Ganglia and ovarian structures in Golgi-Cox staining
In the present study, Golgi-Cox staining was used for the first time to identify the ganglia network of mouse ovaries. Also, with the help of this method of staining and serial cryo-sectioning technique and three-dimensional reconstruction of ovarian slices, the parameters of gangliogenesis relationship with ovarian structures were compared. Briefly, in 2D images, the ganglia structures were stained black and the ovarian tissue was stained brown (Figure 1). Conversely, in 3D images, the ganglia structures were recolored red and the ovarian tissue color was changed to transparent for 3D reconstruction (Figure 2).
In addition, the neural network was detectable in this color in black, but with continuous filament structures in 2D (Figure 1A) and 3D images (Figure 2A). The ganglia were also recognizable as a network of tree dendrites from a cell body between the theca and granulosa cell layers around all types of follicles (Figure 1B). In corpora lutea, these ganglia were scattered throughout the corpus luteum structure (Figure 1A). The size, the shape and the number of branches were structurally different between ganglia but all of them were multipolar (Figure 1C).
After reconstruction of ovarian structures by 3D method, scattering of ganglia between follicles and corpus luteum was observed (Figure 2B). The segmentation of the ganglia after the segmentation of the follicular structures made it possible to image the spatial relationship between both ganglia networks and reproductive structures (Figure 2C). The spot algorithm for measuring follicles and corpora lutea completely segmented the ovarian structures (Figures 2B and 2D). The cell algorithm also segmented the network structures of the ganglia (Figure 2C). Ganglia and neurons were observed in all parts of the ovarian tissue. Nerve tissue density especially neural filaments was higher in the medulla of ovaries than the cortex.
Follicular growth and increase of ganglia number
In the 2D study, the total number of ganglia increased during follicular growth (p<0.05; Figures 3A and 3B). In both diestrus and estrus ovaries, the total number of ganglia in the antral follicles was higher than in the secondary follicles (p=0.03 and p=0.001, respectively; Figure 3A and Figure 3B). Investigating changes in the number of ganglia relative to increasing follicle area in the 2D study, it was observed that the ratio of ganglia number to follicle area in secondary follicles was higher than antral follicles (p =0.001, Figure 4C). However, the number of ganglia in secondary follicles in the estrus and diestrus stages was not significantly different (p>0.05, Figure 3D), but the number of ganglia in antral follicles in estrus stage was higher than diestrus stage (p=0.03, Figure 3E).
On the other hand, in the 3D study, the total number of ganglia increased during follicular growth, as well as 2D study (p<0.05; Figure 3F). Indeed, the total number of ganglia in the antral follicles was higher than the secondary follicles (p<0.001; Figure 3F). Furthermore, the ratio of ganglia number to follicle area in the secondary follicles and the antral follicles was not different, which was in contrast with the 2D study findings (p>0.05; Figure 3G).
Follicular growth and increase of ganglia area
In 2D study, the total area of ganglia increased during follicular growth (p<0.05; Figures 4A and 4B). Total area of ganglia in the antral follicles was higher than the secondary follicles in both diestrus and estrus ovaries (p=0.02 and p<0.001, respectively; Figures 4A and 4B). In addition, the ratio of ganglia area to ganglia number between the antral follicles and the secondary follicles was not different (p>0.05, Figure 4C). Also, the ratio of ganglia area to area of structures in the secondary follicles was higher than the antral follicles (p=0.02, Figure 4D). Furthermore, the area of ganglia in the secondary follicles in diestrus ovary was higher than the estrus ovary (p=0.001, Figure 4E). Also, the area of ganglia in the antral follicles in diestrus ovary was higher than the estrus ovary (p=0.006, Figure 4F).
In the 2D study, there were positive correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and increase in ovarian structures’ area; and increase in ganglia number and increase in ovarian structures’ area (p=0.0001, Table 1). In addition, in the secondary follicles, there were positive correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and increase in secondary follicles’ area and increase in ganglia number and increase in secondary follicles’ area (p=0.0001, Table 1). In contrast, in the antral follicles, there was no correlation between these three groups (p>0.05, Table 1).
