Large-scale detection and characterization of interchromosomal rearrangements in normozoospermic bulls using massive genotype and phenotype data sets.

In this paper, we developed a highly sensitive approach to detect interchromosomal rearrangements in cattle by searching for abnormal linkage disequilibrium patterns between markers located on different chromosomes in large paternal half-sib families genotyped as part of routine genomic evaluations. We screened 5571 families of artificial insemination sires from 15 breeds and revealed 13 putative interchromosomal rearrangements, 12 of which were validated by cytogenetic analysis and long-read sequencing. These consisted of one Robertsonian fusion, 10 reciprocal translocations, and the first case of insertional translocation reported in cattle. Taking advantage of the wealth of data available in cattle, we performed a series of complementary analyses to define the exact nature of these rearrangements, investigate their origins, and search for factors that may have favored their occurrence. We also evaluated the risks to the livestock industry and showed significant negative effects on several traits in the sires and in their balanced or aneuploid progeny compared with wild-type controls. Thus, we present the most comprehensive and thorough screen for interchromosomal rearrangements compatible with normal spermatogenesis in livestock species. This approach is readily applicable to any population that benefits from large genotype data sets, and will have direct applications in animal breeding. Finally, it also offers interesting prospects for basic research by allowing the detection of smaller and rarer types of chromosomal rearrangements than GTG banding, which are interesting models for studying gene regulation and the organization of genome structure.

Genome-wide analysis of hybridization in wild boar populations reveals adaptive introgression from domestic pig.

The admixture of domestic pig into French wild boar populations has been monitored since the 1980s thanks to the existence of a cytogenetic difference between the two sub-species. The number of chromosomes is 2n = 36 in wild boar and 2n = 38 in pig, respectively. This difference makes it possible to assign the "hybrid" status to wild boar individuals controlled with 37 or 38 chromosomes. However, it does not make it possible to determine the timing of the hybridization(s), nor to guarantee the absence of domestic admixture in an animal with 2n = 36 chromosomes. In order to analyze hybridization in greater detail and to avoid the inherent limitations of the cytogenetic approach, 362 wild boars (WB) recently collected in different French geographical areas and in different environments (farms, free ranging in protected or unprotected areas, animals with 2n = 36, 37 or 38 chromosomes) were genotyped on a 70K SNP chip. Principal component analyses allowed the identification of 13 "outliers" (3.6%), for which the proportion of the genome of "domestic" origin was greater than 40% (Admixture analyses). These animals were probably recent hybrids, having Asian domestic pig ancestry for most of them. For the remaining 349 animals studied, the proportion of the genome of "wild" origin varied between 83% and 100% (median: 94%). This proportion varied significantly depending on how the wild boar populations were managed. Local ancestry analyses revealed adaptive introgression from domestic pig, suggesting a critical role of genetic admixture in improving the fitness and population growth of WB. Overall, our results show that the methods used to monitor the domestic genetic contributions to wild boar populations should evolve in order to limit the level of admixture between the two gene pools.

Meiotic Silencing in Pigs: A Case Study in a Translocated Azoospermic Boar.

Carriers of balanced constitutional reciprocal translocations usually present a normal phenotype, but often show reproductive disorders. For the first time in pigs, we analyzed the meiotic process of an autosome-autosome translocation associated with azoospermia. Meiotic process analysis revealed the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Additionally, γH2AX signals were observed on apparently synapsed autosomes other than the SSC1 or SSC15, as previously observed in Ataxia with oculomotor apraxia type 2 patients or knock-out mice for the Senataxin gene. Gene expression showed a downregulation of genes selected on chromosomes 1 and 15, but no upregulation of SSCX genes. We hypothesized that the total meiotic arrest observed in this boar might be due to the silencing of crucial autosomal genes by the mechanism referred to as meiotic silencing of unsynapsed chromatin (MSUC).

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development.

Several studies have demonstrated that overexposure to pesticides can reduce mammalian sperm quality, impairing male fertility. Chlorpyrifos (CPF), a widely used organophosphate pesticide, was shown to impair spermatogenesis by inducing the formation of highly reactive toxic intermediates. To gain further insight into the mechanisms underlying the cytotoxicity and genotoxicity of CPF, bovine spermatozoa were exposed in vitro to environmental CPF concentrations and the motility, in vitro fertilization rates, DNA fragmentation, chromatin alterations, and methylation patterns were assessed. Motility and in vitro fertilization rates were significantly reduced in spermatozoa exposed to CPF, while DNA fragmentation and putative chromatin deconstruction appeared to increase at higher pesticide concentrations. In situ hybridization was carried out with X and Y probes on sperm samples exposed to different CPF concentrations, and subsequent analysis highlighted a significant percentage of spermatozoa with a peculiar morphological malformation, in which a narrowing occurred at the level of the hybridization. Analysis of potential abnormalities in the methylation pattern of NESP55-GNAS and XIST promoters displayed no differentially methylated regions in GNAS promoter relative to the control, whereas spermatozoa exposed to 10 µg/mL CPF had increased methylation variance in one region of imprinted XIST promoter. Our results provide support that CPF can induce a genotoxic effect on spermatozoa, impairig their ability to fertilize and support preimplantation embryo development in vitro. These observations are worrying since altered levels of sporadic methylation in genes of male gametes may affect the success of reproduction and contribute to infertility. Environ. Mol. Mutagen. 60:85-95, 2019. © 2018 Wiley Periodicals, Inc.

