Transcriptome profiling of mice testes following low dose irradiation

Abstract

Background: Radiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis.

Methods: Transcriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi).

Results: We identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster.

Conclusions: The five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

Figure 1

Gene expression profiles of the five transcript clusters during recovery from irradiation. The clusters contain the subset of 988 transcripts changing in expression during recovery from irradiation, which was separated into clusters by PAM. The number of transcripts in each cluster is stated in the title of each plot. The cell specific markers are coloured as following: Blue: Spermatogonia; Red: Spermatocytes; Green: Spermatids; Purple: Somatic cells.

Figure 2

The median z-scaled gene expression of the five transcript clusters during recovery from irradiation. The clusters are coloured as following: Blue: Spermatogonia (cluster 1); Red: Spermatocytes (cluster 2); Green: Early spermatids (cluster 3); Brown: Late spermatids (cluster 4); Purple: Somatic cells (cluster 5).

Figure 3

IHC staining of the testis tissue pi with Leydig and Sertoli cell markers. Tgfbr3 and Hsd3b were used as Leydig cell markers and Vimentin (Vim) was used as a Sertoli cell marker. Indications of Leydig cell hyperplasia are observed at pi day 21–31, whereas the Sertoli cells do not seem affected. A detailed description of the pictures is found in the text.

Figure 4

In silico determination of Leydig cells relative to other testicular cells pi. Areas covered by Leydig cells highly increased following irradiation compared to areas covered by other testicular cells. The peak is observed at pi day 14 where the ratio decreased again.