The gut microbes of a dad are important for baby growth
Reversible placental dysfunction in humans: a metabolite class enrichment study using the Benjamini-Hochberg procedure
Alterations in the expression of genes related to a metabolism called prolactin and steroids and to the genes Hand1 and Syna were highlighted in the analysis. Intriguingly, some of the transcriptional changes can cause placental dysfunction. Certainly, further characterization will be crucial to determine whether effects similar to those observed in the placenta-associated condition pre-eclampsia (which involves maternal hypertension and target-organ injury and can lead to fetal or maternal illness or death) are an underlying cause of disease in this context.
Whether these findings in mice are also relevant to humans remains to be determined. How long it will take for people to recover from antibiotics is an interesting question. The authors’ finding that the negative effect is reversible might prove useful in providing advice on the optimal timing for fertilization, to avoid costs to the offspring.
Metabolite class and superclass enrichment analysis between the treated and control group was calculated using a Fisher’s exact test. The FDR was adjusted for several hypotheses using the Benjamini–Hochberg procedure.
The statistical significance of the features was assessed, using a two-sided t-test, log-scaled data and P values were FDR-corrected for several hypotheses testing. Adjusted P values and foldchanges were visualized using Volcano plots. Mass, retention time and HMDB annotations were retrieved for every feature that showed a significant difference between the treated and the control group.
All statistical analyses and plotting were performed in R v.3.6.2 (ref. 87). To exclude bad sample injection from downstream analyses, the sum of all extracted metabolite features (TIC) was compared between samples (mean = 1.600 × 1010, range = [1.419 × 1010; 1.808 × 1010]) and samples were excluded, if their TIC was not within 3 s.d. from the mean value (0 excluded samples). The threshold was set at 5,000 counts. There are features that are duplicated that fall within the window of a 0.002 a mu or 20ppm relative threshold. retention time window, were considered to be split peaks and therefore collapsed together. Correlation between testis weight and animal body weight was computed, to verify that there was no significant correlation between the two values (cor = 0.1870, P = 0.2477). To consider variation in signal intensity from testis size, the Z-scores were used. It was decided to remove features such as zero variance, singletons and presence in less than 75% of the samples for each class. Finally, feature tables derived from positive and negative mode were collapsed together after checking for exact duplicated features or if feature falling in a 0.002 Da or 20 ppm window existed; if so, the feature with higher average intensity was retained and both annotations were retained. The resulting table included all 30 samples and a total of 10,582 metabolic features.
The first ten bases of the alignment reads were taken out with Cut Adapt (v. 2.3)80. The QUAL of those who were removed had an allele frequency of less than 0.2 or low site coverage. Variant functional region was annotated using ANNOVAR (2020Jne07) annotate_variation.pl85. Structural variations were called with the Delly2 call.
The first 8 and last 2 bds from a single-end read were discarded, and the reads werealigned to millimeters10 with the help of the Bismark method. Cytosine methylation status was extracted from mapped reads using the Bismark methylation extractor tool. Genome-wide methylation calls were analysed using Seqmonk software (1.44.0) with five biological independent replicate datasets for each condition. To identify DMRs, the genome was first binned into sliding tiles containing 50 consecutive CpGs and their methylation status determined using the DNA methylation pipeline. The minimum number of reads is needed to run a read-depth sensitive logistic regression and apply a threshold for an absolute change in DNA methylation. The methylation level at specific genomic features (for example, imprints) was calculated using the DNA methylation pipeline in Seqmonk over target features.
The negative binomial test was used to identify the differences after Benjamani–Hochberg multiple test correction.
Statistical methods for the annotation of mouse transcriptome DEGs and tRNA quantitation with annotated gene clusters
Adaptors were removed using fastx-clipper; fastq files were converted to fasta using a custom perl script and reads of 18–33 nucleotides in length were retained using a custom perl script. Reads were aligned to the mouse genome version mm10 using bowtie, reporting only the best alignment and requiring 0 mismatches (parameters -v 0 -k 1 — best –sam). Alignment sam files were converted to bam files using samtools v.1.9 and bam files were converted to bed files using bedtools v.2. To quantitate miRNAs intersectBed -c was used to count the number of reads overlapping the positions of the known M. musculus miRNAs (from miRbase www.miRBase.org). The tRNAs were obtained by using intersectBed -c to count the number of reads overlapping a bed file documenting predicted mouse tRNA coordinates downloaded from the tRNA scan database (http://gtrnadb.ucsc.edu/genomes/eukaryota/Mmusc10/). The piwi-interacting RNA coordinates were taken from ref. 78 and converted to mm10 using liftover. The piRNAs were quantitated by using intersect -c to intersect the 26 and 32 nucleotides long piRNAs with their corresponding piRNA coordinates.
