Submitted August 31, 2007
Returned for revision September 20, 2007
Accepted January 8, 2008
Toxicology
Methoxyacetic Acid-Induced Spermatocyte Death Is Associated with Histone Hyperacetylation in Rats
Michael G. Wade *,
Alice Kawata ,
Andrew Williams ,
and
Carole Yauk
* To whom correspondence should be addressed. E-mail: mike_wade{at}hc-sc.gc.ca.
Abstract
We evaluated the possible mechanisms by which 2-methoxyacetic acid (MAA) disrupts spermatogenesis using high density microarrays. Levels of mRNA transcripts were determined in total RNA isolated from testes of MAA-treated (650 mg/kg i.p.) or concurrent control rats sacrificed 4, 8, 12 or 24 hrs post exposure (PE). Germ cell death was examined in testis sections using in situ staining for DNA fragmentation. MAA treatment caused increased death of pachytene spermatocytes starting 8 hr PE and increasing dramatically at 12 and 24 hr PE. Microarray results indicated that at 4 hr PE the transcript levels of 7 different genes were significantly over-represented in the testes of MAA exposed animals. One gene (histone H1 zero; H1f0) was significantly over-represented in MAA-treated samples at 4, 8, and 12 hrs PE. As expression of this gene has been associated with increased acetylation of core histones we examined MAA-induced changes in acetylation of histones H4 (histone H4, HISTH4) and H3 (histone H3, HISTH3) in testis nuclear protein. Western blots of acid extracted testis nuclei indicated that the levels of tetraacetyl histone H4 (4acHIST1H4) and of diacetylhistone H3 (2acHIST1H3) were elevated by MAA treatment at 4,8 and 12 hr PE. Using the same antibodies, 4acHIST1H4 and 2acHIST1H3 were localized primarily to elongating spermatids in testis sections from control animals. At 4hr PE, staining for either histone modification was dramatically increased in spermatogonia, and all primary spermatocyte populations except for dividing spermatocytes. MAA treatment of testis nuclear protein extracts from unexposed animals caused both a significant increase in histone acetyltransferase activity and a significant inhibition of histone deacetylase activity suggesting that increased core histone acetylation results from a combination of these complimentary modes of action. Our results indicate that exposure to MAA causes increased acetylation of core histones in several testis germ cell populations including those in prophase of meiosis a large proportion of which die rapidly following this treatment.
Key words:
Testis •
Toxicology •
Meiosis •
Spermatogenesis •
Methoxyacetic acid
