A first insight into the boar sperm transcriptome

PLoS ONE, 2013

Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.

Animal Reproduction Science, 2020

BMC Veterinary Research, 2020

Background Sperm hyperactive motility has previously been shown to influence litter size in pigs, but little is known about the underlying biological mechanisms. The aim of this study was to use RNA sequencing to investigate gene expression differences in testis tissue from Landrace and Duroc boars with high and low levels of sperm hyperactive motility. Boars with divergent phenotypes were selected based on their sperm hyperactivity values at the day of ejaculation (day 0) (contrasts (i) and (ii) for Landrace and Duroc, respectively) and on their change in hyperactivity between day 0 and after 96 h liquid storage at 18 °C (contrast (iii)). Results RNA sequencing was used to measure gene expression in testis. In Landrace boars, 3219 genes were differentially expressed for contrast (i), whereas 102 genes were differentially expressed for contrast (iii). Forty-one differentially expressed genes were identified in both contrasts, suggesting a functional role of these genes in hyperactiv...

Animal Reproduction Science, 2009

The existence of specific messenger RNA remnants contained within freshly ejaculated spermatozoa was described in several species. Those investigations, using high-throughput techniques to screen the population of transcripts in ejaculated spermatozoa, were limited to the probes which mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated spermatozoa of the domestic swine (Sus scrofa), a valuable model for biomedical research. A non-redundant 5 -end complementary DNA library was generated from swine ejaculated spermatozoa. After sequence quality verification, 4562 clones remained. These clones were then clustered and assembled into 514 unique sequences including 188 contigs (36.58%) and 326 singletons (63.42%), representing those clusters containing at least two clones and those clusters without having enough similarity with other clones. These unique gene sequences were annotated in Gene Ontology (GO) hierarchy; they included biological processes (38.7%), molecular functions (39.1%) and cellular components (40.3%). Based on the analysis, a broad spectrum of messenger RNAs existed in swine ejaculated spermatozoa and was closely correlated with nucleic acid binding, structural modifications, and transcriptional regulation. All of these categories are considered to have profound effects on the male reproductive system. Therefore, our work provides initial results on potential spermatozoal gene expression for future studies regarding the tightly regulated spermiogenic processes and later fertilization events.

Theriogenology, 2019

In recent years, genomic and proteomic biomarkers have been identified for the diagnosis of male fertility to overcome the limitations of conventional semen analysis. Owing to the limited genes available so far, the single gene approach is commonly adopted for analyzing the phenotype of interest. However, the single-gene approach is less effective than multiplegene strategies for diagnosing a desirable phenotype. Herein, we investigate the ability of two fertility-related genomic markers (porcine seminal protein (PSP)-I and PSP-II) in spermatozoa to predict boar litter size in addition to conventional semen parameters. First, we examined different semen parameters (motility, motion kinematics, and capacitation status) and gene expression in high-and low-litter size boar spermatozoa. Then, we evaluated the correlation of these parameters with the fertility of 21 Yorkshire boars. Finally, we investigated the efficacy of single/combined markers to predict male fertility using a comprehensive statistical model. Our result showed that there were no significant differences in sperm motility, motion kinematics, or capacitation status, however, the mRNA expression of PSP-I and PSP-II in spermatozoa was significantly different in high-and low-litter size boars. In the individual screening test, the expression of both genes was negatively correlated with boar fertility (r=0-.578 and-0.456, respectively), whereas only hyperactivation (HYP) showed a positive correlation (r=0.444) among the tested semen parameters. As single markers, PSP-I and PSP-II have a better diagnostic power to predict boar fertility, regardless of HYP, in quality assessment analyses. In addition, when these markers were combined, the positive predictive value, negative predictive value, and overall test effectiveness for fertility detection were improved. Surprisingly, when PSP-I and PSP-II were considered together, the deviation of the predicted average litter size between high-and low-litter size boars was 1.77. Based on the findings, we suggest that the use of genomic markers in spermatozoa rather than

Animal reproduction science, 2014

Sperm motility is one of the most widely used parameters in order to evaluate boar semen quality. However, this trait can only be measured after puberty. Thus, the use of genomic information appears as an appealing alternative to evaluate and improve selection for boar fertility traits earlier in life. With this study we aimed to identify SNPs with significant association with sperm motility in two different commercial pig populations and to identify possible candidate genes within the identified QTL regions. We performed a single-SNP genome-wide association study using genotyped animals from a Landrace-based (L1) and a Large White-based (L2) pig populations. For L1, a total of 602 animals genotyped for 42,551 SNPs were used in the association analysis. For L2, a total of 525 animals genotyped for 40,890 SNPs were available. After the association analysis, a false discovery rate q-value ≤0.05 was used as the threshold for significant association. No SNPs were significantly associate...

Systems biology in reproductive medicine, 2018

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; (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...

Animal Reproduction Science, 2010

Seasonal infertility is a well-known problem in the modern swine (Sus scrofa) industry. The molecular mechanisms responsible for thermal effects on spermatogenesis are, however, just beginning to be elucidated. The existence of specific messenger RNA (mRNA) remnants contained within freshly ejaculated sperm has been identified in several species. Investigators have obtained differential RNA profiles of infertile men compared with fertile individuals; however, there are limited to the probes, which are mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated sperm of the domestic swine and uncover important clues regarding the molecular regulation of spermatogenesis under environmental thermo-impacts. We utilized the remnant mRNA collected from swine ejaculated sperm as the target source to detect the global gene expression in summer and in winter by swine sperm-specific oligonucleotide microarray. Sixty-seven transcripts were differentially expressed with statistical differences between seasons of sperm samples collected, including forty-nine in winter (49/67) and eighteen in summer (18/67). There were only 33 of these transcripts that could be annotated to gene ontology hierarchy with the database of Homo sapiens and their functions mostly were involved in variety of metabolic processes. Moreover, these studies also confirmed that significant differences of gene expression profiles were found in swine sperm when comparisons were made between ejaculates collected during the winter and the summer season under the subtropical area such as Taiwan. Even though most of the genes found in our experiments are still poorly understood in terms of their true functions in spermatogenesis, bioinformatics analysis suggested that they are involved in a broad spectrum of biochemical processes including gamete generation. These concordant profiles should permit the development of a non-invasive testing protocol to assess the functional capacity of sperm as well as a new molecular selection scheme for fine breeding swine.

Genetics Selection Evolution, 2020

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 dete...