## 3 Results  
### Expression and Activity of GLO1 in the Ovary of Young and Old Rabbits on Day 6 of Pregnancy  
In ovary samples from young and old rabbits, comparable amounts of GLO1 transcripts, GLO1 protein and GLO1 activity were measured (Figure 1).  
Immunohistochemical staining of the ovary revealed that in young and old rabbits GLO1 was exclusively localised in the cytosol and nucleoplasm, showing a dominant staining in follicles in the oocyte and the surrounding follicular epithelium, as well as the germinal epithelium (Figure 2A). During follicle maturation, the follicular cells differentiate to become several layers of granulosa cells, which showed the most prominent GLO1 staining. Ovarian stromal cells and the theca folliculi showed weaker staining. In the ovaries of old rabbits, tertiary follicles were more intensively stained than those of young ones (Figure 2C), indicating an increased expression of GLO1 at this follicle stage in old animals.  
Figure 1: Quantification of GLO1 mRNA, protein and enzymatic activity in the ovary of young and old rabbits at day 6 p.c. (**A**) GLO1 mRNA levels were quantified by qPCR. (**B**) GLO1 protein was measured by the Simple Western method. Both mRNA and protein amount were normalised to the levels of vinculin. (**C**) Enzymatic GLO1 activity was measured spectrophotometrically in 10 ug ovarian protein lysate. Specific GLO1 activity was calculated relative to GLO1 protein amount within the same sample. Results are shown as box plots. All values are expressed in per cent, with young rabbits = 100% (\(n\geq 9\)).  
Figure 2: Immunohistochemical analysis of GLO1 (**A**) and argpyrimidine (**B**) in the ovary of young and old rabbits. GLO1 and argpyrimidine were visualised through a peroxidase–diaminobenz reaction (brown colour). Nuclei were stained with haematoxylin (blue colour). Oo = Oocyte. Th = Theca folliculi. St = Stromal cells. Fe = Follicular epithelium. \(\rightarrow\) = Germinal epithelium. \(\blacktriangle\) = Granulosa cells. \({}^{\star}\) = Corpus luteum. (**C**) GLO1 and argpyrimidine staining intensities were evaluated independently and blindly by at least two researchers. Staining was graded using a scale from 0 (none) to 5 (very strong). The diagram depicts the staining intensity by follicle stage, calculated as average of oocyte, follicular epithelium/granulosa cell and theca folliculi grading. Relative intensities are expressed as mean \(\pm\) SEM in per cent, with young rabbits = 100% (\(n=6\); \({}^{\star}p=\)0.012).  
The AGE argpyrimidine was colocalised in ovarian sections from young and old rabbits (Figure 2B). For argpyrimidine, immunohistochemical analysis revealed a cytosolic as well as a nucleoplasmatic localisation, particularly in stromal cells, corpus luteum and theca folliculi. Staining patterns were similar to those of GLO1, with the strongest signal being observed in the follicles. Comparing argpyrimidine staining to that of GLO1 at the tertiary stage, a more distinct staining was found in the theca folliculi, while granulosa cell staining was reduced. At all follicle stages, no differences in argpyrimidine distribution between young and old rabbits were observed (Figure 2C).