Name: separation of spermatogenic cell types using sta-put velocity sedimentation
Uploaded: 2013-10-09T21:20:00
Description: Watch this Scientific Journal Video about Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation at JoVE.com

This research was supported by NIH grants GM055360 to SLB and U54HD068157 to RGM. JMB was supported by the T32 Genetics Training Grant at the University of Pennsylvania (GM008216).

The STA-PUT protocol involves three stages: 1) Set up of the apparatus and reagents, 2) Preparation of cell suspension from whole testes, and 3) Cell loading, sedimentation, and fraction collection. When performed by a team of two researchers, the protocol takes eight hours on average.
1. Setting up the STA-PUT Apparatus (Figure 1)
***STA-PUT apparatus should be placed in a 4&#176;C large refrigerator or a cold room that can also accommodate a fraction collector, if ...

<ol>
	<li>Wistuba, J., Stukenborg, J., Luetjens, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Spermatogenesis.">Mammalian Spermatogenesis.</a> Funct. Dev. Embryol. 1, 99-117 (2007).</li><li>Hess, R. A., Cooke, P. S., Hofmann, M. C., Murphy, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+the+regulation+of+the+spermatogonial+stem+cell+niche.">Mechanistic insights into the regulation of the spermatogonial stem cell niche.</a> Cell Cycle. 5, 1164-1170 (2006).</li><li>Dores, C., Alpaugh, W., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+in+vitro+culture+to+in+vivo+models+to+study+testis+development+and+spermatogenesis.">From in vitro culture to in vivo models to study testis development and spermatogenesis.</a> Cell Tissue Res. 349, 691-702 (2012).</li><li>Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+mouse+germ+cell+specification+pathway+in+culture+by+pluripotent+stem+cells.">Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.</a> Cell. 146, 519-532 (2011).</li><li>Easley, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+differentiation+of+human+pluripotent+stem+cells+into+haploid+spermatogenic+cells.">Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells.</a> Cell Rep. 2, 440-446 (2012).</li><li>Bellve, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+culture,+and+fractionation+of+spermatogenic+cells.">Purification, culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li><li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Romrell, L. J., Bellve, A. R., Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Miller, R. G., Phillips, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+cells+by+velocity+sedimentation.">Separation of cells by velocity sedimentation.</a> J. Cell Physiol. 73, 191-201 (1969).</li><li>Bastos, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li><li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602 (2011).</li><li>Grabske, R. J., Lake, S., Gledhill, B. L., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell Physiol. 86, 177-189 (1975).</li><li>Meistrich, M. L., Trostle, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Gassei, K., Ehmcke, J., Schlatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+enrichment+of+undifferentiated+GFR+alpha+1++spermatogonia+from+immature+rat+testis+by+magnetic+activated+cell+sorting.">Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting.</a> Cell Tissue Res. 337, 177-183 (2009).</li><li>Bellve, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenic+cells+of+the+prepuberal+mouse.+Isolation+and+morphological+characterization.">Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization.</a> J. Cell Biol. 74, 68-85 (1977).</li><li>Handel, M. A., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mammalian+meiosis:+regulation,+dynamics+and+impact+on+fertility.">Genetics of mammalian meiosis: regulation, dynamics and impact on fertility.</a> Nat. Rev. Genet. 11, 124-136 (2010).</li><li>Zhao, M., Shirley, C. R., Mounsey, S., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoprotein+transitions+during+spermiogenesis+in+mice+with+transition+nuclear+protein+Tnp1+and+Tnp2+mutations.">Nucleoprotein transitions during spermiogenesis in mice with transition nuclear protein Tnp1 and Tnp2 mutations.</a> Biol. Reprod. 71, 1016-1025 (2004).</li><li>Manku, G., Mazer, M., Culty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+testicular+gonocytes+isolation+and+processing+for+immunocytochemical+analysis.">Neonatal testicular gonocytes isolation and processing for immunocytochemical analysis.</a> Methods Mol. Biol. 825, 17-29 (2012).</li><li>Dehnugara, T., Dhar, S., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro,+short-term+culture+method+for+mammalian+haploid+round+spermatids+amenable+for+molecular+manipulation.">An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation.</a> Mol. Reprod. Dev. 79, 19-30 (1002).</li></ol>

Those who study spermatogenesis rely on animal models for spermatogenic cell samples, as a reliable cell culture system does not yet exist for generating all spermatogenic cell types3. Although spermatogenic cells are readily collected from whole testes, only a mixed population results. This poses a problem for those who wish to study specific subtypes of these cells, such as meiotic cells, round spermatids, and condensing spermatids. Three different methods are currently used to separate these subtypes of ...

