Recombination study by MLH1 immunostaining of mouse spermatocytes after dietary treatments

The dataset is an Excel file with five sheets that contain the following information: Sheet 1 ("1st experiment, 3 strains"): MLH1 foci count per spermatocyte per mice, strain and diet Sheet 2 ("2nd experiment, B6 males"): MLH1 foci count per spermatocyte per C57BL/6 mice treated with two diets (2nd experiment). Columns indicate the mouse ID and number of spermatocytes analyzed in parenthesis. Sheet 3 ("intercrossover distances"): Interfocus distances in control mice (maintenance diet) of 3 strains. Values are shown as percentage of synaptonemal complex length. Sheet 4 ("synaptonemal c. length, 1st"): Total autosomal length of synaptonemal complexes per strain, control groups (maintenance diets) Sheet 5 ("synaptonemal c. length, 2nd"): Total autosomal length of synaptonemal complexes per diet in C57BL/6 mice (2nd experiment)We performed two studies: in the initial one, adult males from the three strains were analyzed for the effect of two diets on recombination (undernourishment (reduction to 50% daily intake) and breeding diets (Teklad Global 18% Protein Rodent Diet)) provided during 24 days relative to a control group kept ad libitum with maintenance diet (Teklad Global 14% Protein Rodent Maintenance Diet). After the 24-day diet period, adult male mice were euthanized by cervical dislocation and weighed. After removing and weighing the testes, chromosome spreads for immunostaining as previously described (Anderson et al. 1999; de Boer et al. 2009; Milano et al. 2019). MLH1 immunostaining allows for identification of about 90% of mammalian crossover sites (Anderson et al. 1999; Cole et al. 2012). All slides were imaged on a Zeiss LSM 710 confocal microscope and analyzed using Zeiss Zen lite software. Only mid and mid-late pachytene stage spermatocytes were scored. For each spermatocyte, we counted the number of foci localizing to the SC of the 19 autosomes (Anderson et al. 1999); total SC length and interfocus distances were also measured in autosomes only.Meiotic recombination is a critical process for sexually reproducing organisms. This exchange of genetic information between homologous chromosomes during meiosis is important not only because it generates genetic diversity, but also because it is often required for proper chromosome segregation. Consequently, the frequency and distribution of crossovers are tightly controlled to ensure fertility and offspring viability. However, in many systems it has been shown that environmental factors can alter the frequency of crossover events. We have explored for the first time the effect of dietary changes on crossover frequency per nucleus. Our study was performed in spermatocytes of 3 mouse inbred strains by analyzing the number and position of crossovers along the synaptonemal complexes, as well as the length of such synaptonemal complexes, by immunostaining with antibodies against MLH1 (which allows the identification of the crossover sites) and SYCP3 (a component of the synaptonemal complex). Our results show that male recombination rate is sensitive to dietary changes, and this sensitivity depends on the genetic background in mice. This is first to report a nutrition effect on genome-wide levels of recombination.Peer reviewe

Diet effects on mouse meiotic recombination: a warning for recombination studies

Meiotic recombination is a critical process for sexually reproducing organisms. This exchange of genetic information between homologous chromosomes during meiosis is important not only because it generates genetic diversity, but also because it is often required for proper chromosome segregation. Consequently, the frequency and distribution of crossovers are tightly controlled to ensure fertility and offspring viability. However, in many systems, it has been shown that environmental factors can alter the frequency of crossover events. Two studies in flies and yeast point to nutritional status affecting the frequency of crossing over. However, this question remains unexplored in mammals. Here, we test how crossover frequency varies in response to diet in Mus musculus males. We use immunohistochemistry to estimate crossover frequency in multiple genotypes under two diet treatments. Our results indicate that while crossover frequency was unaffected by diet in some strains, other strains were sensitive even to small composition changes between two common laboratory chows. Therefore, recombination is both resistant and sensitive to certain dietary changes in a strain-dependent manner and, hence, this response is genetically determined. Our study is the first to report a nutrition effect on genome-wide levels of recombination. Moreover, our work highlights the importance of controlling diet in recombination studies and may point to diet as a potential source of variability among studies, which is relevant for reproducibility.E.d.l.C.-E. received financial support through the program “Plan Propio de Investigacion” of the University of Castilla-La Mancha (2018/11744), cofunded by the European Regional Development Fund (FEDER, UE).Peer reviewe

