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The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 μm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival.

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 10 8 /ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 10 8 /ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 10 8 /ml) was higher compared to that of blastocysts (5.68 × 10 8 /ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos., Competing Interests: The RAT-ChIP method is subject to a patent application (P1433PC00) by TO, AK and AS.

Background: Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA., Methods: We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR., Results: We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication., Conclusion: Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches., Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%)., Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography-mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone-protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-to-protamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition., (© 2017 The Authors.)

The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish.

A significant proportion of mammalian fertilization is mediated through the proteomic composition of the sperm surface. These protein constituents can present as biomarkers to control and regulate breeding of agricultural animals. Previous studies have addressed the bovine sperm cell apical plasma membrane (PM) proteome with nitrogen cavitation enrichment. Alternative workflows would enable to expand the compositional data more globally around the entire sperm's surface. We used a cell surface biotin-labeling in combination with differential centrifugation to enrich sperm surface proteins. Using nano-LC MS/MS, 338 proteins were confidently identified in the PM-enriched proteome. Functional categories of sperm-egg interaction, protein turnover, metabolism as well as molecular transport, spermatogenesis, and signal transduction were represented by proteins with high quantitative signal in our study. A highly significant degree of enrichment was found for transmembrane and PM-targeted proteins. Among them, we also report proteins previously not described on bovine sperm (CPQ, CD58, CKLF, CPVL, GLB1L3, and LPCAT2B) of which CPQ and CPVL cell surface localization was further validated. A descriptive overview of the bovine sperm PM integral and peripheral proteins is provided to complement future studies on animal reproduction and its relation to sperm cell surface. All MS data have been deposited in the ProteomeXchange with identifier PXD001096 (http://proteomecentral.proteomexchange.org/dataset/PXD001096)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, we describe the deep sequencing and structural analysis of the Holstein breed bull genome. Our aim was to receive a high-quality Holstein bull genome reference sequence and to describe different types of variations in its genome compared to Hereford breed as a reference. We generated four mate-paired libraries and one fragment library from 30 μg of genomic DNA. Colour space fasta were mapped and paired to the reference cow (Bos taurus) genome assembly from Oct. 2011 (Baylor 4.6.1/bosTau7). Initial sequencing resulted in the 4,864,054,296 of 50-bp reads. Average mapping efficiency was 71.7 % and altogether 3,494,534,136 reads and 157,928,163,086 bp were successfully mapped, resulting in 60 × coverage. This is the highest coverage for bovine genome published so far. Tertiary analysis found 6,362,988 SNPs in the bull's genome, 4,045,889 heterozygous and 2,317,099 homozygous variants. Annotation revealed that 4,330,337 of all discovered SNPs were annotated in the dbSNP database (build 137) and therefore 2,032,651 SNPs were novel. Large indel variations accounted for the 245,947,845 bp of the variation in entire genome and their number was 312,879. We also found that small indels (number was 633,310) accounted for the total variation of 2,542,552 nucleotides in the genome. Only 106,768 small indels were listed in the dbSNP. Finally, we identified 2,758 inversions in the genome of the bull covering in total 23,099,054 bp of genome's variation. The largest inversion was 87,440 bp in size. In conclusion, the present study discovered different types of novel variants in bull's genome after high-coverage sequencing. Better knowledge of the functions of these variations is needed.

The aim of our study was to create a high-quality Holstein cow genome reference sequence and describe the different types of variations in this genome compared to the reference Hereford breed. We generated one fragment and three mate-paired libraries from genomic DNA. Raw files were mapped and paired to the reference cow (Bos taurus) genome assemblies bosTau6/UMD&#95;3.1. BioScope (v1.3) software was used for mapping and variant analysis. Initial sequencing resulted in 2,842,744,008 of 50-bp reads. Average mapping efficiency was 78.4 % and altogether 2,168,425,497 reads and 98,022,357,422 bp were successfully mapped, resulting in 36.7X coverage. Tertiary analysis found 5,923,230 SNPs in the bovine genome, of which 3,833,249 were heterozygous and 2,089,981 were homozygous variants. Annotation revealed that 4,241,000 of all discovered SNPs were annotated in the dbSNP database and 1,682,230 SNPs were considered as novel. Large indel variations accounted for 48,537,190 bp of the entire genome and there were 138,504 of them. The largest deletion was 18,594 bp and the largest insertion was 13,498 bp. Another group of variants, small indels (n = 458,061), accounted for the total variation of 1,839,872 nucleotides in the genome. Only 92,115 small indels were listed in the dbSNP and therefore 365,946 small indels were novel. Finally, we identified 1,876 inversions in the bovine genome. In conclusion, this is another description of the Holstein cow genome and, similar to previous studies, we found a large amount of novel variations. Better knowledge of these variations could explain significant phenotypic differences (e.g., health, production, reproduction) between different breeds.

In present study we describe the sequencing and annotated analysis of the individual genome of Estonian. Using SOLID technology we generated 2,449,441,916 of 50-bp reads. The Bioscope version 1.3 was used for mapping and pairing of reads to the NCBI human genome reference (build 36, hg18). Bioscope enables also the annotation of the results of variant (tertiary) analysis. The average mapping of reads was 75.5% with total coverage of 107.72 Gb. resulting in mean fold coverage of 34.6. We found 3,482,975 SNPs out of which 352,492 were novel. 21,222 SNPs were in coding region: 10,649 were synonymous SNPs, 10,360 were nonsynonymous missense SNPs, 155 were nonsynonymous nonsense SNPs and 58 were nonsynonymous frameshifts. We identified 219 CNVs with total base pair coverage of 37,326,300 bp and 87,451 large insertion/deletion polymorphisms covering 10,152,256 bp of the genome. In addition, we found 285,864 small size insertion/deletion polymorphisms out of which 133,969 were novel. Finally, we identified 53 inversions, 19 overlapped genes and 2 overlapped exons. Interestingly, we found the region in chromosome 6 to be enriched with the coding SNPs and CNVs. This study confirms previous findings, that our genomes are more complex and variable as thought before. Therefore, sequencing of the personal genomes followed by annotation would improve the analysis of heritability of phenotypes and our understandings on the functions of genome., (Copyright © 2011 Elsevier B.V. All rights reserved.)