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes1. Briefly, stem-like spermatogonia that reside near the epithelium of the seminiferous tubule divide and differentiate into spermatocytes, which then undergo meiotic divisions. After meiosis is complete, the resulting haploid cells, or round spermatids, undergo spermiogenesis, a differentiation process that involves the shedding of cytoplasm and compaction of the nucleus. Spermatids gradually develop a flagellum and undergo elongation and condensation of the nucleus, producing elongating and then condensing spermatids, respectively. The end products are spermatozoa, which are released into the lumen of the seminiferous tubule and ultimately into the epididymis where they mature further.
Because the process of spermatogenesis relies on special hormonal and molecular conditions in the testes, a reliable in vitro culture system for the entire process of spermatogenesis has not yet been developed2,3. Culture methods have been developed for creating "primordial germ cell-like cells" and haploid, "round spermatid-like cells" from stem cells, but these methods are not yet able to generate large numbers of these cells and fail to produce later spermatogenic cell types4,5. Fortunately, the spermatogenic cell types differ significantly in size, which allows for a single-cell suspension obtained from whole testes to be separated with a liquid gradient. The STA-PUT method, demonstrated here, uses a linear BSA gradient and simple sedimentation to separate spermatogenic cells based on size and mass6-9.
The STA-PUT method has several advantages over the other two most widely used methods to separate spermatogenic cell types: FACS and elutriation10-13. The STA-PUT apparatus requires only several pieces of specialized glassware assembled in a cold room or large refrigerator. Thus, it is less expensive than using a cell sorter or an elutriator. The STA-PUT method yields higher amounts of cells per cell type and testis than can be sorted by FACS in a comparable time frame, although the purity of each cell population is not as high as those obtained with FACS11. Cell sorting utilizing magnetic beads (magnetic activated cell sorting, MACS) has recently been successfully employed for enrichment of spermatogonia from a mixed testicular cell population, but it is currently unsuitable for separating spermatocytes or spermatids due to lack of knowledge of appropriate surface markers14. An additional advantage of the STA-PUT method over FACS or MACS is the ability to isolate viable cells suitable for subsequent culture because, in contrast to most FACS protocols, it does not require any DNA or other types of staining. For studies that require large yields of spermatogenic cells types at ~90% purity, the STA-PUT is an ideal method.

<table border="0" cellpadding="0" cellspacing="0" width="856">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">BSA</td>
			<td style="width:109px;">Affymetrix/USB</td>
			<td style="width:255px;">10857 100MG</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">0.5%, 2%, and 4% BSA solutions</td>
			<td colspan="2" style="width:364px;">Dissolve 0.25 g, 11 g, and 22 g (respectively) in total volumes of 50 ml, 550 ml, and 550 ml of 1x KREBS (respectively).</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="91">
			<td height="91" style="height:91px;width:311px;">KREBS (10x)</td>
			<td colspan="2" style="width:364px;">3.26 g KH2PO4 + 139.5 g NaCl +5.89 g MgSO4/7H20 + 40 g Dextrose + 3.78 g CaCl2/2H20 + 7.12 g KCl, bring to 2 L with ddH20</td>
			<td style="width:183px;">Autoclave/filter and store at 4 &#176;C for several months.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">KREBS (1x)</td>
			<td colspan="2" style="width:364px;">Dissolve 4.24 g NaHCO3 in 100 ml ddH20. Add 200 ml 10x KREBS. Bring to 2 L with ddH20.</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Collagenase</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">C9891-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">DNAse</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">DNEP-5MG</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Trypsin</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">T9201-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Triton-X100</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Formaldehyde (~37%)</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">TABLE 2: EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Equipment</td>
			<td style="width:109px;">Company</td>
			<td style="width:255px;">Catalog #</td>
			<td style="width:183px;">Comments</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:311px;">Complete STA-PUT apparatus</td>
			<td style="width:109px;">ProScience Glass Shop</td>
			<td style="width:255px;">STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)</td>
			<td style="width:183px;">Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">10 &#956;m mesh filter</td>
			<td style="width:109px;">Fisherbrand</td>
			<td style="width:255px;">22363549</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Mouse dissection tools</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:311px;">14 ml round bottom fraction collection tubes with caps</td>
			<td colspan="3" style="width:547px;">Preference of researcher </td>
		</tr>
		<tr height="22">
			<td colspan="3" height="22" style="height:22px;width:547px;">Need approximately 100 tubes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:311px;">Fraction collector</td>
			<td style="width:109px;">GE Healthcare Life Sciences</td>
			<td style="width:255px;">Model: Frac-920, Product code: 18-1177-40</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Microscope slides, cover glass</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Light/Fluorescent Microscope</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
	</tbody>
</table>

separation of spermatogenic cell types using sta-put velocity sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The ideal result from the STA-PUT procedure is a fairly noticeable separation of cells from the testes based on cell size and density. While cells isolated from the testes are sedimenting through the BSA gradient, several distinct bands of cells can be observed. Any clumps of cells tend to sink to the bottom of the gradient and will not contaminate the other fractions. A little further up the gradient will be the large somatic and meiotic cells. Farther up the gradient still will be smaller round spermatids. At the ...

Watch this Scientific Journal Video about Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation at JoVE.com

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

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