180 EFFECT OF Dnmt1p mRNA KNOCK DOWN ON Dnmt1 PROTEIN TRANSLATION IN MOUSE TESTIS

Abstract

We have reported that there was a significant reduction (P < 0.05) in the relative amount of Dnmt1p mRNA in spermatozoa from aged male mice (Kato et al. 2007 Reprod. Fertil. Dev. 19, 277 abst.). The reduction of mRNA levels of Dnmt1p in spermatozoa would lead to altered epigenetic modification of the genome. Dnmt1p is one of 5′ exon alternative isoforms of Dnmt1 and its mRNA is specifically expressed in the pachytene spermatocyte. However, the function of Dnmt1p still has not been elucidated. In this study, we tried to elucidate the function of Dnmt1p in the male mouse reproductive system. This was accomplished by suppressing the expression level of Dnmt1p in the whole testis by short hairpin RNA (shRNA) expression, which was specifically designed from mouse Dnmt1p mRNA. Dnmt1p cDNA was cloned from total RNA extracted from a piece of testis from a C57BL/6J male mouse. Four shRNA expression vectors were constructed and the knock-down efficiency of each shRNA expression vector was evaluated by flow cytometry. From these results, the 589 shRNA expression vector was picked out for further experimentation. The 589 shRNA expression vector was linearized and injected into the pronuclei of C57BL/6J mouse embryos. After injection, the embryos were cultured for 24 h and cleavage was evaluated. Cleaved embryos were transferred into oviducts of recipient ICR mice. After 18 to 19 days, fetuses were delivered by C-section. Two weeks after the birth, the existence of the 589 shRNA expression gene construct in its genome was evaluated by PCR. From founders with 589 shRNA expression gene construct in its genome, finally 1 TG strain was established and used for further experimentation. Two F₂ -generation male mice with the 589 shRNA expression gene construct, 2 F₂-generation male mice without the 589 shRNA expression gene construct and 1 C57BL/6J wild-type male mouse were used for evaluating the expression level of Dnmt1p mRNA in the whole testis by quantitative PCR. Then, thin sections of testis derived from the F₂-generation mouse, which showed a suppressed expression level of Dnmt1p, was evaluated by immunostaining for Dnmt1 protein. The survival rate of mouse embryos after gene injection was 76.8% (202/263) and the cleavage rate of gene-injected embryos was 69.8% (141/202). The developmental rate of transferred embryos to the birth was 19% (27/139). The rate of newborn mice with the 589 shRNA expression gene construct was 37% (10/27). The fertility of established TG strain mouse was normal and there was no abnormality in the thin section figure of testis stained with hematoxylin-eosin double staining method. The relative expression level of Dnmt1p mRNA in the whole testis of the F₂ TG mouse was ∼25% of C57BL/6J wild-type male mouse (P < 0.05, Student's t-test). There was Dnmt1 protein in seminiferous tubules, especially in spermatids of C57BL/6J wild-type male mouse. However, there was no Dnmt1 protein in seminiferous tubules of the F₂ TG mouse. From these results, it was concluded that the expression of Dnmt1p mRNA was associated in some way with the translation of the Dnmt1 protein in the mouse testis.