Analysis of Meiotic Segregation Pattern and Interchromosomal Effects in a Bull Heterozygous for a 3/16 Robertsonian Translocation.

Robertsonian translocations are the most frequent chromosomal rearrangements detected in cattle. Here, we report on the detection of a new Robertsonian translocation between chromosomes BTA3 and BTA16. This rob(3;16) was dicentric, suggesting that its occurrence was recent. FISH analysis of decondensed sperm nuclei revealed a relatively low rate of unbalanced gametes produced by adjacent segregation (5.87%). In addition, and for the first time in bovines, a significant interchromosomal effect (ICE) was detected for 2 different autosomes: BTA17 (global disomy + nullisomy rate of 9%) and BTA20 (1.8%). These results suggest that ICE should be taken into consideration when assessing the putative effect of Robertsonian translocations on reproduction.

Meiotic Synapsis and Gene Expression Altered by a Balanced Y-Autosome Reciprocal Translocation in an Azoospermic Pig.

Meiotic sex chromosome silencing (MSCS) has been argued as a prerequisite for normal meiotic cell division progression during the synaptic prophase I stage. Furthermore, irregular asynapsis of autosomal axes at meiosis may be encompassing the lack of transcriptional activity normally observed for the X and Y sex chromosomes. Therefore, any chromosomal rearrangement compromising the normal mechanism of MSCS and/or the contrary, the normal meiotic transcriptional activity of autosomal chromosomes, may be observed as a meiotic and concomitant spermatogenesis arrest. Previously, we have described a Y-autosome translocation t(Y;13)(p1.3;q3.3) in an azoospermic boar. Its chromosome synapsis behavior by synaptonemal complex immunostaining and FISH analyses is documented here. Histone γH2AX protein foci appeared to be located at unsynapsed chromosomal segments (e.g., X chromosome univalents or unpaired multivalent segments), although interestingly a high proportion of primary spermatocytes showed full paired synaptonemal complex-multivalent configurations which were devoid of a γH2AX focus signal, indicating meiotic chromosome silencing. RT-qPCR analysis of testicular expression showed downregulation of 3 SSC13 genes (MLH1, SOX2, UBE2B) and upregulation of SSCY genes (ZFY, SRY). The irregularity of the normal transcription pattern in case of these genes with proven roles in the testis is in agreement with the cytological observations and could contribute to the observed phenotype.

Intraindividual Variation of Meiotic Recombination Parameters in Pig Spermatocytes: A Preliminary Study.

Meiotic recombination parameters like crossover (CO) rate or synaptonemal complex (SC) length are known to vary strongly between individuals and between cells from the same individual. The origins of this variability remain elusive, and little is known about the variations that might occur between different samples and/or over time within the same individual. To document this question, pachytene cells from 3 boars of the Large White breed were analyzed twice, at a 1-year interval, using immunocytological techniques. CO rate, SC length, and MLH1 inter-foci distances varied significantly between the 3 individuals. CO rate and SC length differed significantly between the 2 sampling periods for 1 individual. However, no significant differences were observed between the 2 samples for CO distribution and inter-foci distances in the 3 boars studied.

Corrigendum: Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

This corrects the article DOI: 10.1038/srep27059.

Identification of a t(3;4)(p1.3;q1.5) translocation breakpoint in pigs using somatic cell hybrid mapping and high-resolution mate-pair sequencing.

Reciprocal translocations are the most frequently occurring constitutional structural rearrangements in mammalian genomes. In phenotypically normal pigs, an incidence of 1/200 is estimated for such rearrangements. Even if constitutional translocations do not necessarily induce defects and diseases, they are responsible for significant economic losses in domestic animals due to reproduction failures. Over the last 30 years, advances in molecular and cytogenetic technologies have led to major improvements in the resolution of the characterization of translocation events. Characterization of translocation breakpoints helps to decipher the mechanisms that lead to such rearrangements and the functions of the genes that are involved in the translocation. Here, we describe the fine characterization of a reciprocal translocation t(3;4) (p1.3;q1.5) detected in a pig line. The breakpoint was identified at the base-pair level using a positional cloning and chromosome walking strategy in somatic cell hybrids that were generated from an animal that carries this translocation. We show that this translocation occurs within the ADAMTSL4 gene and results in a loss of expression in homozygous carriers. In addition, by taking this translocation as a model, we used a whole-genome next-generation mate-pair sequencing approach on pooled individuals to evaluate this strategy for high-throughput screening of structural rearrangements.

Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors.

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.

Azoospermia and Testicular Hypoplasia in a Boar Carrier of a Novel Y-Autosome Translocation.

