Integrated analysis of lncRNA, miRNA and mRNA expression profiles reveals regulatory pathways associated with pig testis function.

Long noncoding RNA (lncRNA) and microRNA (miRNA) are known to play pivotal roles in mammalian testicular function and spermatogenesis. However, their impact on porcine male reproduction has yet to be well unraveled. Here, we sequenced and identified lncRNA and miRNA expressed in the testes of Chinese indigenous Banna mini-pig inbred line (BMI) and introduced Western Duroc (DU) and Large White (LW) pigs. By pairwise comparison (BMI vs DU, BMI vs LW, and DU vs LW), we found the gene expression differences in the testes between Chinese local pigs and introduced Western commercial breeds were more striking than those between introduced commercial breeds. Furthermore, we found 1622 co-differentially expressed genes (co-DEGs), 122 co-differentially expressed lncRNAs (co-DELs), 39 co-differentially expressed miRNAs (co-DEMs) in BMI vs introduced commercial breeds (DU and LW). Functional analysis revealed that these co-DEGs and co-DELs/co-DEMs target genes were enriched in male sexual function pathways, including MAPK, AMPK, TGF-ß/Smad, Hippo, NF-kappa B, and PI3K/Akt signaling pathways. Additionally, we established 10,536 lncRNA-mRNA, 11,248 miRNA-mRNA pairs, and 62 ceRNA (lncRNA-miRNA-mRNA) networks. The ssc-miR-1343 had the most interactive factors in the ceRNA network, including 20 mRNAs and 3 lncRNAs, consisting of 56 ceRNA pairs. These factors played extremely important roles in the regulation of testis function as key nodes in the interactive regulatory network. Our results provide insight into the functional roles of lncRNAs and miRNAs in porcine testis and offer a valuable resource for understanding the differences between Chinese indigenous and introduced Western pigs.

Late Magmatic Activity in the Tarim Large Igneous Province: Evidence from the Puchang Granites.

The magmatic evolution sequence and tectonic activity background of the Permian Tarim large igneous province were discussed based on the research of petrogeochemistry, chronology, and zircon Lu-Hf isotope of two types of granite distributed in the Puchang V-Ti-magnetite deposit in the northwestern margin of Tarim. The results show that the two types are characterized by high silica, alkali, and potassium content. Both types of granites are enriched with light rare earth elements, including porphyritic granites with a negative anomaly of Eu and biotite monzogranites with a weak positive anomaly of Eu. The trace elements generally show that the large-ion lithophile elements are enriched relative to the high-field-strength elements, P and Ti are obviously depleted, and Rb, U, and K are relatively enriched. The zircon U-Pb age of porphyritic granites is 275.7 ± 1.0 Ma, the εHf(t) value ranges from -6.6 to -3.3, and the tDM2 ranges from 1469 to 1684 Ma. The zircon U-Pb age of biotite monzogranites is 268.9 ± 1.4 Ma. The εHf(t) value ranges from -1.2 to 0.5, and the tDM2 ranges from 1232 to 1340 Ma. The two types of granite were formed in a postcollisional extensional environment at the end of the early Permian, which is related to the Permian Tarim mantle activity and asthenosphere upwelling. The porphyritic granite was derived from an enriched lithospheric mantle; the biotite monzogranite was formed by the melting of mantle with crust contamination; and basalt and intrusive complexes in the research area were formed in an extensional intraplate rift environment.

Long- and short-read RNA sequencing from five reproductive organs of boar.

The production of semen in boars involves multiple reproductive glands, including the testis (Tes), epididymis (Epi), vesicular gland (VG), prostate gland (PG), and bulbourethral gland (BG). However, previous studies on boar reproduction primarily focused on the testis, with little attention paid to the other glands. Here, we integrated single-molecule long-read sequencing with short-read sequencing to characterize the RNA landscape from five glands of Banna mini-pig inbred line (BMI) and Diannan small-ear pigs (DSE). We identified 110,996 full-length isoforms from 22,298 genes, and classified the alternative splicing (AS) events in these five glands. Transcriptome-wide variation analysis indicated that the number of single nucleotide polymorphisms (SNPs) in five tissues of BMI was significantly lower than that in the non-inbred pig, DSE, revealing the effect of inbreeding on BMI. Additionally, we performed small-RNA sequencing and identified 299 novel miRNAs across all glands. Overall, our findings provide a comprehensive overview of the RNA landscape within these five glands, paving the path for future investigations on reproductive biology and the impact of inbreeding on pig transcriptome.

Molecular characteristics and transcriptional regulatory of spermatogenesis-related gene RFX2 in adult Banna mini-pig inbred line (BMI).

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.

Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing†.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Septin14, a gene specifically expressed in the testis and seminal vesicle of the Banna mini-pig inbred line (BMI).

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.