Principle component analyses of transcriptomes were computed in seqmonk and R statistical software using all expressed genes as input. These were defined as having an RPKM > 0.25 in at least two replicates.
The DEGs were calculated using the DESeq2 package and a multiple-testing adjusted P value. Final DEGs were generated using a fold change filter of more than 2.
For each cell type identified in the dataset, the FindMarkers function was used to identify DEGs between nABX and CON cells using logfc.threshold = 0.25 and p_val_adj < 0.01.
The annotated dataset was made using unique expressed marker genes. The clusters showing no genes were ignored the same way as above. The germ and somatic cells were separated to make a more fine- grained annotations.
The Seurat package recommended the use of the canonical correlation analysis approach for the nABX and CON samples.
The testes-to-body weight ratio was used to evaluate the effect of nABX treatment on sires. Furthermore, to assess the impact on reproductive organs, testes were carefully collected from CON and nABX-treated mice, weighed and the testes-to-body weight ratio difference between the two groups was calculated.
As described previously54, this method of F1 offspring generation follows five critical steps: (1) superovulation of oocyte-donor females; (2) preparation of fresh sperm from sire males; (3) collecting isogenic oocyte from donor females; (4) in vitro sperm–oocyte fertilization; (5) embryo transfer to pseudopregnant CD1 or C57BL/6J surrogate mothers. The process was performed on a machine.
Source: Paternal microbiome perturbations impact offspring fitness
Microbiota transfer and oviductation of single female surrogate mice with nABX at precohousing and postmating
In this experiment, male from each group were cohoused at a 1:1 ratio in a single cage with a treatment-naive. During the mating period, both groups were maintained on regular diet and sterile water ad libitum. We collected faeces at precohousing and 4 postmating to assess the effects of exposure/cohabitation with nABX. The copulatory plug from the female’s vaginal area was samples to determine if microbiota transfer occurs at mating. Using swabs, control samples were collected from the working area and sterile sharp-toothed tweezers were used to collect plug samples.
Sperm–oocyte fertilization: from capacitated sperm in TYH-MBCD drops, a 10 μl of sperm suspension was taken from the peripheral part of the pre-incubation drops which contain the most motile sperm. nABX-treated mice had their sperm transferred to the center of the drops in the fertilization dish. Sperm–Oocytes fertilization dishes were cultured at 37 C under 5% CO2 for 3–4 h. The presumptive zygotes were washed multiple times with 150 l of M2 medium and cultured overnight in four NUNC plates with 500 l of KSOM medium. Two-cell stage embryos were transferred to the oviducts of pseudo Pregnant CD1 or C57BL/6J female surrogate mice.
embryo transfer was carried out as described. Vasectomized males were mated with 6–8-week-old CD1 females or C57BL/6J (27–30 g) to produce 0.5 days postcoitum (dpc) pseudopregnant recipients. Plug-positive females were anesthetized after being selected. To transfer two-cell stage embryos, the following subsequent steps were applied: the dorsal midline opened, ovary body wall incised and held outside the body, after breaking the bursa the tip of the capillary inserted into the infundibulum to transfer ±20 embryos per single pseudopregnant CD1 or C57BL/6J mouse and finally a gentle push applied through mouth pipette to dislodge embryos from the glass capillary tube into the infundibulum of one oviduct.
The vasques were collected in a flow-hood with slight modifications. To prevent cross contamination, one experimental animal (CON or nABX) was placed in the hood per sample collection. To ensure maximum sterilization, the experimenter wore a sterile gown, gloves, mask and scrubbed their hands with 70% ethanol and followed the following procedures. The mice were put into a flow-hood that had been previously cleaned with 70% Ethanol and 2% Virkon and then 2 h of ultraviolet radiation. To collect seminal vesicles aseptically, mice were euthanized by cervical dislocation and their abdominal areas were cleaned with 70% ethanol and incised with sterilized tweezers and scissors. They took the seminomas and put them in a sterile 2ml Eppendorf tube, then stored them in dry ice until they were ready to use for profiling. Note, dissection tools used in this sample collection were used only once per mouse.
Blood samples were collected from the microvettes and then put into a bin for 10min at 5000rpm to get the blood to clot.
