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Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Histone deacetylase inhibition leading to testicular atrophy||KeyEvent||Shihori Tanabe (send email)||Open for citation & comment||WPHA/WNT Endorsed|
Key Event Description
Spermatocytes are differentiated from spermatogonial stem cells via random proliferation, differentiation, and synchronized mitoses with several stages [Rooij, 2001]. In each step of the spermatogonial differentiation, different molecular mechanisms are activated in the testis [Rooij, 2001; de Kretser et al., 2016]. The stem cell factor (SCF) genes are involved in differentiation into A1 spermatogonia. The expression of cyclin D2 is regulated in the epithelial stage VIII when the aligned spermatogonia differentiate into A1 spermatogonia [Rooij, 2001]. Upon the apoptosis of spermatogonia, overexpression of the apoptosis-inhibiting proteins Bcl-2 and Bcl-xL and deficiency of the apoptosis-inducing protein Bax have been shown to cause an accumulation of spermatogonia in the testis, leading to apoptosis of all cells [Rooij, 2001].
How It Is Measured or Detected
Traditional spermatocytes assessment includes sperm count and concentration (haemocytometer, automated image-based system), morphology and motility (microscope, automated image-based system) and viability (for example propidium iodide staining of necrotic cells, TUNEL assay staining apoptotic cells). In addition, there are functional tests such as assays for genetic integrity (e.g. via measurement of DNA fragmentation/integrity -Halosperm kit or reactive oxygen species) and fertilization defects (through various measures of sperm-zona pellucida (ZP) interaction, such as measurement of ZP-receptor binding).
The sperm-containing fluid was squeezed out of the cauda, and suspended in medium containing HEPES buffer and bovine serum albumin, and incubated at 37ºC for 20 min. The number of spermatozoa was determined by a haematocytometer [Zindy et al., 2001].
Testicular sperm counts and daily sperm production were determined by counting the total number of spermatids per testis and divided by the testicular weight to give the results in spermatids per gram of testis [Oishi, 2001].
For the testis cell analysis, fresh testes were dispersed using two-stage enzymatic digestion and incubated in BSA containing collagenase and DNase I [Wade et al., 2006]. The seminiferous tubules were further digested and cells were fixed in ice-cold 70% ethanol [Wade et al., 2006]. Relative proportions of spermatogenic cell populations were assessed in fixed cells using a flow cytometric method [Wade et al., 2006]. The principle of the test is that spermatogenic cells, as they differentiate from normal diploid spermatogonial stem cells through to mature spermatozoa with a highly condensed haploid complement of DNA, progress through various intermediate stages with differing nuclear DNA content and cellular content of mitochondria. Relative proportions of cells in each population were calculated with WinList software [Wade et al., 2006].
Domain of Applicability
There are pieces of evidence of spermatocyte depletion in different species.
・Mature sperm counts were decreased and the residual spermatozoa had reduced motility and decreased viability (Mus musculus) [Zindy et al., 2001].
・The sperm counts in the cauda epididymis of rats were significantly decreased (Rattus norvegicus) [Oishi, 2001].
・Spermatocyte death can be induced in Sprague-Dawley rats (Rattus norvegicus) [Wade et al., 2008].
de Kretser, D.M. et al. (2016), "Endocrinology: Adult and Pediatric (Seventh Edition)", W.B. Saunders, Chapter 136-Spermatogenesis, pages 2325-2353.e9, Editors: J. Larry Jameson, Leslie J De Groot, David M. de Kretser, Linda C. Giudice, Ashley B. Grossman, Shlomo Melmed, John T. Potts, Gordon C. Weir
Oishi, S. (2001), "Effects of butylparaben on the male reproductive system in rats", Toxicol Indust Health 17:31-39
Rooij, D.G. (2001), "Proliferation and differentiation of spermatogonial stem cells", Reproduction 121:347-354
Wade, M.G. et al. (2006), "Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers", Toxicol Sci 89:304-313
Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831
Zindy, F. et al. (2001), "Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18Ink4c and p19Ink4d", Mol Cell Biol 21:3244-3255