Enhanced bovine genome annotation through integration of transcriptomics and epi-transcriptomics datasets facilitates genomic biology.

BACKGROUND: The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. RESULTS: A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. CONCLUSIONS: These validated results show significant improvement over current bovine genome annotations.

Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF).

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45 h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.

The Dynamic of PRAMEY Isoforms in Testis and Epididymis Suggests Their Involvement in Spermatozoa Maturation.

The preferentially expressed antigen in melanoma, Y-linked (PRAMEY) is a cancer/testis antigen expressed predominantly in bovine spermatogenic cells, playing an important role in germ cell formation. To better understand PRAMEY's function during spermatogenesis, we studied the dynamics of PRAMEY isoforms by Western blotting (WB) with PRAMEY-specific antibodies. The PRAMEY protein was assessed in the bovine testicular and epididymal spermatozoa, fluid and tissues, and as well as in ejaculated semen. The protein was further examined, at a subcellular level in sperm head and tail, as well as in the subcellular components, including the cytosol, nucleus, membrane, and mitochondria. RNA expression of PRAMEY was also evaluated in testis and epididymal tissues. Our WB results confirmed the previously reported four isoforms of PRAMEY (58, 30, 26, and 13 kDa) in the bovine testis and spermatozoa. We found that testicular spermatozoa expressed the 58 and 30 kDa isoforms. As spermatozoa migrated to the epididymis, they expressed two additional isoforms, 26 and 13 kDa. Similarly, the 58 and 30 kDa isoforms were detected only in the testis fluid, while all four isoforms were detected in fluid from the cauda epididymis. Tissue evaluation indicated a significantly higher expression of the 58 and 13 kDa isoforms in the cauda tissue when compared to both the testis and caput tissue (p < 0.05). These results indicated that testis samples (spermatozoa, fluid, and tissue) expressed predominantly the 58 and 30 kDa PRAMEY isoforms, suggesting their involvement in spermatogenesis. In contrast, the 26 kDa isoform was specific to epididymal sperm and the 13 kDa isoform was marked in samples derived from the cauda epididymis, suggesting their involvement in sperm maturation. Results from the sperm head and tail experiments indicated that the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda, suggesting this isoform may have a significant role in tail function. Additionally, the 13 kDa isoform increased significantly (p < 0.05) in the cytosol during epididymal passage and tended to increase in other subcellular components. The expression of PRAMEY in the sperm subcellular components during epididymal maturation suggests the involvement of PRAMEY, especially the 13 kDa isoform, in sperm motility.

The PRAME family of cancer testis antigens is essential for germline development and gametogenesis†.

Preferentially expressed antigen in melanoma (PRAME) belongs to a group of cancer/testis antigens that are predominately expressed in the testis and a variety of tumors, and are involved in immunity and reproduction. Much of the attention on PRAME has centered on cancer biology as PRAME is a prognostic biomarker for a wide range of cancers and a potential immunotherapeutic target. Less information is available about the PRAME family's function (s) during gametogenesis and in the overall reproduction process. Here, we review the current knowledge of the PRAME gene family and its function in germline development and gametogenesis. Members of the PRAME family are leucine rich repeat proteins, localized in nucleus and cytoplasm, with multifaceted roles in germ cells. As transcriptional regulators, the PRAME family proteins are involved in germline development, particularly in the maintenance of embryonic stem cell pluripotency, development of primordial germ cells, and differentiation/proliferation of spermatogenic and oogenic cells. The PRAME family proteins are also enriched in cytoplasmic organelles, such as rough endoplasmic reticulum, Golgi vesicle, germinal granules, centrioles, and play a role in the formation of the acrosome and sperm tail during spermiogenesis. The PRAME gene family remains transcriptionally active in the germline throughout the entire life cycle and is essential for gametogenesis, with some members specific to either male or female germ cells, while others are involved in both male and female gametogenesis. A potential molecular mechanism that underlies the function of PRAME, and is shared by gametogenesis and oncogenesis is also discussed.