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Role and Gonadotrophic Regulation of X-Linked: RESULTS(2)

RESULTS(2)FSH Increases XIAP Expression in Cultured Ovarian Follicles

To determine if XIAP expression is regulated by the gonadotropin during follicular development and atresia in vitro, changes in XIAP content and apoptosis in sections of cultured follicles were examined by IHC and TUNEL, respectively (Fig. 5A). Whereas follicles cultured in the absence of FSH for 2 and 4 days showed low XIAP immu-noreactivity and detectable apoptotic signal, addition of FSH (100 ng/ml) to the culture medium markedly increased XIAP expression (Fig. 5A) and decreased the apoptotic signal (Fig. 5A). In addition, gonadotropin also significantly increased follicular XIAP content (P < 0.05) (Fig. 6) and suppressed apoptotic DNA fragmentation (P < 0.05) (Fig. 6). Viagra professional Canadian pharmacy

Effects of XIAP Down-Regulation and Overexpression in Cultured Follicles

Follicles injected with adenoviral XIAP antisense cDNA (MOI = 20) and cultured in the presence of FSH (5 ng/ml) exhibited lower XIAP immunointensity compared to gonadotropin-treated follicles injected with adenoviral LacZ (Fig. 5B). The XIAP down-regulated follicles also had a stronger TUNEL-positive signal compared to the LacZ control (Fig. 5B). The XIAP antisense also markedly decreased XIAP contents (P < 0.05) (Fig. 7) and significantly increased DNA fragmentation (P < 0.05) (Fig. 7). In addition, infection of the follicles with XIAP antisense cDNA significantly attenuated the FSH-induced follicular growth as indicated by a marked decrease in DNA content (P < 0.05) and follicular volume (P < 0.05) but was ineffective in the absence of gonadotropin (Fig. 8). Two-way ANOVA shows a significant FSH effect (P < 0.001), antisense effect (P < 0.001), and interaction between these factors (P < 0.001), brought about by the observation that the XlAP antisense was more effective in follicles cultured in the presence than in the absence of gonadotropin.
Fig5Role and Gonadotrophic-5
FIG. 5. Effects of FSH (A) and XIAP gene manipulation (B) on rat follicle cultures showing XIAP content and apoptosis. A) Follicles were cultured in the absence or presence of FSH (100 ng/ml) for 2 or 4 days. B) Follicles were injected with adenoviral LacZ (control, MOI = 20) or full-length XIAP sense (MOI = 3) or antisense (MOI = 20) cDNA, and FSH (5 ng/ml) was added to the cultures 24 h thereafter. The follicles were cultured with gonadotropin for another 3 days, fixed, and paraffin-sectioned. The TUNEL assay (green) and IHC of XIAP (red) were performed as described in Materials and Methods. Different sizes of the labels ”XIAP” and ”TUNEL” signify relative intensities of signals. An image from 16 representative follicles is shown for each treatment group.

Fig6Role and Gonadotrophic-6
FIG. 6. Effects of FSH on rat follicular XIAP content and apoptotic DNA fragmentation. Follicles were cultured in the absence (CTL) or presence of FSH (100 ng/ml) for 4 days. The XIAP and tubulin contents were determined by Western blot analysis, and apoptosis was determined by 3′ end-labeling of DNA fragments. Representative images (A) and quantitative analysis (B) of follicular XIAP contents (normalized against respective tubulin levels and expressed as fold of control) and DNA fragmentation (expressed by the ratio of

Fig7Role and Gonadotrophic-7
FIG. 7. Effects of XIAP gene manipulation on rat follicular XIAP content and apoptotic DNA fragmentation. Follicles were injected with adenoviral LacZ (LacZ; MOI = 20) or full-length XIAP sense (XIAP-S; MOI = 3) or antisense (XIAP-AS; MOI = 20) cDNA, and FSH (5 ng/ml) was added to the cultures 24 h thereafter. The follicles were cultured with gonadotropin for another 3 days. The XIAP and tubulin contents were determined by Western blot analysis, and apoptosis was determined by 3′ end-labeling of DNA fragments. Representative images (A) and quantitative analysis (B) of follicular XIAP contents (normalized against respective tubulin levels and expressed as fold of control) and DNA fragmentation (expressed by the ratio of