Three-parameter flow cytometric analysis of rat spermatogenesis

Abstract

Mammalian spermatogenesis is a complex process which is not yet fully understood. In this paper we describe the analysis of rat spermatogenesis by means of 3‐parameter flow cytometry. Since the analysis of DNA content only provides sufficient information for the identification of 4 cell populations, additional parameters were combined with propidium iodide (PI) staining. Immunostaining of the intermediate filament vimentin allowed the identification of somatic (vimentin positive) and germ (vimentin negative) cells. Utilizing the combination of DNA and vimentin staining, we have been able to quantitate the somatic cells present in a testicular cell suspension and to analyze somatic and germinal cells separately. Furthermore, the addition of mitochondrial staining with the fluorochrome nonyl acridine orange (NAO) allowed several cell subpopulations within each ploidy group to be distinguished. After 3‐color staining and subsequent cell sorting, 11 testicular cell subpopulations could be identified: somatic cells, and 10 subtypes of germinal cells. The method described in this paper represents a valuable tool for the evaluation of spermatogenesis in both normal and perturbed situations. Cytometry 27:161–168, 1997. © 1997 Wiley‐Liss, Inc.

Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis

“…The number of contaminating germ cells was reduced to a very low level (about 10%) by several cycles of fragmentation of the cell clusters through a 19-gauge syringe needle, followed by sedimentation. Vimentin immunoreactivity was used to determine the percentage of germ cells (vimentin negative) contaminating the (vimentin positive) Sertoli cell preparations (Franke et al 1979, Suter et al 1997, Kopecky et al 2005). An aliquot of each Sertoli cell preparation was cytospun onto aminoalkylsilanized slides.…”

Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats

“…injection of sodium pentobarbital (University Hospital Pharmacy, Norway) and necropsies were performed. Testes were removed and weighed, and hereafter a longitudinal deep incision was made into the rete testis and approximately 5 mm-thick blocks of tissue were dissected and immediately placed in 2 ml TNE buffer (0.15 M NaCl, 0.01 M Tris/HCl and 1 mM EDTA, pH 7.4) on crushed ice for FCM analysis (Suter et al 1997). Of the other testis, similar fractions from the same region were fixed in Bouin's fixative for 24 h prior to storage in 70% ethanol.…”

Assessment of quantitative dual-parameter flow cytometric analysis for the evaluation of testicular toxicity using cyclophosphamide- and ethinylestradiol-treated rats.

“…Thus, sensitivity is the major limitation of the FCM analysis in evaluating testicular toxicity. Several improved methods have been reported using in combination another parameter such as the somatic-specific antibody (Hittmair et al, 1994), the amount of mitochondria (Petit et al, 1995), or both of them (Suter et al, 1997a(Suter et al, , 1997b(Suter et al, , 1998a(Suter et al, , and 1998b.…”