RNA interference mediated pten knock-down inhibit the formation of polycystic ovary

Abstract

Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

Fig. 1

Lentiviral vectors for pten shRNA

Fig. 2

Appearances of ovaries in the control groups and the model group. a, b Indicate external ovarian appearance and tissue morphological appearances (HE ×40), respectively. A day 0 control, B day 12 control, C model. Black arrow primordial follicle, red arrow preantral follicle, yellow arrow antral follicle, blue arrow cystic follicle

Fig. 3

Immunohistochemical results of PTEN protein expression in the ovaries of the control and model groups (×40). A–C Indicate primordial follicles, preantral follicles, and antral follicles, respectively

Fig. 4

Expressions of PTEN in the ovaries of the control and model groups. a, b Indicate the pten mRNA expression revealed by RT-PCR ($\overline{x}$ ± s, n = 3) and Expressions of PTEN protein by western-blot, respectively. 1, 2, and 3 represent day 0 control, model, and day 12 control, respectively

Fig. 5

Changes of pten mRNA levels in ovaries after pten-shRNA1, pten-shRNA2, and pten-shRNA3 interference by RT-PCR. M marker, C control, 1 pten-shRNA1, 2 pten-shRNA2, 3 pten-shRNA3

Fig. 6

Changes of polycystic ovarian morphology after pten shRNA lentivirus transfection. A–C showed the model control group, the empty vector + model group and the pten shRNA + model group, respectively

Fig. 7

Expressions of PTEN after transfecting the pten shRNA lentivirus into the ovaries of the control and model groups. 1, 2, and 3 represent day 0 control, model, and day 12 control, respectively. a Expression of GFP in ovaries after pten shRNA lentivirus transfection (×40). b Expressions of pten mRNA revealed by RT-PCR. c PTEN protein expression revealed by western-blot ($\overline{x}$ ± s, n = 3)

Fig. 8

Morphological observations of ovarian tissue after pten shRNA lentivirus transfection (HE ×40). Note: 1, 2 and 3 referred to the model control group, the empty vector + model group and the pten shRNA + model group, respectively