De NovoTestosterone Production in Luteinizing Hormone Receptor Knockout Mice after Transplantation of Leydig Stem Cells

Mesenchymal stem cells or Leydig cell progenitors are rare and difficult to isolate from adult testes. The property of differential efflux of Hoechst 33342 dye by the multi-drug-like transporter enriches murine hematopoietic stem cells from bone marrow. Our work on testicular cell transplantation suggests that the "Hoechst dim" side population (SP) also contains Leydig stem cells or progenitors that proliferate and differentiate into mature functional Leydig cells. We harvested testicular cells from cryptorchid ROSA26 mice, stained them with Hoechst dye, and isolated the cell population that excludes the dye using flow cytometry. Mice with targeted deletion of the LH receptor (LHR) gene were used as the recipients of the transplanted cells. These mice are hypogonadal and infertile. Both testicular SP and non-SP cells were transplanted into the interstitium of the LHR knockout recipients' testes. Serial serum testosterone assays revealed a significant increase in the circulating testosterone levels and restoration of spermatogenesis in the LHR-knockout recipients transplanted with the SP cells compared with that of those transplanted with non-SP. A SP cell concentration- and time-dependent increase in circulating testosterone was observed. This demonstrates the successful transplantation of functional putative Leydig stem cells into a hypogonadal recipient. The increase in testosterone concentration indicates the de novo synthesis of androgen by the transplanted SP cells. This method offers a novel technique to isolate Leydig stem cells and to study Leydig cell development.