Few sex-autosome chromosome abnormalities have been documented in domestic animal species. In humans, Y-autosome chromosome abnormalities may occur at a rate of 1/2,000 live births, whereas in the domestic pig only 2 Y-autosome reciprocal translocations have been previously described. During a routine cytogenetic screening of young boars, we identified a new Y-autosome translocation carrier, which after puberty showed semen devoid of sperm and testicular hypoplasia with spermatogenesis arrest. Whole chromosome painting by FISH analysis corroborated the reciprocal nature of the chromosomal exchanges between the Y chromosome and SSC13. The possible causes for the observed meiotic arrest of the carrier are reviewed.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex.

This study was designed to determine the ability of computer-assisted sperm morphometry analysis (CASA-Morph) with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa (SX and SY, respectively). Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average (P < 0.001) although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed (mixed SX and SY) and sexed (SX) semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa (P < 0.05). We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

Meiotic pairing and gene expression disturbance in germ cells from an infertile boar with a balanced reciprocal autosome-autosome translocation.

Individuals carrying balanced constitutional reciprocal translocations generally have a normal phenotype, but often present reproductive disorders. The aim of our research was to analyze the meiotic process in an oligoasthenoteratospermic boar carrying an asymmetric reciprocal translocation involving chromosomes 1 and 14. Different multivalent structures (quadrivalent and trivalent plus univalent) were identified during chromosome pairing analysis. Some of these multivalents were characterized by the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Gene expression in spermatocytes was studied by RNA-DNA-FISH and microarray-based testis transcriptome analysis. Our results revealed a decrease in gene expression for chromosomes 1 and 14 and an up-regulated expression of X-chromosome genes for the translocated boar compared with normal individuals. We hypothesized that the observed meiotic arrest and reproductive failure in this boar might be due to silencing of crucial autosomal genes (MSUC) and disturbance of meiotic sex chromosome inactivation (MSCI). Further analysis revealed abnormal meiotic recombination (frequency and distribution) and the production of a high rate of unbalanced spermatozoa.

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.

Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements.

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

Genome-Wide Study of Structural Variants in Bovine Holstein, Montbéliarde and Normande Dairy Breeds.

High-throughput sequencing technologies have offered in recent years new opportunities to study genome variations. These studies have mostly focused on single nucleotide polymorphisms, small insertions or deletions and on copy number variants. Other structural variants, such as large insertions or deletions, tandem duplications, translocations, and inversions are less well-studied, despite that some have an important impact on phenotypes. In the present study, we performed a large-scale survey of structural variants in cattle. We report the identification of 6,426 putative structural variants in cattle extracted from whole-genome sequence data of 62 bulls representing the three major French dairy breeds. These genomic variants affect DNA segments greater than 50 base pairs and correspond to deletions, inversions and tandem duplications. Out of these, we identified a total of 547 deletions and 410 tandem duplications which could potentially code for CNVs. Experimental validation was carried out on 331 structural variants using a novel high-throughput genotyping method. Out of these, 255 structural variants (77%) generated good quality genotypes and 191 (75%) of them were validated. Gene content analyses in structural variant regions revealed 941 large deletions removing completely one or several genes, including 10 single-copy genes. In addition, some of the structural variants are located within quantitative trait loci for dairy traits. This study is a pan-genome assessment of genomic variations in cattle and may provide a new glimpse into the bovine genome architecture. Our results may also help to study the effects of structural variants on gene expression and consequently their effect on certain phenotypes of interest.

Sperm DNA methylation analysis in swine reveals conserved and species-specific methylation patterns and highlights an altered methylation at the GNAS locus in infertile boars.

Male infertility is an increasing health issue in today's society for both human and livestock populations. In livestock, male infertility slows the improvement of animal selection programs and agricultural productivity. There is increasing evidence that epigenetic marks play an important role in the production of good-quality sperm. We therefore screened for specific or common epigenetic signatures of livestock infertility. To do so, we compared DNA methylation level in sperm DNA from fertile and infertile boars. We evaluated first the global level of sperm DNA methylation and found no difference between the two groups of boars. We then selected 42 loci of interest, most of them known to be imprinted in human or mice, and assessed the imprinting status of five of them not previously described in swine tissues: WT1, CNTN3, IMPACT, QPCT, and GRB10. DNA methylation level was then quantified in fertile and infertile boars at these 42 loci. Results from fertile boars indicated that the methylation level of the selected loci is highly conserved between pig, human, and mice, with a few exceptions, including the POU5F1 (OCT4) promoter and RTL1. Comparison between fertile and infertile boars revealed that one imprinted region, the GNAS locus, shows an increase in sperm DNA methylation in three out of eight infertile boars with low semen quality. This increase in DNA methylation is associated with an altered expression of the genes belonging to the GNAS locus, suggesting a new role for GNAS in the proper formation of functional gametes.

Meiotic recombination analyses of individual chromosomes in male domestic pigs (Sus scrofa domestica).

For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes) were identified by immunostaining and fluorescence in situ hybridization (FISH). The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers), on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells) and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18) and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.