Single-molecule PCR analysis of an unstable microsatellite for detecting mutations in sperm of mice exposed to chemical mutagens

Abstract

Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded simple tandem repeats (ESTRs), has been the most efficient method for studying germline mutation induction in endogenous sequences to date. However, the long length of these sequences makes mutation detection imprecise and laborious, and they have been characterized only in mice. Here, we explore the use of unstable microsatellite sequences that can be typed with high precision by capillary electrophoresis as alternative loci for detecting germline mutations. We screened 24 microsatellite loci across inbred mouse strains and identified Mm2.2.1 as the most polymorphic microsatellite locus. We then optimized SM-PCR of Mm2.2.1 to detect mutations in sperm. SM-PCR analysis of sperm from untreated B6C3F1 and Muta(™)Mouse samples revealed mutation frequencies that are consistent with rates derived from family pedigree analysis (∼ 5 × 10(-3)). To determine whether this locus can be used to detect chemically induced germline mutations, Muta(™)Mouse males were exposed by oral gavage to a single dose of 100mg/kg of N-ethyl-N-nitrosourea (ENU) or to 100mg/kg of benzo(a)pyrene (BaP) for 28 days alongside vehicle treated controls. Sperm were collected 10 weeks post-ENU exposure to sample sperm exposed as spermatogonial stem cells and 6 weeks post-BaP exposure to sample sperm that were dividing spermatogonia when the exposure was terminated. Both treatments resulted in a significant (approximately 2-fold) increase in mutation frequency in sperm compared to the control animals. The work establishes the utility of this microsatellite for studying mutation induction in the germ cells of mice. Because microsatellites are found in virtually every species, this approach holds promise for other organisms, including humans.

BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility

“…Other studies have associated BaP exposure, not only with the mutagenic and carcinogenic effects on somatic cells (17,19,21), but also with the negative impact on follicular and ovarian development (23,25,26). To determine whether BaP would deteriorate the oocyte quality and weaken the female fertility, we examined oocyte maturation and fertilization processes, two crucial events and key indicators of the high-quality oocyte in female reproduction.…”

“…When BaP is released into the air, it can move into the atmosphere, land, or water (16). In addition to the well-known mutagenic and carcinogenic effects of BaP on the pulmonary cells in both animals and humans (17)(18)(19)(20)(21)(22), accumulating data have indicated that BaP is toxic to the female reproductive system (23)(24)(25)(26)(27). It has been reported that significantly increased levels of BaP are found in the follicular fluid of female smokers, who have more exposure to PAHs than nonsmoking people (28).…”

Mlh1 haploinsufficiency induces microsatellite instability specifically in intestine

“…The extracted DNA was quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA) and diluted to 30 pg/μl concentration in 5 mM Tris-HCl (pH 7.5) supplemented with 5 ng/µl carrier herring sperm (Thermo Fisher Scientific). The MSI assay was performed using single-molecule PCR (SM-PCR) [26][27][28]. Three microsatellites, two mononucleotide repeats A27 and A33 [29], and a dinucleotide repeat D14Mit15 [7] were assayed for MSI.…”

Adverse Outcome Pathway on Alkylation of DNA in Male Pre-Meiotic Germ Cells Leading to Heritable Mutations

“…For expanded simple tandem repeat this involves agarose gel electrophoresis and Southern blotting, whereas for microsatellites sizing is done by capillary electrophoresis. Detailed methodologies for this approach are found in Yauk et al (2002) and Beal et al (2015).…”