Fayomi, Adetunji/ P
(2018)
Stem cells and spermatogenic lineage development in the primate testis.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Stem cells in the primate testis, Adark and Apale, have been designated as “reserve” and “active” stem cells, respectively. It is not clear if Adark participate in steady-state spermatogenesis; little is known about how these descriptions correlate with molecular markers or clone size and there are knowledge gaps on the potential use of primate stem cells for fertility preservation/restoration. We show that Adark are mitotically active, slow-cycling, label-retaining cells during steady-state spermatogenesis, with evidence of self-renewal and differentiation. We found that UTF1, ENO2 and UCHL1 are markers of Adark and Apale spermatogonia during development and they mark undifferentiated spermatogonia in adult Rhesus Testis. We also demonstrate that undifferentiated spermatogonia (UTF1+/cKIT- cells) exist more frequently as clones of 1 or 2, less frequently as clones of 3 or 4, clones greater than 4 are rare. cKIT+ clones of 1 (~20%) and 2 (~15%) cells were also observed. Our novel approach to staging seminiferous epithelium through EDU staining revealed that cKIT+ clones of 1 or 2 cells are not stage dependent. Our data indicate that primate spermatogonia undergo both symmetric (undifferentiated) and asymmetric (differentiating) divisions during steady-state spermatogenesis. For cell-based application of primate stem cell, we show that the concentration of xenotranplantable stem cells was higher in neonatal testis. Using ITGA6, stem and progenitor population were enriched by 18-fold (FACS) and 9-fold (MACS) while stem cell activity was enriched by 5-fold (MACS). We established foundational primate SSC culture conditions. ITGA6-positive cells produced colonies with “grape-like” appearance in culture. ITGA6-positive cells retain phenotype of stem and progenitor cells up to 14 days in culture. For tissue-based application of primate stem cell, we grafted fresh and frozen-thawed prepubertal testicular tissues in the subcutis of the back and scrotal skin. Volume of testicular tissue grafts increased in graft sites. Although weight of recovered tissue grafts from the scrotal area were higher than from the back, spermatogenesis was confirmed in grafts with spermatids/sperm cells in more than 70% of tissue cross-section. Millions of sperm were recovered per graft and sperm were competent to fertilize oocytes, resulting in hatching blastocyst in vitro and pregnancy following embryo transfer.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
19 April 2018 |
| Date Type: |
Publication |
| Defense Date: |
22 March 2018 |
| Approval Date: |
19 April 2018 |
| Submission Date: |
19 April 2018 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
233 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Genetics and Developmental Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Spermatogonial stem cells, Rhesus macaque, Autologous testicular tissue grafting, Primate spermatogonial stem cell culture, stages of seminiferous epithelium primate, spermatogonial stem and progenitor cell enrichment |
| Date Deposited: |
19 Apr 2018 17:21 |
| Last Modified: |
19 Apr 2019 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/34319 |
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