Figure 45.4 Enrichment of Leydig cell progenitors by flow cytometry analysis of the Hoechst dim side population. Goodell and colleagues reported the use of Hoechst dye exclusion to select for a population of stem cells based upon their ability to exclude the dye via the multidrug resistance transporter protein. The cells are sorted into SP (side-population), which extrudes the dye, and non-SP which retains the dye. The SP fraction represents about 0.9% of the total population. Briefly, after the Hoechst dye staining, the cell solution is excited with the UV laser at 350 nm. Duo-wavelength filters in the flow cytometer are used to detect the resulting fluorescence (Dakocytomation, Carpinteria, California).

Lo et al. were able to identify successful transplantation of Leydig stem cells by restoration of spermatogenesis and a significant increase in the levels of circulating testosterone in infertile mice. The ability to isolate Leydig progenitor offers a powerful method to study the adult testicular Leydig stem cell, characterize the phenotype, and discover what signals regulate differentiation, which will ultimately lead to human reproductive therapies.

Recent work by Davidoff et al. describes the progenitors of Leydig cells as vascular smooth muscle cells (VSMCs) and pericytes (PCs). Using an in vivo model of EDS treatment, Leydig cell regeneration was preceded by a proliferation of VSMCs and PCs and their subsequent conversion into steriodogenic Leydig cells. As advances in reproductive technology allow us to isolate and transplant testicular stem cells into infertile men and cancer patients, the ethical responsibility to the patient and society cannot be disregarded. Considerations for all involved parties, including unborn children, must be in place before human therapies can be implemented.

Testicular stem cells and andropause Aging is described as ''the progressive loss of function accompanied by decreasing fertility and increasing mortality with advancing age'' (Kirkwood et al., 2000). Androgen secretion by the testis progressively declines with age. Currently, a variety of drugs are on the market for replacement of testosterone in hypogonadal aging men (Androgel, Androderm). Similarly, loss of fertility due to chemotherapy or radiotherapy (as well as other infertility problems) is a deficiency that cannot be corrected with any available treatment, but recent work with testicular stem cells has given hope for therapies. Specifically, the findings of Atala and colleagues (Machluf et al., 2003) that encapsulated transplanted Leydig cells or granulosa cells (Yoo et al., 2000) can provide the basis for natural hormone replacement in aging. Similarly, germ cell transplantation may allow for restoration of fertility.

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