Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Summary

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

Abstract

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.

Introduction

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 spermati....

Protocol

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°C large refrigerator or a cold room that can also accommodate a fraction collector, if that method of collection is preferred.

1. The night before (or at least a few hours before) you perform the method, wash all equipment (es....

Results

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 .......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSA	Affymetrix/USB	10857 100MG	Alternate can be used.
0.5%, 2%, and 4% BSA solutions	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).	To be made day of the STA-PUT procedure. Filter sterilize.
KREBS (10x)	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	Autoclave/filter and store at 4 °C for several months.
KREBS (1x)	Dissolve 4.24 g NaHCO3 in 100 ml ddH20. Add 200 ml 10x KREBS. Bring to 2 L with ddH20.	To be made day of the STA-PUT procedure. Filter sterilize.
Collagenase	Sigma	C9891-1G
DNAse	Sigma	DNEP-5MG
Trypsin	Sigma	T9201-1G
DAPI	Preference of researcher
Triton-X100	Preference of researcher
Formaldehyde (~37%)	Preference of researcher
TABLE 2: EQUIPMENT
Equipment	Company	Catalog #	Comments
Complete STA-PUT apparatus	ProScience Glass Shop	STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)	Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.
10 μm mesh filter	Fisherbrand	22363549
Mouse dissection tools	Preference of researcher
14 ml round bottom fraction collection tubes with caps	Preference of researcher
Need approximately 100 tubes
Fraction collector	GE Healthcare Life Sciences	Model: Frac-920, Product code: 18-1177-40	Alternate can be used.
Microscope slides, cover glass	Preference of researcher
Light/Fluorescent Microscope	Preference of researcher