abstract
Flow cytometry (FCM) is a powerful tool to evaluate cell DNA content and ploidy levels. We have assessed the accuracy of two protocols of nuclei isolation from paraffinized samples (P1and P2) by comparing FCM results with those obtained using fresh material (F1-F3). After isolation, nuclei were stained with propidium iodide and quantitatively analysed by FCM for changes in germ cell ratios. Results obtained with Protocol P2 were similar to those obtained using the protocol that gave best results for fresh tissues (F2). Protocol P2 was then applied to paraffin embedded testicular samples from ICR-CD1 mice exposed to 1, 2 and 3 mg CdCl2/g bw by single subcutaneous injection, and to 74 and 100 mg PbCl2/kg bw administered in four repeated doses. The highest doses of CdCl2 decreased the number of haploid (1C) cells and increased the number of diploid (2C), S phase and tetraploid (4C) cells. Treatment with PbCl2 did not induce significant changes in testicular cells subpopulations. These results support the usefulness of FCM in evaluating the effect of toxic substances on mouse spermatogenesis, using both fresh and paraffinized material. (c) 2006 Elsevier Inc. All rights reserved.
keywords
MALE REPRODUCTIVE-SYSTEM; RAT; MICE; TESTIS; TOXICITY; EXPOSURE; CHLORIDE; DNA
subject category
Reproductive Biology; Toxicology
authors
Oliveira, H; Loureiro, J; Filipe, L; Santos, C; Ramalho-Santos, J; Sousa, M; Pereira, MD
our authors
Helena Oliveira
Post-Doc Fellowship