Placenta samples collected at E18.5 were fixed in 4% paraformaldehyde at 4 °C, followed by embedding in paraffin and sectioned into 7-μm-thick slices. Sections of samples were mounted on slides and dried on a hot plate. The dried sections were then placed in a container at a temperature of 4 C and put into a solution of anti-mouse VE-cadherin. catalogue no.14-1441-81; 2.5 μg ml−1) and detected by anti-Rat-Alexa Fluor 568 (Thermo Fisher Scientific; catalogue no. A-11088; 4 g cm. Nuclei were counterstained with 4′,6- diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific; catalogue no. D1306; 5 g. Slides were washed before being mounted with ProLong Glass (Thermo Fisher Scientific; catalogue no. P36983) and imaged on a Leica THUNDER Imager Live Cell with a ×20 objective (numerical aperture = 0.8). The areas of maze and whole placenta were measured with the QuPath software.
The concentration of a specific target is determined by the use of ELISA kits. Frozen plasma and testis samples were used for the measurement of leptin (Lep) in F0 sire males, whereas F1 placenta tissues were used for the measurement of placental growth factor (Plgf) and soluble fms-like tyrosine kinase-1 (sFlt1). Mouse leptin kit, mouse PlGF-2 The person is named ELISA. The kit had to be used for the quantative of the three variables after the manufacturer ordered it. Reagents, standard curve dilutions and samples were prepared as described on the kits and all samples, standards and control were assayed in technical duplicate, with several biological replicates. To quantify the target protein concentrations, the data generated were interpolated by nonlinear regression model curve fit.
Source: Paternal microbiome perturbations impact offspring fitness
Quantification of nABX and its residues in distal tissues: A complementary approach. Methods and results for the determination of antibiotics in testis tissues
To screen for antibiotic residues and test whether nABX or its residues are detectable in distal tissues or the circulatory system we took independent and complementary approaches.
Samples for the quantification of antibiotics in testis tissues were prepared as described in the section on Metabolomics below. The samples were resuspended in 20 liters of methanol:water and injected into the mass spectrometer. Calibration curves (twofold dilutions) were prepared using chemical standard resulting in the following ranges of injected amounts: bacteriocin (5.6–90.0 pmol), primaricin (7.0–112.5 pmol) and neomycin (5.3–85.0 pmol). The limits of detection and quantification of antibiotics were determined with the help of linear regression and R.
PremiTest kit (R-Biopharm) was used for the determination of nABX or avaABX residues in testis samples stored at −80 °C, which was performed in accordance with the manufacturer’s instructions for use in meat. The principle of the test is based on thermophilic bacterium spores (Bacillus stearothermophilus) which are highly sensitive to several antibiotics and sulfa compounds. After the testis were frozen, they were washed in 1PBS and then homogenized using a 2ml Dounce tissue grinder, containing 200 l of 1. Sulfurous water was used as negative control and nABX cocktail dilution in water was used as positive control at a concentration of less than the minimum detection limit. 100 l of the sample solution was pipetted onto the agar and used for prediffusion for 20 minutes. After the prediffusion, the sample solution was flushed out of the ampoule by washing twice with water and covered with foil to avoid evaporation. The testampules were placed in the block heater at 64 0.5 C for about 3– 3.5 h when the negative control turned from purple to yellow. The results were deduced on the basis of the kit bicolour indicator at this point, as well as the removed Ampoules.
Fresh faecal samples were collected from each individually housed F0 sire males at several different time points: (1) before nABX administration at day 0; (2) at the end of the 6 week treatment before setting up mating; and (3) during the recovery period following nABX withdrawal. The sample was collected at the end of the F1 child’s life. To collect fresh faecal samples, each parent or offspring for which faeces were to be collected was put into clean, bedding-free autoclaved cages with food and water for 2–3 h, faecal pellets collected with sterilized tweezer and stored immediately in −80 °C freezers until DNA extraction for microbiome profiling.
All samples were stored at −80 °C until processing by 16S rRNA sequencing. The QIAGEN PowerFecal Pro DNA Kit was used to perform microbbacterial DNA removal from faecal specimens, with instructions on how to use the kit. Briefly, faecal samples processed with the DNA extraction kit were added to a PowerBead beating tube and rapidly homogenized on a Vortex Adapter (QIAGEN catalogue no. 13000-V1-24) using Vortex-Genie 2 mixer (Scientific Industries). Once cells are lysed and potential inhibitors removed, total genomic DNA is captured on a silica membrane, washed and eluted for downstream gut microbiota profiling.