On the other hand, in the 3D study, total area of ganglia increased during follicular development (p<0.05; Figure 4G). Area of ganglia in the antral follicles was higher than the secondary follicles, as well as the 2D study (p<0.001, Figure 4G). In contrast with the 2D study, the ratio of ganglia area to follicular area in the antral follicles was not different with the secondary follicles (p>0.05, 4H). However, the ratio of ganglia area to ganglia number in the antral follicles was higher than the secondary follicles (p=0.01, Figure 4I).
In the 3D study, the same as the 2D study, there were positive correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and increase in ovarian structures’ area; and increase in ganglia number and increase in ovarian structures’ area (p=0.0001, Table 2). In addition, in the secondary follicles as well as the 2D study, there were positive correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and increase in secondary follicles’ area; and increase in ganglia number and increase in secondary follicles’ area (p<0.05, Table 2). In contrast with the 2D study in the antral follicles, there were positive correlations between increase in ganglia area and increase in ganglia number; increase in ganglia area and increase in antral follicles’ area; and increase in ganglia number and increase in antral follicles’ area (p<0.01, Table 2).
Corpus luteum development and increase of ganglia parameters
In the 2D study, the total area and number of ganglia increased during corpus luteum development (Figures 2A, 2B, 3A, and 3B). Specifically, the total number of ganglia in the corpora lutea was higher than the antral and secondary follicles in diestrus ovary (p<0.001, Figure 3A). In contrast with diestrus ovary, the total number of ganglia in the antral follicles and the corpus luteum in estrus was not different (p>0.05, Figure 3B). In both diestrus and estrus stages, number of ganglia in corpora lutea was higher than the secondary follicles (p<0.001 and p=0.007, respectively; Figures 3A and 3B).
The ratio of ganglia number to area of structures and also the ratio of ganglia area to ganglia number in the corpora lutea and the antral follicles was not different (p>0.05, Figures 3C and 4C). In contrast, the ratio of ganglia area to ganglia number in the corpora lutea was higher than the secondary follicles (p=0.04, Figure 4C). Also, the ratio of ganglia area to area of structures in the corpora lutea was more than the antral follicles (p=0.02, Figure 4D). Moreover, number of ganglia in the corpora lutea in diestrus and estrus stages was not different (p>0.05, Figure 5A). In contrast, the area of ganglia in the corpora lutea in diestrus ovary was higher than estrus ovary (p=0.01, Figure 5B).
In the 2D analysis, there were positive correlations between area of ganglia and number of ganglia in the corpora lutea (p=0.003, Table 1). Also, positive correlations between area of ganglia and area of structure and number of ganglia and area of structure in the corpora lutea were observed (p=0.004 and p=0.007, respectively; Table 1).
In the 3D study, total number of ganglia in the antral follicles was higher than the corpora lutea in estrus stage (p=0.003, Figure 3F), in contrast with diestrus ovary. The number of ganglia in corpora lutea was higher than secondary follicles in estrus ovary (p<0.001, Figure 3F), the same as diestrus ovary. In contrast with the 2D study, total area of ganglia in the antral follicles was higher than the corpora lutea (p=0.04, Figure 4G) and higher than secondary follicle (p<0.001, Figure 4G). Furthermore, the ratio of ganglia area to ganglia number between the corpora lutea and the antral follicles was not different (p>0.05, Figure 4I). In contrast, the ratio of ganglia area to ganglia number in the corpora lutea was higher than the secondary follicles (p<0.001, Figure 4I). The area of ganglia to area of structures ratio in the corpora lutea was higher than the secondary follicles (p=0.01, Figure 4H). In contrast, this ratio between the corpora lutea and the antral follicles was not different (p>0.05, Figure 4H).
In the 3D analysis, there were positive correlations between area of ganglia and number of ganglia in the corpora lutea (p=0.02, Table 2), area of ganglia and area of structure (p<0.001, Table 2) and number of ganglia and area of structure (p=0.02, Table 2).