Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity

Abstract

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

Figure 1

Fluorescent (top) and autoradiographic visualization (bottom) of recovered oligonucleotide from the UDG initiated-in vitro BER assay. Oligonucleotide standards were loaded spanning the linear range of fluorescent quantification for generation of a standard curve and as a 51 bp size standard (top). A 5′-radiolabeled 51 bp size standard was loaded for autoradiographic visualization.

Figure 2

Linearity of the UDG-BER in vitro assay. Nuclear extracts were prepared from tissues of male 4- to 6-month-old C57BL/6J mice. Data are expressed as means (±SEM) from three replicate assays for each of three nuclear extract preparations obtained from brain (circles), liver (squares) and mixed germ cells (triangles). The amounts of protein selected for subsequent assays are indicated by an asterisk.

Figure 3

Incorporation of various [α-³³P]dNTPs during UDG-BER in vitro. Nuclear extracts were prepared from tissues and cell types obtained from male 4- to 6-month-old C57BL/6J mice. Results are expressed as means (±SEM) for three replicate assays for each of five different nuclear extract preparations. (dCTP, open bars; dATP, hatched bars; dGTP, gray bars; dTTP, closed bars)

Figure 4

UDG-BER in vitro activities for nuclear extracts prepared from select tissues and cell types. Results are presented as means (±SEM) of three replicate assays for each of three nuclear protein extract preparations. Nuclear extracts were prepared from C57Bl/6 (open bars), CD1 (hatched bars) and B₆D₂F₁ (closed bars) male mice. (a) Significantly higher UDG-BER activity than all other tissues and cell types. (b) Significantly higher UDG-BER activity than brain. (c) Significantly higher UDG-BER activity than liver. (d) Significantly higher UDG-BER activity than small intestine and thymocytes.

Figure 5

Western blot analysis of BER proteins. A total of 50 µg of nuclear extract was loaded for brain (B) and mixed germ cells (MGC) and 200 µg of nuclear extract was loaded for liver (L). Bands corresponding to DNA ligase I (130 kDa), DNA ligase III (93 kDa), Xrcc-1 (69 kDa), β-pol (39 kDa) and Ape/Ref-1 (37 kDa) proteins were visualized. A molecular mass protein standard and purified DNA ligases I and III, β-pol and Ape are shown for comparison. Faint bands at ~75 and 80 kDa are due to background binding of secondary antibody.