Abstract PICK1 plays a crucial role in mammalian spermatogenesis. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine PICK1 expression patterns in adult Baoshan pig (BS) testes. We identified the most important transcript ENSSSCT00000000120 of PICK1, obtaining its full-length coding sequence (CDS) spanning 1254 bp. Gene structure analysis located PICK1 on pig chromosome 5 with 14 exons. Protein structure analysis reflected that PICK1 consisted of 417 amino acids containing two conserved domains, PDZ and BAR_PICK1. Phylogenetic analysis underscored the evolutionary conservation and homology of PICK1 across different mammalian species. Evaluation of protein interaction network, KEGG, and GO pathways implied that interacted with 50 proteins, predominantly involved in glutamatergic synapses, amphetamine addiction, neuroactive ligand-receptor interactions, dopaminergic synapses, and synaptic vesicle recycling, and PICK1 exhibited significant correlation with DLG4 and TBC1D20. Functional annotation identified that PICK1 was involved in 9 GOs, including seven cellular components and two molecular functions. ceRNA network analysis suggested BS PICK1 was regulated by seven miRNA targets. Moreover, qPCR expression analysis across 15 tissues highlighted that PICK1 was highly expressed in the bulbourethral gland and testis. Subcellular localization analysis in ST (Swine Tesits) cells demonstrated that PICK1 significantly localized within the cytoplasm. Overall, our findings shed new light on PICK1’s role in BS reproduction, providing a foundation for further functional studies of PICK1. PICK spermatogenesis Here singlemolecule single molecule longread long read shortread short (BS testes ENSSSCT ENSSSCT0000000012 fulllength full length CDS (CDS 125 bp 1 exons 41 domains BARPICK1 BARPICK BAR BAR_PICK1 species KEGG proteins synapses addiction ligandreceptor ligand receptor interactions recycling DLG TBC1D20 TBCD TBC D GOs functions targets Moreover testis Swine Tesits cytoplasm Overall PICK1s PICKs s reproduction ENSSSCT000000001 12 4 BAR_PICK TBC1D2 ENSSSCT00000000 TBC1D ENSSSCT0000000 ENSSSCT000000 ENSSSCT00000 ENSSSCT0000 ENSSSCT000 ENSSSCT00 ENSSSCT0

Abstract RFX2 plays critical roles in mammalian spermatogenesis and cilium maturation. Here, the testes of 12-month-old adult boars of Banna mini-pig inbred line (BMI) were subjected to whole-transcriptome sequencing. The results indicated that the average expression (raw count) of RFX2 gene in BMI testes was 16138.25, and the average expression value of the corresponding transcript ENSSSCT00000043271.2 was 123.1898. The CDS of RFX2 obtained from BMI testes was 2,817 bp (GenBank accession number: OL362242). Gene structure analysis showed that RFX2 was located on chromosome 2 of the pig genome with 19 exons. Protein structure analysis indicated that RFX2 contains 728 amino acids with two conserved domains. Phylogenetic analysis revealed that RFX2 was highly conserved with evolutionary homologies among mammalian species. Other analyses, including PPI networks, KEGG, and GO, indicated that BMI RFX2 had interactions with 43 proteins involving various functions, such as in cell cycle, spermatid development, spermatid differentiation, cilium assembly, and cilium organization, etc. Correlation analysis between these proteins and the transcriptome data implied that RFX2 was significantly associated with FOXJ1, DNAH9, TMEM138, E2F7, and ATR, and particularly showed the highest correlation with ATR, demonstrating the importance of RFX2 and ART in spermatogenesis. Functional annotation implied that RFX2 was involved in 17 GO terms, including three cellular components (CC), six molecular functions (MF), and eight biological processes (BP). The analysis of miRNA-gene targeting indicated that BMI RFX2 was mainly regulated by two miRNAs, among which four lncRNAs and five lncRNAs competitively bound ssc-miR-365-5p and ssc-miR-744 with RFX2, respectively. Further, the dual-luciferase report assay indicated that the ssc-miR-365-5p and ssc-miR-744 significantly reduced luciferase activity of RFX2 3'UTR in the 293T cells, suggesting that these two miRNAs regulated the expression of RFX2. Our results revealed the important role of RFX2 in BMI spermatogenesis, making it an intriguing candidate for follow-up studies. RFX maturation Here 12monthold monthold 12 month old minipig mini (BMI wholetranscriptome whole sequencing raw count 1613825 16138 25 16138.25 ENSSSCT000000432712 ENSSSCT ENSSSCT00000043271 ENSSSCT00000043271. 1231898 123 1898 123.1898 2817 817 2,81 GenBank number OL362242. OL362242 OL . OL362242) 1 exons 72 domains species analyses networks KEGG 4 cycle development differentiation assembly organization etc FOXJ1 FOXJ DNAH9 DNAH TMEM138 TMEM E2F7 EF E F ATR terms CC, CC , (CC) MF, MF (MF) BP. BP (BP) miRNAgene miRNA sscmiR3655p sscmiRp ssc miR 365 5p p sscmiR744 sscmiR 744 ssc-miR-74 respectively Further dualluciferase dual 3UTR UTR 3 T cells followup follow up studies 161382 1613 16138.2 ENSSSCT0000004327 123189 189 123.189 281 81 2,8 OL36224 7 TMEM13 E2F (CC (MF (BP 36 sscmiR74 74 ssc-miR-7 161 16138. ENSSSCT000000432 12318 18 123.18 28 8 2, OL3622 TMEM1 sscmiR7 ssc-miR- 16 ENSSSCT00000043 1231 123.1 OL362 ssc-miR ENSSSCT0000004 123. OL36 ENSSSCT000000 OL3 ENSSSCT00000 ENSSSCT0000 ENSSSCT000 ENSSSCT00 ENSSSCT0

Abstract Septin14 is an important spermatogenesis related gene involved in the pathogenesis of male infertility that has not been well studied. Here, full-length Septin14 cDNA of the Banna mini-pig inbred line (BMI) was cloned using the RACE method and expressed in pig kidney epithelial cells (PK15) and E. coli Rosetta (DE3) cells. Septin14 expression was identified in somatic tissues and testis in different developmental stages. The pig Septin14 CDS is 1,299 bp long, and encodes a peptide (or protein) of 432 amino acids (MW=50.4 kDa). Phylogenetic analysis indicated that pig Septin14 was highly evolutionarily conserved. Subcellular localization of GFP-tagged Septin14 fusion protein revealed that Septin14 was distributed throughout the testicular cells. Among 34 pig tissues, Septin14 mRNA was found specifically in testis and seminal vesicle. In six different postnatal developmental stages, the testicular level of Septin14 mRNA was barely detectable on day 2, while the highest level occurred on day 75. The spatiotemporal expression profile of Septin14, reported herein for the first time in pig, indicated that Septin14 might be involved in the division, development and apoptosis of germ cells. Furthermore, using a pET prokaryotic expression system, we expressed and isolated recombinant 67.9 kDa Septin14 protein.