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Understanding the cell molecular changes occurring as a results of climatic circumstances is crucial in the current days in which climate change and global warming are one of the most serious challenges that living organisms have to face. Sperm are one of the mammals' cells most sensitive to heat, therefore evaluating the impact of seasonal changes in terms of its transcriptional activity can contribute to elucidate how these cells cope with heat stress events. We sequenced the total sperm RNA from 64 ejaculates, 28 collected in summer and 36 collected in autumn, from 40 Manchega rams. A highly rich transcriptome (11,896 different transcripts) with 90 protein coding genes that exceed an average number of 5000 counts were found. Comparing transcriptome in the summer and autumn ejaculates, 236 significant differential abundance genes were assessed, most of them (228) downregulated. The main functions that these genes are related to sexual reproduction and negative regulation of protein metabolic processes and kinase activity. Sperm response to heat stress supposes a drastic decrease of the transcriptional activity, and the upregulation of only a few genes related with the basic functions to maintain the organisms' homeostasis and surviving. Rams' spermatozoids carry remnant mRNAs which are retrospectively indicators of events occurring along the spermatogenesis process, including abiotic factors such as environmental temperature., Competing Interests: The authors have declared that no competing interests exist.

Background: Genetic pressure in animal breeding is sparking the interest of breeders for selecting elite boars with higher sperm quality to optimize ejaculate doses and fertility rates. However, the molecular basis of sperm quality is not yet fully understood. Our aim was to identify candidate genes, pathways and DNA variants associated to sperm quality in swine by analysing 25 sperm-related phenotypes and integrating genome-wide association studies (GWAS) and RNA-seq under a systems biology framework., Results: By GWAS, we identified 12 quantitative trait loci (QTL) associated to the percentage of head and neck abnormalities, abnormal acrosomes and motile spermatozoa. Candidate genes included CHD2, KATNAL2, SLC14A2 and ABCA1. By RNA-seq, we identified a wide repertoire of mRNAs (e.g. PRM1, OAZ3, DNAJB8, TPPP2 and TNP1) and miRNAs (e.g. ssc-miR-30d, ssc-miR-34c, ssc-miR-30c-5p, ssc-miR-191, members of the let-7 family and ssc-miR-425-5p) with functions related to sperm biology. We detected 6128 significant correlations (P-value ≤ 0.05) between sperm traits and mRNA abundances. By expression (e)GWAS, we identified three trans-expression QTL involving the genes IQCJ, ACTR2 and HARS. Using the GWAS and RNA-seq data, we built a gene interaction network. We considered that the genes and interactions that were present in both the GWAS and RNA-seq networks had a higher probability of being actually involved in sperm quality and used them to build a robust gene interaction network. In addition, in the final network we included genes with RNA abundances correlated with more than four semen traits and miRNAs interacting with the genes on the network. The final network was enriched for genes involved in gamete generation and development, meiotic cell cycle, DNA repair or embryo implantation. Finally, we designed a panel of 73 SNPs based on the GWAS, eGWAS and final network data, that explains between 5% (for sperm cell concentration) and 36% (for percentage of neck abnormalities) of the phenotypic variance of the sperm traits., Conclusions: By applying a systems biology approach, we identified genes that potentially affect sperm quality and constructed a SNP panel that explains a substantial part of the phenotypic variance for semen quality in our study and that should be tested in other swine populations to evaluate its relevance for the pig breeding sector.

Transmission Ratio Distortion (TRD), the uneven transmission of an allele from a parent to its offspring, can be caused by allelic differences affecting gametogenesis, fertilization or embryogenesis. However, TRD remains vaguely studied at a genomic scale. We sequenced the diploid and haploid genomes of three boars from leukocytes and spermatozoa at 50x to shed light into the genetic basis of spermatogenesis-caused Allelic Ratio Distortion (ARD). We first developed a Binomial model to identify ARD by simultaneously analysing all three males. This led to the identification of 55 ARD SNPs, most of which were animal-specific. We then evaluated ARD individually within each pig by a Fisher's exact test and identified two shared genes (TOP3A and UNC5B) and four shared genomic regions harbouring distinct ARD SNPs in the three boars. The shared genomic regions contained candidate genes with functions related to spermatogenesis including AK7, ARID4B, BDKRB2, GSK3B, NID1, NSMCE1, PALB2, VRK1 and ZC3H13. Using the Fisher's test, we also identified 378 genes containing variants with protein damaging potential in at least one boar, a high proportion of which, including FAM120B, TDRD15, JAM2 or AOX4 among others, are associated to spermatogenesis. Overall, our results show that sperm is subjected to ARD with variants associated to a wide variety of genes involved in different stages of spermatogenesis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Circular RNAs (circRNAs) are emerging as a novel class of noncoding RNAs which potential role as gene regulators is quickly gaining interest. circRNAs have been studied in different tissues and cell types across several animal species. However, a thorough characterization of the circRNAome in ejaculated sperm remains unexplored. In this study, we profiled the sperm circRNA catalogue using 40 porcine ejaculates. A complex population of 1,598 circRNAs was shared in at least 30 of the 40 samples. Generally speaking, the predicted circRNAs presented low abundances and were tissue-specific. Around 80% of the circRNAs identified in the boar sperm were reported as novel. Results from abundance correlation between circRNAs and miRNAs together with the prediction of microRNA (miRNA) target sites in circRNAs suggested that circRNAs may act as miRNA sponges. Moreover, we found significant correlations between the abundance of 148 exonic circRNAs and sperm motility parameters. Two of these correlations, involving ssc_circ_1458 and ssc_circ_1321, were confirmed by RT-qPCR using 36 additional samples with extreme and opposite sperm motility values. Our study provides a thorough characterization of circRNAs in sperm and suggests that circRNAs hold potential as noninvasive biomarkers for sperm quality and male fertility.

