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.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-11092006-163407 |
| Date | 22 November 2006 |
| Creators | Miller, Stephanie Renee |
| Contributors | Dr. Paul Mozdziak, Dr. Jim Petitte, Dr. William Flowers, Dr. Robert Petters |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-11092006-163407/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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