On the other hand, follicles injected with XIAP sense and subsequently cultured in the low concentration of FSH (5 ng/ml) showed high XIAP immunointensity and less-intense TUNEL signal compared to adenoviral LacZ-in-jected follicles (Fig. 5B). Although a range in the intensity in both XIAP and TUNEL signals was observed within each experimental group, the differences between groups (LacZ vs. XIAP sense vs. XIAP antisense) were obvious. Similarly, XIAP sense infection significantly increased XIAP protein content (P < 0.01) (Fig. 7) and suppressed apoptotic DNA fragmentation (P < 0.05) (Fig. 7). The increase in XIAP expression following adenoviral XIAP sense infection was not associated with any significant changes in DNA content and follicular volume (P > 0.05) (Fig. 8) in the absence of FSH. generic Viagra Canada
In contrast, follicles infected with XIAP sense and cultured in the presence of low concentration of FSH (5 ng/ml) showed a marked increase in DNA content (13.28 ± 1.89 ng/follicle vs. 6.85 ± 0.82 ng/ follicle, n = 36, P < 0.05) and follicular volume (22.88 ± 3.29 nl vs. 10.85 ± 2.02 nl, n = 36, P < 0.05) compared to the FSH-stimulated and LacZ-infected follicles. Analysis of variance demonstrates a significant FSH effect (P < 0.001) and XIAP sense effect (P < 0.001), and an interaction between the factors (P < 0.001) (Fig. 8), because of a greater influence by XlAP overexpression on follicular growth in the presence of gonadotropin.
FIG. 8. Effects of XIAP gene manipulation on follicular DNA content (top panels) and volume (lower panels) with or without FSH. After 1 day of culture in the absence of FSH, 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 in the absence or presence of gonadotropin (5 ng/ml) for another 3 days. Follicular volume change was defined as the growth between Day 0 and 4 of culture. Following confirmation that no interreplicate differences existed in each experiment (oneway ANOVA), individual observations from all replicates were pooled for analysis by two-way (repeated-measure) ANOVA. Values represent the mean ± SEM of a total of 36 follicles from three independent experiments. *P < 0.05 (vs. CTL-LacZ), **P < 0.01 (vs. CTL-LacZ), +P < 0.05 (vs. FSH-LacZ).
TABLE 1. Effects of FSH, LacZ, and XIAP cDNA expression on cell number and apoptosis in primary granulosa cell culture.3
a Values represent mean ± SEM, n = 3 experiments.
b Analysis of granulosa cell number by two-way ANOVA indicates a significant overall FSH effect. Post-hocTukey test: P < 0.05 (XIAP sense or antisense plus FSH vs. XIAP sense or antisense alone).
c Chi-square analysis for apoptosis data (% of apoptosis cell in total 600 pooled attached and detached cells) indicates a very significant FSH effect (P < 0.01).
d Chi-square analysis shows difference between treatments P < 0.05 vs. LacZ in same group (i.e., control or FSH).
FIG. 9. Effects of FSH on granulosa cell XIAP content in vitro. Granulosa cells were cultured for 24 and 48 h in the serum-free RPMI 1640 in the presence of different concentrations of FSH (0-150 ng/ml). The XIAP and tubulin contents were determined by Western blot analysis. Representative images and densitometric data of XIAP contents, normalized against respective tubulin levels and expressed as fold of control, are shown. Because the XIAP content was expressed as fold of control, it was arcsine square root-transformed before two-way ANOVA. Values represent the mean ± SEM of three independent experiments. *P < 0.05 vs. control (no FSH).