Intronic Parent-of-Origin Dependent Differential Methylation at the <em>Actn1</em> Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

<div><p>Parent-of-origin differential DNA methylation has been associated with regulation of the preferential expression of paternal or maternal alleles of imprinted genes. Based on this association, recent studies have searched for parent-of-origin dependent differentially methylated regions in order to identify new imprinted genes in their vicinity. In a previous genome-wide analysis of mouse brain DNA methylation, we found a novel differentially methylated region in a CpG island located in the last intron of the <em>alpha 1 Actinin (Actn1)</em> gene. In this region, preferential methylation of the maternal allele was observed; however, there were no reports of imprinted expression of <em>Actn1</em>. Therefore, we have tested if differential methylation of this region is common to other tissues and species and affects the expression of <em>Actn1</em>. We have found that <em>Actn1</em> differential methylation occurs in diverse mouse tissues. Moreover, it is also present in other murine rodents (rat), but not in the orthologous human region. In contrast, we have found no indication of an imprinted effect on gene expression of <em>Actn1</em> in mice: expression is always biallelic regardless of sex, tissue type, developmental stage or isoform. Therefore, we have identified a novel parent-of-origin dependent differentially methylated region that has no apparent association with imprinted expression of the closest genes. Our findings sound a cautionary note to genome-wide searches on the use of differentially methylated regions for the identification of imprinted genes and suggest that parent-of-origin dependent differential methylation might be conserved for functions other that the control of imprinted expression.</p> </div

Bisulfite sequencing analysis of the <i>Actn1</i> DMR in mouse, rat and human tissues.

<p>Panel A shows bisulfite sequencing results from clones isolated from rat liver, mouse liver, and human hepatocytes. Each horizontal line represents a unique clone. Red and blue lines represent maternal and paternal parent-of-origin, respectively, based on five strain-specific variants. Open circles are unmethylated CpGs, while closed circles are methylated CpGs. Green and yellow circles shown in human hepatocyte clones represent variant rs11557769 and distinguish parental alleles, although parent-of-origin is unknown. Orthologous CpGs are connected by dotted lines (in relation to mouse). Panel B shows bisulfite sequencing results from clones isolated from rat right brain hemisphere (top) and mouse right brain hemispheres (bottom).</p

Maternal methylation of a novel DMR at the <i>Actn1</i> gene in diverse mouse tissues.

<p>A) A detailed map of the novel maternal <i>Actn</i>1 DMR is shown in the lower part. The diagram directly above shows the design for the MS-RFLP and bisulfite sequencing validation assays. Also included in this diagram are the locations of the methylation-sensitive enzyme restriction sites tested with MS-RFLP (<i>BsaAI, EagI</i> and <i>HpaII)</i>, the strain-specific cut sites (<i>AhdI</i> (present in 129S1 but not in PWK, due to SNP rs32640406) and <i>StyI</i> (present in PWK but not in 129S1, due to SNP rs32640412)), and the strain-specific resulting restriction fragments (see Methods). B) MS-RFLP results of four mouse liver samples. The matrix above the gel shows the different conditions for each individual lane. The plus sign (+) indicates addition, while the minus sign (−) indicated no addition of each corresponding endonuclease. C) Percent maternal methylation of an individual CpG (targeted by the <i>BsaAI</i> endonuclease) within different tissues. Circles represent individual (PWK×129S1)F₁ mice, while triangles represent individual (129S1×PWK)F₁ mice. Horizontal bars represent percent maternal methylation averages.</p