groups, the mRNA levels of INHβB, FSHβB, LHβB and GnRHR were up-regulated [P>0.05] in APCs transfected with pshRNAi-2. However,mRNA levels of INHβB and GnRHR were significantly up-regulated, whereas mRNA level of β-glycan was significantly down-regulated. B: The proteins levels of FSH, LH, ACVB, INHB, GnRHR and β-glycan were detected by western blotting. The results showed that FSH, LH, ACVB, INHB, GnRHR proteins were up-regulated by inhibin gene silencing. But the protein of β-glycan was down-regulated by inhibin silencing.

A: Detection of the INHB concentration was done by RIA kit. Compared with pshRNAi-negative and PBS groups, the INHB concentrations was decreased significantly at 24 and 48 h after transfection with pshRNAi-2. B: Similarly, compared with pshRNAi-negative and PBS groups, the ACVB concentrations was increased significantly at 24 and 48 hours after transfection with pshRNAi-2.

A: The mRNA levels of apoptosis related genes were examined by q-PCR. The results demonstrated that Caspase-3 and Bcl-2 was significantly decreased in pshRNA2 group 48 h post-transfection [p<0.05, compared with pshRNA-negative and PBS groups] but the levels of Bax and p53 gene was unlatered [p>0.05]. B: The protein levels of apoptosis related genes were detected by western blotting. The results showed that Caspase-3, Bax and Bcl-2 genes was up-regulated by inhibin gene silencing. But p53 gene was not affected by inhibin silencing.