Transfection of antisense chorionic gonadotropin beta gene into choriocarcinoma cells suppresses the cell proliferation and induces apoptosis.

CONTEXT: Choriocarcinoma cells not only synthesize human chorionic gonadotropin (hCG), but also express LH/CG receptors on the cell membrane. This suggests that the hCG and LH/CG receptors may play a role in regulating the biological function of choriocarcinoma cells in an autocrine/paracrine manner. OBJECTIVE AND METHODS: The objective of this study was to ascertain whether the inhibition of CGbeta gene expression in choriocarcinoma cells affects their proliferation and apoptosis. Expression vector bearing antisense CGbeta gene was transfected into the choriocarcinoma cell line, JAr. CGbeta protein synthesis was monitored by Western immunoblot, and CGbeta mRNA expression was determined by RT-PCR. Cell proliferation was assessed by 3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and nuclear incorporation of 5-bromo-2'-deoxyuridine, and the apoptosis-positive rate was assessed by terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling analysis and nuclear staining with Hoechst 32258. RESULTS: JAr cells transfected with antisense CGbeta gene (JAr-aCGbeta cells) showed a significant decrease in hCG production and cell proliferation compared with untransfected and mock-transfected cells. The apoptosis-positive rate of the JAr-aCGbeta cells significantly increased compared with that of the controls. LH/CG receptor expression in JAr-aCGbeta cells decreased compared with that in controls. By contrast, supplementation of exogenous hCG significantly increased the LH/CG receptor expression and viability of JAr-aCGbeta cells. CONCLUSIONS: These results suggest that hCG, through its binding to the LH/CG receptor, may augment proliferation and inhibit apoptosis in choriocarcinoma JAr cells, and that the introduction of an antisense gene may be a potential approach to the inhibition of choriocarcinoma cell growth.

Abundant expression of sodium-potassium-activated adenosinetriphosphatase alpha 1 subunit in corpus luteum of porcine ovary.

Follicular development is accompanied by the accumulation of follicular fluid. During corpus luteum formation, follicular fluid is diminished and antrum is replaced by lutein cells. These dynamic changes in fluid distribution suggest the existence of control mechanism of fluid transport and membrane permeability. One of the major factors regulating membrane permeability is the sodium-potassium-activated adenosinetriphosphatase (Na(+)-K(+)-ATPase). To elucidate the possible involvement of Na(+)-K(+)-ATPase in follicular growth and luteinization, immunohistochemical localization of Na(+)-K(+)-ATPase alpha1 subunit and enzyme activity in porcine ovary were investigated. In primordial follicles, Na(+)-K(+)-ATPase alpha1 subunit immunostaining was localized only in the oocyte and the surrounding stromal cells. In preantral follicles, immunostaining for Na(+)-K(+)-ATPase alpha1 subunit became apparent in granulosa and theca cells. As the follicle matured, the staining intensity in the oocyte, theca, and granulosa cells increased, which corresponded with the enzyme activity. Na(+)-K(+)-ATPase alpha1 subunit immunostaining became most abundant in granulosa and theca lutein cells in corpus luteum, and decreased in the regressing corpus luteum. Enzyme activity in corpus luteum was significantly higher than that in the follicles. This is the first study indicating that Na(+)-K(+)-ATPase alpha1 subunit expression is augmented in granulosa cells by follicular growth and most abundant in lutein cells in the corpus luteum, suggesting its possible involvement in corpus luteum formation.

Thyroid hormone synergizes with follicle stimulating hormone to inhibit apoptosis in porcine granulosa cells selectively from small follicles.

To investigate the effect of thyroid hormone on the proliferative activity and apoptosis of granulosa cells at the varying stages of follicular growth, porcine granulosa cells obtained from small (1-2 mm), medium (3-5 mm) and large (6-11 mm) follicles were cultured under a serum-free condition in the presence or absence of follicle stimulating hormone (FSH; 20 ng/ml), with or without triiodothyronine (T3; 10-8M). Relative viability, proliferative activity, and apoptosis of cultured granulosa cells were evaluated with 3-(4.5-dimethylahiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assay, Ki67 expression and activated caspase-3 protein expression, respectively. MTT assay showed that T3 had no significant effect on the relative viability of granulosa cells regardless of the follicle size. Ki67-positive rate in small follicle granulosa cells was augmented by treatment with FSH whereas it was not affected by T3. Furthermore, FSH treatment decreased activated caspase-3 protein-positive rate of small follicle granulosa cells. Relative to the treatment with FSH alone, concomitant treatment with FSH and T3 resulted in further decrease in caspase-3 protein-positive rate in small follicle granulosa cells. Treatment with T3 alone did not affect the caspase-3 protein-positive rate. These results suggest that thyroid hormone synergizes with FSH to inhibit apoptosis in small follicle granulosa cells without affecting the proliferative potential of those cells.