DNA analysis and sorting of rat testis cells using two‐parameter flow cytometry

Abstract

~~ ~~By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell Key terms: Two-parameter flow cytometry, compartments could be distinguished without cell sorting, octaploid cells, rat spermpre-enrichment of the samples. Cells in these atogenesis compartments were identified by sorting and subsequent microscopic examination.Flow cytometric (FCM) analysis of human (13,171 and animal (24,7,8,11,15) + M levels of DNA are primary spermatocytes and spermatogonia (12). Meistrich and associates (12) reported distinguishing seven subpopulations of testicular cells that were identified by FCM and cell sorting.Because of the highly complex effects of both physiological and pharmacological stimuli on the subpopulations of germ cells, a more detailed analysis of these subpopulations would be useful in FCM measurement of changes in testicular function. A possible solution to this problem is offered by the fact that the chromatin structure of the various germ cells as well as the size and shape of their nuclei vary widely in the different stages of maturation from spermatogonia to elongated spermatids (1). These differences can be measured using simple DNA staining if the peak and the area of the DNA fluorescence signal are analyzed as separate parameters in a two-parameter FCM system.The aim of the present study is to identify the different populations observed by two-parameter FCM analysis of would be more useful in giving detailed information on the various phases of the complex process of spermatogenesis in comparison with the single parameter usually used. MATERIALS AND METHODSAll experiments were performed with 3-mo-old male outbred Wistar rats (Cpb:WU) each weighing approximately 300 gm. Cell DispersalThe testis was dissected free from fat and connective tissue. After decapsulation, the tissue was finely minced with scissors in 15 ml phosphate buffer (pH = 7.4; P043-23.8 mg/ml) and sieved through a 50-pm mesh filter. The mincing and preparations were done at room temperature. This cell suspension was passed through a 25-gauge, 1-inch needle, centrifuged (370 x g) for 10 min, and resuspended in 10 ml phosphate buffer by vortexing.Debris was removed by filtration through a nylon filter (pore size: 50 pm). The suspension was again centrifuged (370 x g) for 10 min and the supernatant discarded. All aliquots were fixed in 50% ethanol (-ZOO C) and stored at 4" C until analysis.

Developmental Schedule of the Postnatal Rat Testis Determined by Flow Cytometry1

“…For example, differences in transcriptional activity (mRNA content) within the 2d cells, as well as within the 4d cells, were reported by Janca et al [16]. As to the 1d cells in which two peaks of fluorescence were apparent, it has been well documented that fluorescence emitted from haploid cells is distributed within a wide range, with two predominant peaks bordering that range: one representing round spermatids and one representing elongated spermatozoa [11,14,19]. This wide fluorescence distribution is a consequence of the chromatin condensation state that dictates propidium iodide intercalation efficiency [15].…”

“…Flow cytometry of testicular cell suspensions was utilized as another useful tool for studying mammalian spermatogenesis. Various fluorescent dyes were used to stain DNA (and/or other components) of testicular cells, and the stained cells were analyzed or sorted according to the intensity of fluorescence emission, which correlates with DNA content [11][12][13][14][15][16][17][18][19]. This technique has also been used as a diagnostic tool to assess spermatogenesis as well as development of testicular cancer in human patients [20][21][22].…”

A New Approach for the Analysis of Testicular Cells Using a Laser Scanning Cytometer

“…A major limitation in the study of genes and factors involved in germ cell differentiation has been the lack of an efficient in vitro system that supports this differentiation process [8,13]. Flow cytometry (FC) shows great potential for application to the assessment of sper-…”

“…It allows morphological evaluation of events gated according to fluorescence intensity with light and/or fluorescence microscopy [6]. In this paper, the application of LSC to the evaluation of spermatogenesis is described.Testicular cells were isolated from C57BL/6J mice (CLEA Japan, Tokyo, Japan) according to the Kwon Medicine, Chonbuk National University, Jeonju, Korea Analysis of biochemical events and the genes expressed at various postnatal developmental stages in the testis of mammals is great importance for understanding spermatogenesis [8].Mammalian spermatogenesis, the process of male gamete production, is a complex, highly organized process that can be divided into three main stages [7,10]: 1) mitotic proliferation of spermatogonial stem cells and premeiotic differentiation of primary spermatocytes; 2) progression of meiotic spermatocytes to haploid round spermatids via two successive divisions; and 3) spermiogenesis, a cellular and nuclear reorganization process that turns spermatids into spermatozoa.A major limitation in the study of genes and factors involved in germ cell differentiation has been the lack of an efficient in vitro system that supports this differentiation process [8,13]. Flow cytometry (FC) shows great potential for application to the assessment of sper-…”

Flow Cytometry for Gene Expression Studies in Mammalian Spermatogenesis

“…Testis developmental schedule and cell composition for different mammalian species such as mouse [Meistrich et al, 1978;Janca et al, 1986;Petit et al, 1995], rat [Clausen et al, 1982;Van Kroonenburgh et al, 1985;Suter et al, 1997;Malkov et al, 1998], hamster [Vigodner et al, 2003[Vigodner et al, , 2004, pig [Oskam et al, 2008], cat [Neubauer et al, 2004], several primates [Aravindan et al, 1990;Aslam et al, 2002;Wistuba et al, 2003] including man [Blanchard et al, 1991;Hittmair et al, 1992], and others, have been assessed by FC (see fig. 1 A as an example).…”