Iron is essential to fertility as it is required for germ cell division, differentiation and metabolism. DMT1 is expressed in the apical membranes of duodenal enterocytes and is responsible for the non-transferrin-mediated absorption of dietary iron. DMT1 is also expressed in vesicular membranes and participates in transferrin-mediated iron acquisition by cells. It is highly expressed in the kidney and is thought to play a role in renal iron handling. The aim of this study was to determine the pattern of DMT1 expression in the rat testis.

Peroxidase immunohistochemistry using an affinity-purified anti-DMT1 polyclonal antibody targeted to recognise all DMT1 isoforms (Ferguson et al. 2001) was used to study the cellular distribution of DMT1. Western analysis was used to quantify the levels of DMT1 in the testis of humanely killed 5-, 15-, 25-, 35-day-old and adult rats.

DMT1 immunoreactive species were detected throughout development in the seminiferous tubules but not in the interstitium. In the 5- and 15-day-old rats, immunostaining was equally widespread throughout each tubule and could be localised to the cytoplasm of the Sertoli cells. This was the case in the 25- and 35-day-old animals but more punctate areas could be detected.

In the adult rat immunostaining was specific to each of the 14 stages of the cycle of the seminiferous epithelium and as a result tubules appeared heterogeneous, unlike in the immature rat. Staining could be localised to the nuclei and cytoplasm of both Sertoli and germ cells. The most intense immunoreactivity appeared at Stages VII and VIII and could be attributed mainly to the deep staining of the mature spermatozoa, suggesting that these cells have a high requirement for iron as they prepare to leave the seminiferous tubules.

Western analysis detected an immunoreactive protein band of around 60 kDa in the membrane fractions at all ages tested. At 15, 25 and 35 days and in the adult rat a second higher weight band was detected of around 70 kDa, revealing expression of a different isoform at these ages. Deglycosylation experiments with N-glycosidase showed that the major DMT1 isoform present (55 kDa) was different from that in the kidney (50 kDa).

The expression profile of DMT1 in the development of the rat testis was found to be cell specific and highly co-ordinated to the spermatogenic cycle. This suggests an important role for DMT1 in spermatogenesis and implies that the germ cells have a need for a precisely timed supply of iron. This stage-specific nature of expression means that DMT1 plays a central role in male fertility. This may be applicable when considering conditions of abnormal iron regulation.

This work was funded by the BBSRC, The Royal Society and The Wellcome Trust