The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPure TM ; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome., Abbreviations: FPKM: fragments per kilobase of transcript per million mapped reads; KRT1: keratin 1; miRNA: micro-RNA; miscRNA: miscellaneous RNA; Mt rRNA: mitochondrial ribosomal RNA; Mt tRNA: mitochondrial transference RNA; OAZ3: ornithine decarboxylase antizyme 3; ORT: osmotic resistance test; piRNA: Piwi-interacting RNA; PRM1: protamine 1; PTPRC: protein tyrosine phosphatase receptor type C; rRNA: ribosomal RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRR: sperm recovery rate; tRNA: transfer RNA.

Having been debated for many years, the presence and role of spermatozoal RNAs is resolving, and their contribution to development is now appreciated. Data from different species continue show that sperm contain a complex suite of coding and noncoding RNAs that play a role in an individual's life course. Mature sperm RNAs provide a retrospective of spermatogenesis, with their presence and abundance reflecting sperm maturation, fertility potential, and the paternal contribution to the developmental path the offspring may follow.Sperm RNAs delivered upon fertilization provide some of the initial contacts with the oocyte, directly confront the maternal with the paternal contribution as a prelude to genome consolidation. Following syngamy, early embryo development may in part be modulated by paternal RNAs that can include epidydimal passengers. This provides a direct path to relay an experience and then initiate a paternal response to the environment to the oocyte and beyond. Their epigenetic impact is likely felt prior to embryonic genome activation when the population of sperm delivered transcripts markedly changes. Here, we review the insights gained from sperm RNAs over the years, the subtypes, and the caveats of the RNAs described. We discuss the role of sperm RNAs in fertilization and embryo development, and their possible mechanism(s) influencing offspring phenotype. Approaches to meet the future challenges as the study of sperm RNAs continues, include, elucidating the potential mechanisms underlying how paternal allostatic load, the constant adaptation of health to external conditions, may be relayed by sperm RNAs to affect future generations.

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements. These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars. [ABSTRACT FROM AUTHOR]

The microbiome plays a key role in homeostasis and health and it has been also linked to fertility and semen quality in several animal species including swine. Despite the more than likely importance of sperm bacteria on the boar's reproductive ability and the dissemination of pathogens and antimicrobial resistance genes, the high throughput characterization of the swine sperm microbiome remains scarce. We carried RNA-seq on 40 ejaculates each from a different Pietrain boar and found that a proportion of the sequencing reads did not map to the Sus scrofa genome. The current study aimed at using these reads not belonging to pig to carry a pilot study to profile the boar sperm bacterial population and its relation with 7 semen quality traits. We found that the boar sperm contains a broad population of bacteria. The most abundant phyla were Proteobacteria (39.1%), Firmicutes (27.5%), Actinobacteria (14.9%) and Bacteroidetes (5.7%). The predominant species contaminated sperm after ejaculation from soil, faeces and water sources (Bacillus megaterium , Brachybacterium faecium , Bacillus coagulans). Some potential pathogens were also found but at relatively low levels (Escherichia coli , Clostridioides difficile , Clostridium perfringens , Clostridium botulinum and Mycobacterium tuberculosis). We also identified 3 potential antibiotic resistant genes from E. coli against chloramphenicol, Neisseria meningitidis against spectinomycin and Staphylococcus aureus against linezolid. None of these genes were highly abundant. Finally, we classified the ejaculates into categories according to their bacterial features and semen quality parameters and identified two categories that significantly differed for 5 semen quality traits and 13 bacterial features including the genera Acinetobacter , Stenotrophomonas and Rhodobacter. Our results show that boar semen contains a bacterial community, including potential pathogens and putative antibiotic resistance genes, and that these bacteria may affect its reproductive performance. • A pilot RNA-seq study in 40 Pietrain ejaculates to characterize the porcine sperm microbiome. • Most bacteria in our pig sperm samples were contaminants from soil, water and faeces. • Potential pathogens were scarce and the most abundant was Escherichia coli. • Resistance genes against chloramphenicol, spectinomycin and linezolid were predicted. • LINK-HD draw two sample clusters defined by 13 bacterial features and 5 semen traits. [ABSTRACT FROM AUTHOR]

Understanding the molecular basis of cell function and ultimate phenotypes is crucial for the development of biological markers. With this aim, several RNA-seq studies have been devoted to the characterization of the transcriptome of ejaculated spermatozoa in relation to sperm quality and fertility. Semen quality follows a seasonal pattern and decays in the summer months in several animal species. The aim of this study was to deeply profile the transcriptome of the boar sperm and to evaluate its seasonal changes. We sequenced the total and the short fractions of the sperm RNA from 10 Pietrain boars, 5 collected in summer and 5 five sampled in winter, and identified a complex and rich transcriptome with 4,436 coding genes of moderate to high abundance. Transcript fragmentation was high but less obvious in genes related to spermatogenesis, chromatin compaction and fertility. Short non-coding RNAs mostly included piwi-interacting RNAs, transfer RNAs and microRNAs. We also compared the transcriptome of the summer and the winter ejaculates and identified 34 coding genes and 7 microRNAs with a significantly distinct distribution. These genes were mostly related to oxidative stress, DNA damage and autophagy. This is the deepest characterization of the boar sperm transcriptome and the first study linking the transcriptome and the seasonal variability of semen quality in animals. The annotation described here can be used as a reference for the identification of markers of sperm quality in pigs. [ABSTRACT FROM AUTHOR]