Assessment of Nycodenz Gradient on Enrichment and Culture of Perinatal Porcine Spermatogonial Stem Cells

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Title: Assessment of Nycodenz Gradient on Enrichment and Culture of Perinatal Porcine Spermatogonial Stem Cells
Author: Miller, Stephanie Renee
Advisors: Dr. Paul Mozdziak, Committee Member
Dr. Jim Petitte, Committee Member
Dr. William Flowers, Committee Member
Dr. Robert Petters, Committee Chair
Abstract: The objective of this study was to assess the effectiveness of a Nycodenz gradient enrichment method to enrich a dissociated single cell suspension of porcine testicular cells for spermatogonia, and to observe the separated fractions from the gradient over a 14-day culture period for cell viability and number of spermatogonia in culture. Two germ cell specific genes, VASA and DAZL, were utilized for detection of spermatogonia using immunohistochemistry. The control group included cultures generated from the enzymatic digestion of porcine testes prior to the enrichment protocol for each replicate. The NycoDenz gradient consistently separated the isolated cell suspension into three distinct layers and a pellet, all of which were assessed for spermatogonial enrichment. Testis cells were isolated and seeded in culture on day 0. Cell viability and percent of spermatogonia was assessed on day 0, 7, and 14 of culture. Viability was determined using trypan blue exclusion assay and quantified using a hemocytometer. Spermatogonia were morphologically identified as round, plump cells with a large amount of cytoplasm. Visualization of spermatogonia was facilitated by immunostaining with DAZL and VASA polyclonal antibodies and cells exhibiting morphological characteristics in addition to bright, concentrated fluorescence were counted as spermatogonia.
Date: 2006-11-22
Degree: MS
Discipline: Animal Science
URI: http://www.lib.ncsu.edu/resolver/1840.16/2680


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