Abstract: PD08-11
Sources of Funding: This work was supported in part by the Urology Care Foundation Research Scholar Award to RR.

Introduction

Leydig cell loss or dysfunction is associated with impaired testosterone production. Exogenous testosterone supplementation can be used to treat low testosterone, however it has several adverse effects including infertility due to negative feedback on the hypothalamic-pituitary-gonadal axis. We studied testosterone production in mouse models following autograft in skin with Leydig stem cells isolated from testes.

Methods

A total of 10 wild-type adult C57/BL6 mice were included in the study. Orchiectomy was conducted in seven mice (4 experimental and 3 negative controls) and the remaining three were used as positive controls. Leydig stem cells were harvested from testis by collagenase/trypsin digestion. Cells from each mouse were allowed to grow separately in the media containing DMEM, FBS (10%), P/S, ITS, Dexamethasone, EGF, PDGF-AA. After 10 days following orchiectomy, 1 X 106 cells from four animals were autografted in the subcutaneous tissue. After four weeks, grafts and blood were harvested. We evaluated testosterone production, graft morphology, and expression of Leydig cell markers.

Results

We successfully isolated and cultured up to 1 million Leydig stem cells / testis from all 7 animals. These cells were differentiated and converted into functional adult Leydig cells in vitro. Stem cell property of cultured cells was confirmed by IF and qPCR in which the expression of PDGFR-? was high in regular media vs differentiation induction media and expression of 3BHSD was low in regular media vs differentiation induction media. The autografts were able to survive in animals for at least one month. H&E staining showed the presence of Leydig stem cells subcutaneously. Testosterone levels were almost doubled in autograft mice as compared to negative controls.

Conclusions

Our results indicate that Leydig stem cells can be isolated and cultured from wild-type mice. Leydig stem cell autograft can a novel therapeutic approach to increasing serum testosterone while simultaneously preserving fertility.

Funding

This work was supported in part by the Urology Care Foundation Research Scholar Award to RR.