Presentation Authors: HIMANSHU ARORA*, Marilia Sanches Santos Rizzo Zutti, Bruno Nahar, Joshua M Hare, RANJITH RAMASAMY, MIAMI, FL
Introduction: 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 (HPG) axis. We evaluated the feasibility of subcutaneously autografting Leydig stem cells (LSC) in combination with Sertoli and myoid cells to increase testosterone. Furthermore, we evaluated the mechanism essential for survival of LSC in extra-testicular milieu.
Methods: We performed orchiectomy in sixteen adult C57/BL6 mice (subcutaneous autograft (n=8); negative controls(n=5); positive control(n=3) â€“ testosterone pellet) and five mice were used as sham 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 with matrigel (1:1). Grafts were harvested at two weeks (n=3 autografted) and after four weeks (n=18), and blood was collected. We evaluated testosterone production, graft morphology, and expression of Leydig cell markers. Furthermore, to evaluate Hedgehog signalling on graft differentiation, LSCâ€™s in-vitro were treated with Vismodigib (Hedgehog inhibitor) and SAG (Hedgehog inducer). Expression of Hedgehog signalling markers and LSC differentiation marker was evaluated by qRT-PCR. Additionally, LSCâ€™s, upon treatment with Vismodigib and SAG were subcutaneously autografted in mice for 4 weeks.
Results: Four weeks after subcutaneous autograft of LSCs in combination with Sertoli and myoid cells in castrate mice, the cells in graft expressed 3B-HSD, SOX-9 and a-SMA. Serum testosterone in castrated mice that received autograft was significantly higher compared to mice that did not receive autograft (22.4+/-1.9 VS 11.9+/-0.8 Ng/DL, p < 0.05). Importantly, mice that received LSC autograft maintained production of LH and FSH with levels higher than mice that received testosterone pellet implant (LH 3.09+/-1.47 VS 0.01+/-0.01 ng/ml) (FSH 102.6+/-28.1 VS 51.7+/-7.4ng/ml) respectively. Furthermore, T levels consistently increased at 15, 30 and 60 days following subcutaneous autograft (12.01+/-0.87ng/DL (neg CTL) vs 15.1+/-1.50ng/DL (15 days Autograft) vs 22.26+/-1.33ng/DL (30 days Autograft) vs 37.3+/-9.6ng/DL (60 days Autograft)), demonstrating differentiating LSC within the autograft. In addition, we found that levels of 3BHSD were induced upon SAG (DHH inducer) treatment in-vitro conditions. Immunostaining autografts (4 weeks), containing LSCs treated with SAG before subcutaneous implantation, showed higher levels of SOX9, and a- SMA establishing that Hedgehog signalling induces survival of adjacent testicular cells that are required for graft survival and LSC differentiation.
Conclusions: Our results demonstrate that subcutaneous autograft of LSC in combination with Sertoli cells and myoid cells can increase serum testosterone without inhibiting circulating LH and FSH in castrate mice. Extratesticular LSC appear to be regulated by Hedgehog signalling. Leydig stem cell autograft can a novel therapeutic approach to increase serum testosterone while simultaneously preserving hypothalamic-pituitary-gonadal axis. Factors involved in hedgehog signalling can be utilized to enhance graft efficacy.
Source of Funding: This work was supported in part by the Urology Care Foundation Research Scholar Award and Stanley Glaser Award to RR.