Figure 1.
Histological picture of one representative ovary from 35-day old control (A) and GalTase- treated (B) rats. The control ovary exhibits the presence of follicles at different stages of maturity. The GalTase treated ovary exhibits an infantile appearance with a deficient follicular reserve and preponderance of atretic follicles. 1. Primordial, 2. Growing, 3. Antral, and 4. Atretic follicles. Each image is one representative of five independent ovaries of the respective groups; bar = 500 µm.
Figure 2.
Quantification of different types of follicles per ovary.
The figure represents overall reduction in the number of follicles of all stages and increase in atretic follicles in the GalTase-Ab exposed group as compared to the control. Values are expressed as mean±SEM. The numbers of follicles of all stages are significantly lower (P<0.0001) in the GalTase-Ab exposed group as compared to respective follicle type of the control group.
Figure 3.
Comparison of postnatal growth rates between control rats transplanted with neonatal ovary (COT) or a wedge of fat (CFT) and GalTase-ab-exposed rats transplanted with neonatal ovary (GOT) or wedge of fat (GFT). All the groups exhibited comparable increase in their body weight till 9th post-natal week. Each point represents the mean value of at least 5 rats in each group.
Figure 4.
The timing of vaginal opening.
Onset of puberty, as marked by the day of vaginal opening, in different groups of rats is presented with respect to age (4A) and body weight (4B). Transplantation of neither fat (CFT), nor ovary (COT) impacts the day of vaginal opening in the control group. But as compared to CFT and COT, the fat transplanted sub-group of the GalTase-exposed rats (GFT) exhibit significantly (P<0.0001) delayed vaginal opening. The timing of vaginal opening, however, is significantly (P<0.0001) advanced in the ovary-transplanted rats of the GalTase-exposed group (GOT). Due to delayed onset of puberty, the body weight on the day of VO is significantly higher in the GFT group with respect to all other groups (P<0.0001). Values are presented as individual datum with mean.
Figure 5.
Histological sections demonstrate marked follicle stimulation in the host ovary (host) of the GalTase-exposed rats transplanted (trans) with ovary (GOT) as compared to unstimulated, dormant or atretic follicles in those with fat transplantation (GFT). Each image is one representative of five independent ovaries of the respective groups; bar = 500 µm.
Figure 6.
Immuno-fluorescence detection of apoptosis by TUNEL assay.
The representative confocal images of isolated granulosa cells (Fig. 6A) demonstrate higher proportion of TUNEL positive cells (green) in the GFT ovary (Fig. 6A-a) as compared to the GOT-H (Fig. 6A-b) and GOT-T ovaries (Fig. 6A-c) that show little or no TUNEL positive cells. Each image is one representative of 3 replicates from 3 independent pools of cells, each representing 6-8 rats of the respective groups. The histogram (Fig. 6B) shows the mean percentage ±SEM of apoptotic cells counted in 9 microscopic fields at ×100. There are 3 randomly selected fields per slide, and each slide is represented by independent pool of cells. GFT vs. GOT-H: P<0.0001; GFT vs. GOT-T: P<0.0001.
Figure 7.
Hoechest staining of granulosa cells.
Confocal images (Fig. 7A) of isolated granulosa cells from GFT ovaries (Fig.7A-a) appear shrunken and irregularly shaped with fragmented nuclei (←), while the those isolated from GOT-H (Fig.7A-b) and GOT-T (Fig.7A-c) ovaries exhibit regular contour with round and large nuclei indicating healthy cellular condition. Each image is one representative of 9 replicates, 3 replicates from each pool of cells and each pool represents 6-8 rats of the respective groups. The histogram (Fig. 7B) shows the mean percentage ±SEM of apoptotic cells counted in 9 microscopic fields at ×100. There are 3 randomly selected fields per slide, and each slide is represented by granulosa cells from 6-8 rats of the respective group. GFT vs. GOT-H: P<0.0001; GFT vs. GOT-T: P<0.0001.
Figure 8.
Ovarian oxidative stress markers.
