bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.313908; this version posted September 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Insights into the mechanism of bovine spermiogenesis based on comparative transcriptomic studies Xin Li 1, Chenying Duan 2, Ruyi Li 2, Dong Wang 1,* 1 Institute of Animal Science, Chinese Academy of Agricultural Sciences, 100193 Beijing, China 2 College of Animal Science and Technology, Jilin Agricultural University, 130118 Changchun, China * Corresponding author: Dong Wang E-mail: [email protected] Keywords: Spermiogenesis, Differentially expressed genes, Homology trends analysis, Protein-regulating network, ADP-ribosyltransferase 3 (ART3) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.313908; this version posted September 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. (2), resulting in a tremendous waste. At the same Abstract time, about 15% of the couples of childbearing age worldwide are affected by infertility, of To reduce the reproductive loss caused by which 50% are due to male factors (3), and even semen quality and provide theoretical guidance sperm with normal morphology can cause for the eradication of human male infertility, infertility (4). Therefore, it is necessary to differential analysis of the bovine transcriptome explore the mechanism of spermiogenesis, among round spermatids, elongated spermatids, identify important regulatory pathways and and epididymal sperm was carried out with the important functional genes to improve the semen reference of the mouse transcriptome, and the quality and yield of herd sire, and overcome the homology trends of gene expression to the problem of male infertility (2,5). mouse were also analysed. First, to explore the Since histones are gradually replaced by physiological mechanism of spermiogenesis protamine in the process of spermiogenesis, that profoundly affects semen quality, chromosomes are highly condensed, resulting in homological trends of differential genes were the overall shrinkage of the nucleus. At the same compared during spermiogenesis in dairy cattle time, the spermatid gradually polarises, and the and mice. Next, the Gene ontology (GO), Golgi bodies progressively aggregate and transfer Kyoto Encyclopedia of Genes and Genomes to one end of the spermatid, and finally become (KEGG) pathway enrichment, protein-protein specialised into the acrosome, forming the sperm interaction network (PPI network), and head together with the nucleus. The centrioles bioinformatics analysis uncovered the move in the direction opposite to the head and regulation network of acrosome formation extend outward to become the skeleton of the during the transition from round to elongated sperm tail, while mitochondria gradually spermatids. In addition, processes that regulate specialise to form a ring-shaped mitochondrial gene expression during spermiogenesis from sheath, which is attached to the outside of the elongated spermatid to epididymal sperm, such axon filament in the midpiece of the sperm tail. as ubiquitination, acetylation, deacetylation, At the same time, the cytoplasmic residue glycosylation, and the functional gene ART3 gradually fell off. Following these changes, the may play an important role during round spermatid eventually develops into the spermiogenesis. Therefore, its localisation in tadpole-like sperm with head and tail, which are the seminiferous tubule was investigated by mature and stored for a short time in the immunofluorescent analysis, and its structure epididymis (Figure 1). and function were also predicted. This study However, abnormal expression of some provides important data for revealing the important functional genes during mystery of life during spermiogenesis resulting spermiogenesis affects the morphology (6) and from acrosome formation, histone replacement, structure of the sperm, endanger the function of and the fine regulation of gene expression. the sperm, and lead to male sterility (7). The quality standard for frozen bovine semen (GB4143-2008) stipulates that the proportion of Introduction morphologically abnormal spermatozoa in qualified bovine semen should be less than 18%, With the extensive application of artificial while the Real-World Health Organization insemination, breeders have progressively Human Semen Analysis Laboratory Technical focused on semen requirements for the quality Manual (Fifth Edition) stipulates that the and quantity of excellent bulls. Approximately 10 proportion of morphologically abnormal to 60 billion sperms can be obtained from bulls spermatozoa in human semen should be less than when the semen is collected at a frequency of 4%. Therefore, understanding the regulatory three times a week (1). Considering the presence mechanism of spermiogenesis and mining of 20 million sperms per straw and a calving rate important functional genes have become the of 53% (2), each bull can theoretically breed focus of exploration, prevention, and control of about 13,000 to 78,000 offspring yearly. the causes of male breeding difficulty (8,9). However, due to semen quality issuses caused by Therefore, we performed transcriptome insufficient spermiogenesis, the pregnancy rate of analysis of round spermatids, elongated each straw of frozen semen varied by about 20% 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.25.313908; this version posted September 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. spermatids, and epididymal sperms of cattle. In samples R1, R2, R3, elongated spermatid order to identify differentially expressed genes samples E1, E2, E3, and epididymal sperm that play an important role in spermiogenesis, the samples M1, M2, and M3 were clustered together. same stage mouse transcriptome sequencing data The PCA results (figure 2B) showed that the sum were introduced as a reference. Homology of PC1 (83.9%) and PC2 (12.9%) of mice was comparison analysis between species was used to 96.8%, among which round spermatid samples obtain bovine genes with the same expression SRR3395024, SRR3395025, and SRR3395026, tendency in mice. Through PPI network, GO and elongated spermatid samples SRR3395030, KEGG pathway enrichment, and bioinformatic SRR3395031, SRR3395032, and epididymal analysis of these genes, we initially obtained the sperm samples SRR4423201, SRR4423202, and protein interaction networks and pathways SRR4423204 were clustered together. related to spermiogenesis, such as acrosomal and This showed that the transcripts of various protamine replacement histones. At the same types of spermatids from bulls and mice showed time, the structure and function of the important high intra-group repeatability, and the differences functional gene ART3 were predicted, and the between the groups were large, so that the localisation of the ART3 protein in the samples were representative. seminiferous tubules was analysed by immunofluorescence. These results provide quantitative real-time PCR (qPCR) validation important clues and a theoretical basis for further of the bull RNA-Seq results exploration of the spermiogenesis mechanism, The relative expression of 11 randomly and play a positive role in improving the selected genes was higher in round and elongated reproductive potential of cattle and promoting the spermatids than in sperm, which was consistent exploration of human infertility mechanisms. with the variations from RNA-seq analysis (Fig.3). Among these genes, the relative Results expression levels of LMTK2 and MIGA2 showed no significant difference (p >0.05) in the sperm Analysis of sequencing results of bull deformation stage; the relative expression level of spermatids and sperm DNAL1 was significantly reduced from round After the quality control of the original spermatid to elongated spermatid (p ＜ 0.05); sequencing data, the number of clean reads for ART3, HIP1, SDHA, and YBX2 showed each sample was more than 66.61 million, which significant reduction in relative expression levels accounted for more than 76.7% of the original from elongated spermatid to epididymal sequencing data, and the Clean Q30 Bases ratio spermatid (p <0.05). The relative expression of each sample was higher than 91%, which levels of TEKT2 and PRKAR1A were showed that the sequencing data could be used in significantly reduced from both round spermatid subsequent experiments (Table 1). After quality to elongated spermatid and elongated spermatid control, the data were compared with the bovine to epididymal sperm (p <0.05); the relative reference genome, the alignment ratio of clean expression level of OAZ3 was significantly reads was higher than 86% for each sample increased from round to elongated spermatid (p (Table 1), showing that the sequencing results <0.05), and a significant decline from elongated could cover most of the reference genome, and spermatid to epididymal sperm (p <0.01). The further analyses could reveal the biological qPCR results validated the RNA-Seq results, information