Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. In rodents, the SSC pool is comprised of type Asingle (As) spermatogonia that balance self-renewing and differentiating divisions to exactly meet the biological demand of the spermatogenic system. Spermatogenesis is highly conserved in mammals and it is tempting to extrapolate rodent SSC data to higher species, including humans. However, in humans and nonhuman primates (NHPs), spermatogenesis arises from Adark (Ad) and Apale (Ap) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between species highlight the need to investigate the biology of NHP SSCs, which have greater relevance to human physiology. To facilitate the investigation of spermatogonial stem cells in primate testes, an in vivo functional assay is needed. In preliminary studies, we developed primate- to-nude mouse xenotransplantation as a biological assay to evaluate SSC function. In addition, we identified a busulfan treatment regimen that causes long- term infertility in rhesus macaques to facilitate autologous and homologous testis cell transplantation. This model will be used to test the full regenerative potential of rhesus SSCs and stem cell/niche interactions. We will exploit the rhesus-to-nude mouse xenotransplantation assay in the current application to characterize SSC activity in juvenile and adult (Aims 1) and identify phenotypic characteristics of rhesus SSCs using a fluorescence-activated cell sorter (Aim 2). Progress in Aim 2 will enable the isolation, enrichment and molecular characterization of rhesus SSCs. Because of evolutionary distance between rhesus and mouse, rhesus SSCs will engraft and persist in mouse seminiferous tubules, but will not produce complete spermatogenesis. Therefore, autologous and homologous rhesus-to-rhesus transplantations will be performed in Aim 3 to test the full regenerative potential of putative SSC-enriched rhesus testis cell populations identified in Aim 2.
This aim will also provide information about the quality of the stem cell niche in busulfan-treated testes, the immunological tolerance of autologous vs. homologous donor cells, and the effect of cryopreservation on SSC regenerative potential. Finally, our progress characterizing NHP SSCs (from Aims 1-3) will guide our initial studies in Aim 4 to characterize human spermatogonial stem cells. These studies will provide fundamental information about stem cells and the stem cell niche in nonhuman primate testes and lay the groundwork to translate these discoveries for future investigations of the biology and therapeutic potential of human SSCs. Project Narrative The proposed studies will provide the experimental framework to study the biology and molecular characteristics of rhesus spermatogonial stem cells (SSCs) and translate the results to facilitate the investigation of human SSCs. The studies will also establish a nonhuman primate model of infertility and examine the regenerative potential of SSCs to establish donor spermatogenesis and/or restore fertility. Responsible development of these ideas in the rhesus model may have implications for treating some cases of male infertility.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD055475-05
Application #
8298669
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Moss, Stuart B
Project Start
2008-09-15
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$702,860
Indirect Cost
$143,334
Name
Magee-Women's Research Institute and Foundation
Department
Type
DUNS #
119132785
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Valli, Hanna; Phillips, Bart T; Shetty, Gunapala et al. (2014) Germline stem cells: toward the regeneration of spermatogenesis. Fertil Steril 101:3-13
Valli, Hanna; Sukhwani, Meena; Dovey, Serena L et al. (2014) Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells. Fertil Steril 102:566-580.e7
Skaznik-Wikiel, Malgorzata E; McGuire, Megan M; Sukhwani, Meena et al. (2013) Granulocyte colony-stimulating factor with or without stem cell factor extends time to premature ovarian insufficiency in female mice treated with alkylating chemotherapy. Fertil Steril 99:2045-54.e3
Gassei, Kathrin; Orwig, Kyle E (2013) SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes. PLoS One 8:e53976
Zhang, Zhengxing; Zhang, Siliang; Huang, Xin et al. (2013) Rapid assembly of customized TALENs into multiple delivery systems. PLoS One 8:e80281
Dovey, Serena L; Valli, Hanna; Hermann, Brian P et al. (2013) Eliminating malignant contamination from therapeutic human spermatogonial stem cells. J Clin Invest 123:1833-43
Hermann, Brian P; Sukhwani, Meena; Salati, Jennifer et al. (2011) Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions. Hum Reprod 26:3222-31
Hermann, Brian P; Phillips, Bart T; Orwig, Kyle E (2011) The elusive spermatogonial stem cell marker? Biol Reprod 85:221-3
Ochalski, Melanie E; Shuttleworth, Jennifer J; Chu, Tianjiao et al. (2011) Thalidomide treatment attenuates chemotherapy-induced gonadal toxicity. Fertil Steril 95:819-22
Hermann, Brian P; Sukhwani, Meena; Hansel, Marc C et al. (2010) Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 139:479-93

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