Data demonstrate that the generation of malondialdehyde (MDA) that marks lipid peroxidation (Fig. 8A) is significantly lower, and the ovarian SOD (Fig. 8B) and catalase activity (Fig. 8C) are markedly higher in both the host (GOT-H) as well as transplanted ovary (GOT-T) of the GOT group, as compared to that of the fat transplanted group (GFT). Results are expressed as mean ±SEM of five independent determinations. For all parameters, GOT-H vs. GFT: P<0.05, GOT-T vs. GFT: P<0.001.
Figure 9.
Detection of ROS generation by dichlorofluorescein assay.
The representative confocal images demonstrate higher level of ROS production in the GFT ovarian granulosa cells (Fig. 9A), but the cells isolated from the host (GOT-H) ovary (Fig. 9B), like those of the transplanted ovary (GOT-T) (Fig. 9C), demonstrate little or no ROS positive cells. Each image is one representative of 7–9 replicates, 2–3 replicates from independent granulosa cell pool, and each pool represented by ovaries from 6–8 rats.
Figure 10.
Potential-dependent mitochondrial JC1 staining.
Images of mitochondrial JC-1 fluorescence in granulosa cells are presented in Figs. 10 A–C. Granulosa cells obtained from follicle-deficient ovaries in the GFT group (Fig. 10A) show green fluorescence indicating disruption of mitochondrial membrane potential. The follicle-deficient host ovary (GOT-H) (Fig. 10B) exhibits shifting from green to reddish orange fluorescence and re-establishment of mitochondrial membrane potential following transplantation of ovary (GOT-T) (Fig. 10C) that also shows reddish orange florescence in most of the cells due to strong JC-1 aggregation denoting high mitochondrial membrane potential. Each image is one representative of 9 replicates; 3 replicates from each granulosa cell pool, and each pool represented by ovaries from 6-8 rats.
Figure 11.
Evaluation of apoptosis with annexin V.
Images of annexin V and propidium iodide (PI) fluorescence in granulosa cells are presented in Figs. 11 A–C. The granulosa cells from GFT group of rats (Fig. 11A) are clearly positive for both annexin V and PI fluorescence, which is indicative of loss of membrane integrity that characterizes apoptotic death. The cells isolated from follicle-deficient ovary (GOT-H) after ovary transplantation shows lesser annexin V binding, but without PI fluorescence (Fig.11B). The granulosa cells from the transplanted ovary (GOT-T) (Fig. 11C) also show very limited annexin V binding and no PI fluorescence. Each image is one representative of 9 replicates; three replicates from each granulosa cell pool, and each pool represented by ovaries from 6–8 rats.
Figure 12.
Immunoblot and densitometric analysis of p53, bax, bcl2 and caspase3.
Images of representative immunoblots (Fig. 12A) demonstrate decreased expression level of pro-apoptotic factors p53, bax and caspase3, and increased expression of anti-apoptotic factor, bcl2, in both the host and transplanted ovaries of the GOT group, as compared with the expression of the corresponding factors of the follicle-deficient ovary of the GFT group. The histograms (Fig. 12B) represent the densitometric analyses with relative intensity of the bands normalized to loading control, β-actin.
Figure 13.
Relative mRNA expression of gdf9, bmp15 and kit ligand as assessed by real-time RT-PCR.
The expression levels of gdf9, bmp15 and kit ligand are normalized with internal control, β-actin, and expressed as fold-change with respect to GFT group. The expression level of gdf9 is 1.61-fold higher in GOT-H and 2.76-fold higher in GOT-T, while the expression of bmp15 increased by 1.24-fold in GOT-H and 1.87-fold in GOT-T ovaries. The expression of kit ligand in the GOT-H and GOT-T is 1.62-fold and 2.69-fold higher, respectively. Data are presented as mean ±SEM of five independent determinations, each from individual rats of the corresponding gtoup (**p<0.05 vs. GFT; ***p<0.001 vs. GFT).
Figure 14.
Fluorescence image of kit ligand.
The expression level of kit ligand (←) in both the host and transplanted (trans) ovaries of the GOT group (Fig. 14B) is remarkably higher than that of follicle-deficient-ovary of the GFT group (Fig. 14A). Each image is one representative of five replicates, each from independent rats of the respective groups.