The testis has two main functions, the synthesis of steroid hormones and the production
of spermatozoa. The adult testis contains three main somatic cell types, namely Sertoli
cells, Leydig cells and peritubular myoid cells, as well as germ cells at all stages of
maturation. Interactions between these cells and the steroid hormones produced by the
testis are responsible for the regulation and maintenance of spermatogenesis and
fertility. Depletion or exposure to high levels of oestrogens, or androgens, both have an
adverse impact on male reproductive function. In the testis, as well as in other organs,
steroid hormone action is mediated by ligand-activated receptors. A single androgen
receptor (AR) and two oestrogen receptors (ERα and ERß) have been identified.The aims of this study were to investigate the role of steroid hormones, in particular
oestrogens, in murine spermatogenesis. A major focus of these investigations was the
role played by ERß in the modulation of germ cell and somatic cell function. Studies
were conducted both using a transformed murine Sertoli cell line (SK11), which has
maintained a differentiated Sertoli cell phenotype and spermatogonial stem cells, which
were successfully isolated and characterised. Steroid hormone receptor status, steroid
responsiveness and the impact of targeted deletion using RNAi were all assessed.Characterisation of the SKI 1 cell line, which was cultivated under conditions which
maintained them in an undifferentiated or differentiated state, revealed they retain many
features of Sertoli cells in vivo. ERß mRNA and protein were shown to be expressed in
the SK11 cells both in the undifferentiated and differentiated states. Transient
transfections using ERß or ARE-luciferase reporter constructs and stimulation with
steroid ligands revealed that the cells contained functional steroid hormone receptors.
Knockdown of ER|3 mRNA and protein was achieved in the cells after targeted deletion
using a short hairpin RNAi containing vector; this blunted the ability of the cells to
respond to oestrogen.Isolation of spermatogonial stem cells was carried out using immunomagnetic beads.
The stem cell population were shown to express Oct-4 and GFRα-1 mRNAs, both of
which are stem cell markers, but not c-kit, which is a marker of differentiated germ
cells. Taqman Q-RT-PCR demonstrated that the stem cell population expressed ERß.
Oct-4 mRNA expression was shown to be reduced by RNAi; this induced the cells to
undergo differentiation in vitro characterised by increased expression of c-kit.In conclusion, the current studies have extended our understanding of the impact of
steroid hormones on testicular function and have revealed for the first time that
spermatogonial stem cells are ERß positive. The SKI 1 cell line has been found to
provide a suitable model system for the study of steroid regulation of Sertoli cell
function. In addition, the use of RNAi has provided an exciting new avenue by which to
manipulate gene expression levels in testicular germ and somatic cells
