Mammalian spermatozoa deliver various classes of RNAs to the oocyte during fertilization, and many of them may regulate fertility. The objective of the present study was to determine the composition and abundance of spermatozoal transcripts in fresh bull semen. The entire transcriptome of the spermatozoa from bulls (n = 3) was sequenced using two different platforms (Ion Proton and Illumina) to identify the maximum number of genes present in the spermatozoa. The bovine spermatozoa contained transcripts for 13,833 genes (transcripts per million, TPM > 10). Both intact and fragmented transcripts were found. These spermatozoal transcripts were associated with various stages of spermatogenesis, spermatozoal function, fertilization, and embryo development. The presence of intact transcripts of pregnancy-associated glycoproteins (PAGs) in the spermatozoa suggest a possible influence of sperm transcripts beyond early embryonic development. The specific regions (exon, intron, and exon-intron) of the particular spermatozoal transcripts might help regulate fertilization. This study demonstrates that the use of two different RNA-seq platforms provides a comprehensive profile of bovine spermatozoal RNA. Spermatozoal RNA profiling may be useful as a non-invasive method to delineate possible causes of male infertility and to predict fertility in a manner that is more effective than the conventional methods.

With artificial insemination (AI) and other precision dependent assisted reproductive technologies (ART) being followed in large scale in human and animal reproduction, assessing semen quality and fertilizability is under continuous scrutiny. Various tests have been developed to predict semen quality, but so far no single, highly reliable test is available. In this regard, transcriptomic profiling of spermatozoa assumes significance as it carries the information about spermatogenesis, sperm function, and paternal roles in post-fertilization events. Human spermatozoal transcriptome profiling has been carried out on a large number of individuals to predict the semen quality. A study in human indicated that the outcome of some idiopathic couples seeking reproductive care could be helped using transcriptomic profiling of spermatozoa. Such studies have a direct impact on the bovine dairy industry, wherein AI is practiced. Limited studies in bovine spermatozoal transcriptome profiling have revealed that the spermatozoa contain various classes of RNA, like in human. Approximately 13,000 bovine genes yield a series of spermatozoal transcripts, of which most are fragmented in nature. Their abundance is indicative of the timing of events associated with spermatogenesis, e.g., PRM1, IGF1, BMP2; sperm function, TSSK6, CRISP, HSFY2; fertility, UBE2D3, Integrin-β, LDC-1; and embryonic development, miR34c-5p, BCL2L11, BRCA1. The most abundant translated bovine transcripts are BSP3 and SPATA18, and are involved in regulation of germ cell development and the maintenance of chromatin integrity during spermatogenesis respectively. The presence of transcripts associated with placental development, e.g., placental associated glycoproteins (PAGs) have suggested their possible influence beyond early embryonic development. Changes in transcript levels like RPL31 and PRKCE that increase, and PRM1 that decreases, during cryopreservation need to be defined in order to optimize cryopreservation and fertility yield. Spermatozoal transcriptome profiling with validation studies are warranted in large numbers of animals to elucidate their significance for selecting fertile bulls for the breeding program. Abbreviations: AI: artificial insemination; BSE: breeding soundness evaluation; cfs-mRNA: cell-free seminal mRNA; piRNA: PIWI-interacting RNA; tRNA: transfer RNA; fg: femtogram; TPM: transcripts per million reads; RPKM: reads per kilobase million; rRNA: ribosomal RNA; mt-RNA: mitochondrial RNA; lncRNA: long non-coding RNA; sncRNA: small noncoding RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; miRNA: microRNA; snaR: small NF90-associated RNAs; SINES: short interspersed nuclear elements; LINES: long interspersed nuclear elements; MER: medium reiterated sequence; F1 offspring: filial 1 offspring; PAGs: placental associated glycoproteins; TCP: Transcription factor T complex protein; BSP3: bovine seminal plasma protein 3; SCNT: somatic cell nuclear transfer; qPCR: quantitative (real-time) polymerase chain reaction; SSH: suppression subt...

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This study aimed to identify sperm proteomic signatures regulating sperm functions and fertility by: (i) comparing the sperm electrophoretic protein profiles and identifying the differentially abundant proteins among breeding bulls differing in fertility status and (ii) elucidating the possible role of one of the identified novel proteins, PEBP4 on sperm function and fertility. The grouping of bulls as fertile (n = 6) and low fertile (n = 6) was performed based on bull fertility index and infertile (n = 6) based on semen rejection rate (>33%). The sperm motility, fructolysis index, acrosomal reaction, intracellular calcium levels, and seminal plasma fructose and calcium levels were studied among fertility groups. The differentially expressed sperm proteins observed in single- and two-dimensional gel electrophoresis (2DE) were identified using Nano-LC-MS/MS. In the fertile bulls, the expression levels of calmodulin (CALM1), spermadhesinZ13 (SPADH2), and phosphatidylethanolamine-binding protein 4 (PEBP4) were significantly (p < 0.05) higher than in other fertility groups. In bovine, expression of PEBP4 a novel seminal protein was not observed in spermatozoa of infertile bulls. When the bulls were grouped based on the presence (n = 8) or absence (n = 10) of PEBP4 protein in spermatozoa, a positive significant (p < 0.05) association of this protein with the percentage of motile, type-A spermatozoa, and sperm fructose uptake was observed. Further, PEBP4 was localized in elongated spermatids, Leydig cells, excurrent duct system, and principal piece of spermatozoa. These findings suggest a crucial role for the PEBP4 protein in spermiogenesis, epididymal sperm maturation, and sperm motility. This first study in bovine indicates the positive association of PEBP4 in regulating sperm maturation, functions, and fertility and could be a potential marker for predicting semen quality and fertility.