FSH Stimulates Ovarian Follicular Growth In Vitro
Follicles cultured for up to 6 days in the absence of FSH exhibited minimal growth (Day 6 vs. Day 0), as evidenced by an absence of change in follicular size (follicular volume: 3.4 ± 0.55 nl vs. 2.6 ± 0.22 nl, n = 43), cell number equivalence (2.8 ± 0.35 vs. 1.5 ± 0.15, n = 46, as determined by Alamar Blue reduction), and DNA content (2.5 ± 0.39 ng/follicle vs. 1.2 ± 0.18 ng/follicle, n = 40) (Fig. 2A). Addition of FSH (100 ng/ml) to the culture medium significantly increased these parameters (follicular volume: 33.6 ± 3.26 nl, n = 43; cell number equivalence: 11.2 ± 2.35, n = 46; DNA content: 21.5 ± 3.39 ng/follicle, n = 40; P < 0.002 vs. control) (Fig. 2A). The increases in cell number equivalence, follicular volume, and daily growth rate were maximal on Day 3 of culture (Fig. 2, A and B). Viagra super active Canadian pharmacy
Histological examination of the HPS-stained follicles previously cultured over 6 days in the presence of gonadotropin indicated a well-preserved follicular structure (Fig. 4) containing granulosa cells, theca cells, and intact basement membrane (for Day 2, see Fig. 4B; for Day 6, see Fig. 4C). Theca cells on Day 6 of culture (Fig. 4C) appeared more cuboidal compared to those of follicles of similar stage in situ (Fig. 4D), and freshly isolated follicles (for Day 0, see Fig. 4A). When in vitro FSH exposure (100 ng/ml) was delayed for 2 days (i.e., FSH present on Days 3-6), the follicles were also responsive to the gonadotropin, although overall follicular growth and the daily growth rate were markedly decreased (P < 0.002) (Fig. 2).
FIG. 1. Linear relationship between Alamar Blue reduction, follicular volume, and DNA content. Follicles (n = 20) of different size (volume, 2.5-25 nl) were cultured individually for 24 h. Alamar Blue reagent was added during the last 3 h of culture (1:10 [v/v]). Follicular volume and DNA content were determined following Alamar Blue reduction assay.
FIG. 2. FSH increases follicular growth in vitro. Rat follicles were cultured for 6 days, with FSH (100 ng/ml) being added at the end of Day 0 or Day 2 of culture. Alamar Blue reagent was added during the last 3 h of culture on each day. Control cultures received no FSH. At the end of the daily culture period, spent medium was collected for spectrophotometer determination (optical density, 570 nm/630 nm) of cell number equivalence. Changes in DNA content and follicular size were determined as described in Materials and Methods. Follicular volume change on Day “n” of culture (A) is defined as the volume difference between Day ”n” and Day 0, whereas daily follicular growth of Day ”n” (B) is defined as that between Day ”n” and Day ”n – 1.” Following confirmation that no interreplicate differences existed in each experiment (one-way 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 43 follicles from three independent experiments. *P < 0.05, **P < 0.01 vs. control (no FSH).
FIG. 3. Validation of adenovirus gene delivery in cultured rat ovarian follicles. Adenoviral LacZ-infected follicles (MOI = 20; 3 days) were washed, fixed, stained in X-gal containing buffer, and incubated in series with increasing glycerol concentrations, as described in Materials and Methods. Shown are follicles without (A) and with (B) a full cycle of glycerol treatment (10%, 20%, 40%, and 60% [v/v]). These results indicate that adenoviral infection was successful. In addition, follicular sections were incubated with rabbit anti-p-galactosidase (C and D) and HRP-conjugated anti-Myc antibody (E and F), as described in Materials and Methods. Magnification X40 (C and E) and X100 (D and F).
FIG. 4. Comparison of follicular morphology at Day 0 (A), Day 2 (B), and Day 6 (C) of culture in the presence of FSH and of ovary in vivo (D). The HPS-stained follicular section shows thecal cells (TC), granulosa cells (GC), oocyte (OC), and basement membrane (Д).