Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa

Abstract

BACKGROUND: The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the offspring. METHODS: Adult male rats were given CPA in one of two schedules: (i) subchronic, 4 days-day 1 (100 mg kg 21) and days 2-4 (50 mg kg 21 per day) or (ii) chronic-daily (6.0 mg kg 21 per day). Animals were euthanized on days 14, 21 or 28. RESULTS: The effects of CPA on epididymal sperm chromatin structure were germ-cell-phase specific; mid-spermiogenic spermatids were most sensitive. The acridine orange DNA denaturation assay showed significant increases in susceptibility to denaturation (P < 0.01). Chromatin packaging assessment revealed 1,4-dithiothreitol-dependent chromomycin A3 DNA binding and less condensed, protaminedeficient sperm; the total thiol (P < 0.001) and protamine contents (P < 0.01), measured using monobromobimane and the HUP1N protamine 1 antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization. CONCLUSIONS: Paternal exposure to CPA alters sperm chromatin structure, as well as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development.

The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment

“…For example, M540 bodies, which represent an interference in flow cytometry and contain apoptotic markers (Marchiani et al, 2007), were excluded and a standardized fixation protocol (Muratori et al, 2009) was introduced. Additionally, pretreating the samples with the disulphide-bridge-reducing agent dithiothreitol resulted in relaxation of the compact chromatin structure for TdT to access the DNA (Codrington et al, 2007). Using this technique, Mitchell and co-workers (2011) significantly enhanced the sensitivity of the TUNEL assay.…”

Isolation and identification of proteins from swine sperm chromatin and nuclear matrix

“…The extraction of chromatin and the nuclear matrix followed the methodology adapted from Codrington et al (2007). The isolated heads were resuspended in 500 μl of a solution containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 5 μl of protease inhibitor cocktail (Sigma Aldrich P8340/011M4000) and vortexed for 10 min at room temperature.…”

Kemoterapötiklerin erkek üreme sistemi üzerindeki yan etkileri ve koruyucu stratejiler

“…Öte yandan gelişim düzenine göre spermatogonia; köken, proliferatif (çoğalabilen) ve farklılaşan hücreler olmak üzere 3 sınıfa da ayrılabilmektedir. Kemoterapötiklerin erkek üreme sistemi üzerindeki yan etki mekanizması kesin olarak bilinmemekle birlikte spermatogenik hücrelerde doğrudan (17,30,31) ve/ veya oksidatif stresle ilişkili olarak dolaylı (20,32,33) • ) gibi reaktif nitrojen türlerinden oluşmaktadır (37). Organizmanın bütün hücre, doku, organ ve sistemlerinde serbest radikallerin oluşum hızı ile bunların enzimatik [Glutatyon-peroksidaz (GSH-Px), süpe-roksit dismutaz (SOD), katalaz] ve enzimatik olmayan [Glutatyon (GSH), vitamin A, E, C ve selenyum gibi] antioksidanlar tarafından ortadan kaldırılma hızı bir denge içerisinde-dir ve bu durum "oksidatif denge" olarak adlandırılır (28).…”