Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals |
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Authors: | R Suresh G R Aravindan N R Moudgal |
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Institution: | (1) Primate Research Laboratory, Center for Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, 560 012 Bangalore, India |
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Abstract: | Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome
(ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells
of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with
ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and
treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells
with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as
C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and
elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species
revealed little variation in the relative quantities of cells with 2C (8–11%), S-phase (6–9%), and 4C (6–9%) amount of DNA.
Though the spermatid cell populations exhibited variations (1C:31–46%, HCl:7–20% and and HC2:11–25%) they represented the
bulk of germ cells (70–80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to 1C
(1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA
flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by
analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms
in germ cell proportions observed following treatment, fore.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple
steps in germ cell transformation |
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Keywords: | Testicular germ cells mammals DNA flow cytometry germ cell transformation kinetics |
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