324 EFFECT OF AGING ON AMOUNTS OF DNA METHYLTRANSFERASE mRNA IN MOUSE SPERMATOZOA

Abstract

The spermatozoon is a specially differentiated cell designed to carry a haploid male genome into an oocyte at fertilization. It recently was reported that a matured spermatozoon contains several kinds of mRNAs and these are delivered into the oocyte at fertilization (Ostermeier et al. 2004 Nature 429, 154). The physiological role of paternally derived mRNAs is not clear; however, there is a report that the DNA methyltransferase (Dnmt) mRNA level in spermatozoa from male rats exposed to ethanol was significantly reduced (Bielawski et al. 2002 Alcohl. Clin. Res. 26, 347–351). The reduction of mRNA levels of Dnmt in spermatozoa would lead to altered epigenetic modification of the genome. Because factors such as age may affect spermatozoa mRNA levels, this study evaluated the effect of individual aging on the expression levels of Dnmts during spermatogenesis. This was accomplished by determining expression levels of Dnmts in the whole testis and in spermatozoa from young and aged mice by quantitative reverse-transcription-PCR. Seven- (young) and 68- (aged) week-old C57BL/6N male mice (n = 3/group) were sacrificed by cervical dislocation and whole testes and matured spermatozoa were collected. Total RNA was extracted and purified from each sample. In this study, 5 Dnmts (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a reference gene, were examined for expression levels in whole testis and spermatozoa using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Shiga, Japan) and the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Real-Time PCR runs for each Dnmt and GAPDH were repeated 3 times using different RNA batches from different individuals. The GAPDH expression level was used to normalize the expression levels of each Dnmt. Data were analyzed by Student's t-test. Relative expression levels of each Dnmt in testis from aged males compared to that of young males were 0.94, 1.15, 0.91, 1.15, and 1.14 (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l, respectively). There was no difference in the expression levels of the 5 Dnmts examined between testes from aged and young males. On the other hand, the relative amounts of each Dnmt mRNA in spermatozoa from aged males compared to that of young males were 0.87, 0.01, 0.54, 1.07, and 1.75 (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l, respectively). There was a significant reduction (P < 0.05) in the amount of Dnmt1p mRNA. The reason why the amount of Dnmt1p mRNA in spermatozoa from aged male mice showed such reduction is not clear. There was no difference in the relative expression levels of Dnmt1p in testis irrespective of male age. Dnmt1p is only translated in the spermatocyte during the pachytene stage in meiosis and its physiological role is not clear. To elucidate this male, age-related reduction of the amount of Dnmt1p mRNA in spermatozoa would clarify part of physiological role of Dnmt1p. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan.