Stereological assessment of sexual dimorphism in the rat liver reveals differences in hepatocytes and Kupffer cells but not hepatic stellate cells

Abstract

There is long-standing evidence that male and female rat livers differ in enzyme activity. More recently, differences in gene expression profiling have also been found to exist; however, it is still unclear whether there is morphological expression of male/female differences in the normal liver. Such differences could help to explain features seen at the pathological level, such as the greater regenerative potential generally attributed to the female liver. In this paper, hepatocytes (HEP), Kupffer cells (KC) and hepatic stellate cells (HSC) of male and female rats were examined to investigate hypothesised differences in number, volume and spatial co-localisation of these cell types. Immunohistochemistry and design-based stereology were used to estimate total numbers, numbers per gram and mean cell volumes. The position of HSC within lobules (periportal vs. centrilobular) and their spatial proximity to KC was also assessed. In addition, flow cytometry was used to investigate the liver ploidy. In the case of HEP and KC, differences in the measured cell parameters were observed between male and female specimens; however, no such differences were detected for HSC. Female samples contained a higher number of HEP per gram, with more binucleate cells. The HEP nuclei were smaller in females, which was coincident with more abundant diploid particles in these animals. The female liver also had a greater number of KC per gram, with a lower percentage of KC in the vicinity of HSC compared with males. In this study, we document hitherto unknown morphological sexual dimorphism in the rat liver, namely in HEP and KC. These differences may account for the higher regenerative potential of the female liver and lend weight to the argument for considering the rat liver as a sexually dimorphic organ.

Keywords: Kupffer cells; dimorphism; hepatic stellate cells; hepatocytes; liver; stereology.

Figure 1

Overview of the methods used in this study in thin and thick liver sections and in frozen pieces.

Figure 2

Thin liver section immune‐stained against glial fibrillary acidic protein for detecting hepatic stellate cells (HSC). The relative volume of HSC was estimated by counting points falling within HSC and within the reference space (whole liver). To avoid counting an excessive number of points, two different point densities were used: the sparser points (in yellow) quantified the whole liver. Scale bar: 4 μm.

Figure 3

Thick liver section immune‐stained against glial fibrillary acidic protein and ED2 for detecting hepatic stellate cells (HSC, black arrows) and Kupffer cells (KC, open arrows), respectively. Cells were counted if their nucleus was in focus below 4 μm and above or equal to 24 μm in the z‐axis (section depth), if they were inside the inclusion (green) lines, or not touching the exclusion (red) lines. Scale bar: 6 μm.

Figure 4

Histogram of the number‐weighted mean nuclear volume of hepatocytes in males (yellow) and female (blue). The volume of diploid nuclei was estimated to be 225 ± 36 μm³, whereas that of tetraploid nuclei was 447 ± 52 μm³.