To derive the relative abundance of 16S rRNA gene copy number in mice faeces, a standard curve was generated using serial dilutions of the control sample.
Targeted amplification of the 16S rRNA V4 region (primer sequences F515 5′-GTGCCAGCMGCCGCGGTAA-3′ and R806 5′-GGACTACHVGGGTWTCTAAT-3′ (ref. The KAPA Hi Fi HotStartPCR mix is a two-step barcoded protocol that requires minor modifications from the manufacturer’s instructions. PCR products were pooled, purified using size-selective SPRIselect magnetic beads (0.8 left-sized) and then sequenced at 2 × 250 base pairs (bp) on an Illumina MiSeq (Illumina) at the Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg.
The reads were trimmed, denoised and passed through a process to remove chimaeric PCR artifacts. 61). The resulting amplicon sequence variants were then clustered into operational taxonomic units (OTUs) at 98% sequence similarity using an open-reference approach: reads were first mapped to a preclustered reference set of full-length 16S rRNA sequences at 98% similarity using MAPseq62. Reads that did not confidently map were aligned to bacterial and archaeal secondary structure-aware rRNA models using Infernal63 and clustered into OTUs with 98% average linkage using hpc-clust64, as described previously65. More than half of the spurious OTS were removed by noise filters, which claimed that samples retained at least 1,000 reads and taxa were common in at least two samples.
Local sample diversities were calculated as OTU richness, exponential Shannon entropy and inverse Simpson index (corresponding to Hill diversities of order 0, 1 and 2 (ref. 66) as average values of 100 rarefaction iterations to 5,000 reads per sample. Between-sample community diversity was calculated as Bray–Curtis dissimilarity. Trends in community composition were quantified using ordination methods (principal coordinate analysis, distance-based redundancy analysis) and tested using permutational multivariate analysis of variance (PERMANOVA67, as implemented in the R package vegan68.
Source: Paternal microbiome perturbations impact offspring fitness
High sensitivity roll circle amplification and library preparation for cryotranslation of testis spermatodine on superFrost Plus adhesion slides
The protocol was followed as described in ref. The score is 69. Briefly, mRNA transcripts were targeted from testis tissue cryosectioned at 10 μm thickness mounted on SuperFrost Plus adhesion slides. Sections were fixed in 3% formaldehyde, permeabilized with 0.1 M HCl for 5 min and washed with PBS. Using random compounds, the research team were able to reverse transcribed the mRNA on the 34th of March at 37 C. Following reverse transcription, tissue sections were washed with PBS and hybridized with 10 nM/PLP at a final concentration of 30 nM/PLP. After sections were washed with PBS, rolling circle amplification was performed overnight at 30 °C using phi29polymerase and exonuclease I (Thermo). After incubation, reagents were removed, samples washed twice in PBS and the SecureSeal chamber was removed with forceps, followed by hybridizing Bridge-probes (10 nM) for 1 h at room temperature in hybridization buffer (2× SSC, 20% formamide) in the dark, on a rocker. The detection probes were washed twice with PBS and stained once with DAPI after being hybridized in the dark. Sections were washed with PBS, mounted with about 20 l of Fluoromount-G mounting medium, and then stored at 4 C until imaged. Images were taken with a slide scanner and analysed using an interactive map.
The testicular single-cells resuspended in PBS + 0.04% BSA were adjusted to load on the 10x Chromium chip with a concentration of 1,100 cells. Cell capturing and library preparation were carried out according to instructions in the kit. v.3.1 (Dual Index) User Guide). In brief, about 6,000 cells were targeted for capture per sample; after complementary DNA synthesis, 12–14 cycles were used for library amplification. Illumina NextSeq 2000 was used to pool and sequence the libraries. P2 flowcell (100CYC) sequencing protocol.
The purification of sperm genomicDNA was achieved using the DNeasy Blood & Tissue Kit. The sequence of tasks that involved the DNA removal was as follows. The sperm samples were stored at a temperature of 80 C. Tris pH 8 is 20 mM. It is 200 mM. The sample was tested for sperm lysis at 56C on Eppendorf’s thermomixer and contained 80 mM dithiothreitol and 12 l ml1 of K, which was added to the sample. After incubation, the user-developed protocol DY03 (QIAGEN) for purification of total DNA from animal sperm was applied. BS-Seq libraries were constructed according to the manufacturer’s instructions using TruSeq DNA Methylation Library Prep Kit (Illumina). Amplified libraries were multiplexed and sequenced on an Illumina NextSeq 500 (PE75).
The gDNA was taken from the F1 offspring of control or nABX using the manufacturer’s instructions. To make sure that the integrity number was more than 7, an evaluation of the quantity and quality of DNA was done. The NEBNext Ultra II DNA Library Prep kit was following the guidelines of the manufacturer. The libraries were arrayed on an illumined Hiseq 4000 and put into a deep state.
Source: Paternal microbiome perturbations impact offspring fitness
Mass Profiler software for metabolic feature extraction from liquid chromatography-mass spectrometry data: application to the particle profiling (LC-MS) process
All chemicals for liquid chromatography–mass spectrometry (LC–MS) analysis including water and acetonitrile (LC–MS grade) were purchased from Fisher Scientific. Standards for online mass calibration were purchased from Agilent Technologies.
The MassHunter Qualitative Analysis Software (Agilent Technologies, v.10.0) was used to extract metabolic feature from the acquired LC–MS data. The following settings were implemented, the peak filter of absolute height: 5,000 counts, limit assigned charge states to 1 and only H+ charged molecules were included with compound quality scores greater than 80%. Peak alignment and identification were done using the Mass Profiler Professional with default parameters: mass tolerance of 20 ppm and retention time tolerance of 0.2 min or 2%. Extracted and aligned features were exported as .csv file for further data analysis
Source: Paternal microbiome perturbations impact offspring fitness
Nested t-test of F1-fetoplacental ratio and labyrinth of a genome-wide study of unmatched offspring with a given number of fathers
The statistical analyses were performed using graphical software and R.3.6.2. The replicate number is dependent on the number of uniquely exposed fathers (N) not on the number of offspring (n) which is greater, this is known as nested analyses. There is a single N value for each individual that comes from the same father, even if we generate n > 150 offspring per experiment to capture effect size and partially penetrant phenotypes. For example, a nested t-test compares the means of two unmatched groups (all F1 offspring from control or dysbiotic fathers), for which there is a nested factor in those treatment groups (shared father amongst each litter). This is necessary as using individual offspring as independent variables in intergenerational studies will lead to inflated alpha error rates and spurious significance. Testes-to-body weight ratio, fetoplacental ratio and labyrinth zone were analysed by two-tailed unpaired t-test. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed using with Baptista–Pike method and the statistical significance of the ORs determined using chi-squared test. The Kaplan–Meier method was applied to generate survival analysis curves. The curve fit for the Hyperbola model was acceptable.
Raw reads were aligned and mapped using the count module in 10x Genomics Cellranger 6.1.2 (ref. 75) to the mm10 transcriptome assembly (2020-A) with default parameters.
All subsequent steps were performed in R (v.4.1.2) using the Seurat package76. The cells wereFILTERED based on a number of unique genes detected and a number of mitochondria. The samples were clustered using the default clustering approach. In both conditions, clusters having no uniquely expressed genes (using the FindMarkers function with logfc.threshold = 0.5 and min.pct = 0.5) were discarded.
Source: Paternal microbiome perturbations impact offspring fitness
Study of intergenerational effects of paternal testicular leptin deficiency in C57BL/6J mice at the European Molecular Biology Laboratory
The guidelines and protocol for the laboratory animal management and ethics committee of the European Molecular Biology Laboratory were used for all experiments carried out with mice. 20190708_JH and the Italian Ministry of Health under authorization code no. There is a PR for this year’s and the year after. The primary mouse was created from the inbred C57BL/6J strain while the CD1 IGS or C57BL/6J dams were used as surrogate mothers. C57BL/6J mice deficient in leptin (ob/ob mice) were used to study the intergenerational effects of paternal testicular leptin deficiency. Mice were housed under a 12 h light/dark cycle (from 07:00 to 19:00), with ad libitum access to regular diet and water. Standard chow contained 18.5% protein, 5.3% fat, 4% fibre and other nutritional additives in pellet form and is suitable for long-term maintenance, breeding, lactation and gestation periods (NFM18, Mucedola).
The method of F1 offspring generation follows five critical steps: superovulation of oocyte-donor females, preparation of fresh sperm from sire males, collection of isogenic oocyte from donor females, collecting isogenic oocytes from donor females, in vitro sperm–oocyte fertilization, embryo transfer to pseudopregnant CD1 or C57BL/6J surrogate mothers. PremiTest kit was used for determination of nABX residues in testis samples stored at 80 C.