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 minipig 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-beta/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.

【目的】Dickkopf样顶体蛋白1(DKKL1)是一种重要的顶体蛋白，为发掘其重要功能及潜在的临床价值，以版纳微型猪近交系(BMI)为对象，研究其睾丸组织中DKKL1的分子结构、转录调控特征和蛋白质功能。【方法】通过全转录组测序获得BMI DKKL1的表达量，使用逆转录聚合酶链反应(RT-PCR)获得DKKL1编码区序列并分析其基因结构和蛋白质特征，使用UniProt数据库对DKKL1进行功能注释并分析DKKL1与微小RNA(miRNA)、长链非编码RNA(lncRNA)间的竞争性内源RNA(ceRNA)转录调控。【结果】获得的BMI DKKL1编码区全长为702 bp,位于基因第6号染色体上，有5个外显子和4个内含子；蛋白质功能分析表明，DKKL1包含233个氨基酸，具有疏水性，含磷酸化位点、信号肽和较多的无规则卷曲亚结构；系统进化和同源性分析表明，DKKL1氨基酸序列在哺乳动物间高度保守；蛋白互作分析显示，DKKL1与含螺旋结构域的蛋白155(CCDC155)、转录增强缔合域蛋白2(TEAD2)、RAS癌基因家族成员11B(RAB11B)等多种蛋白互作；基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析表明，这些蛋白富集在典型Wnt信号等通路上；GO功能注释表明，DKKL1在睾酮生物合成过程的负向调控、透明带穿透、顶体泡生成等方面具有重要功能；ceRNA转录调控网络分析表明，DKKL1被miR-15a和miR-15b靶向调控。【结论】本研究获得了BMI睾丸全转录数据，阐明了DKKL1的分子特征、蛋白质功能并构建了转录调控网络。

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. © 2023. Springer Nature Limited.

目的CatSper1是精子特定的电压门控Ca^(2+)通道蛋白,在精子生成和受精过程中具有重要作用。本研究旨在分析CatSper1基因在版纳微型猪近交系(BMI)的表达调控模式、序列特征与潜在的生物学功能。方法取成年BMI公猪睾丸进行转录组测序;采用

利用RT-PCR技术从版纳微型猪近交系睾丸组织中扩增出GSKIP全长编码序列并分析其分子特征,对GSKIP基因进行功能注释,分析其与非编码RNA(miRNA和lnRNA)间的调控关系,并构建ceRNA转录调控网络,进一步分析GSKIP蛋白的基本功能,构建多物种进

为研究AURKC基因在版纳微型猪近交系(Banna mini-pig inbred line, BMI)精子发生中的功能特性及表达调控,本研究利用RNA-seq技术对BMI 12月龄成年公猪的睾丸进行全转录组测序;采用RT-PCR技术从BMI睾丸获得AURKC全长编码序列,并分析其分

【目的】获得版纳微型猪近交系(Banna mini-piginbredline,BMI)精子发生相关基因PHF7的分子特征,构建其转录调控网络。【方法】通过RNA-seq技术对BMI睾丸进行全转录组测序;利用RT-PCR技术从BMI睾丸分离PHF7全长编码序列并分析其分子特

Inbred pigs are promising animal models for biomedical research and xenotransplantation. Established in 1980, the Banna minipig inbred (BMI) line originated from a sow and its own male offspring. It was selected from a small backcountry minority Lahu village, where records show that no other pig breed has ever been introduced. During the inbreeding process, we perfomed extreme inbreeding over 23 consecutive generations using full-sibling or parent-offspring mating. In order to investigate the inbreeding effects in BMI pigs across generations over the past 40 years, in this study we conducted a genome-wide SNP genotyping of the last 10 generations, representing generations 14-23. In total, we genotyped 57,746 SNPs, corresponding to an average decrease in heterozygosity rate of 0.0078 per generation. Furthermore, we were only able to identify 18,216 polymorphic loci with a MAF larger than 0.05, which is substantially lower than the values in previous reports on other pig breeds. In addition, we sequenced the genome of the first pig in the twenty-third generation (inbreeding coefficient 99.28%) to an average coverage of 12.4x to evaluate at the genome level the impact of advanced inbreeding. ROH analysis indicates that BMI pigs have longer ROHs than Wuzhishan and Duroc pigs. Those long ROH regions in BMI pigs are enriched for distinct functions compared with the highly polymorphic regions. Our study reveals a genome-wide allele diversity loss during the progress of inbreeding in BMI pigs and characterizes ROH and polymorphic regions as a result of inbreeding. Overall, our results indicate the successful establishment of the BMI line, which paves the way for further in-depth studies.

旨在获得猪类无精症缺失基因DAZL cDNA全长序列,阐明该基因的序列、mRNA组织表达模式、蛋白质结构特征及亚细胞定位情况。本研究以版纳微型猪近交系（Banna mini-pig inbred line, BMI）10月龄成年公猪为研究对象,屠宰收集组织样,利用RACE和RT-PCR技术获得DAZL基因cDNA全长序列;使用实时荧光定量PCR（qPCR）技术检测其mRNA多组织表达谱;在线分析蛋白质的结构特点和保守结构域;用体外细胞转染技术鉴定其在ST猪睾丸细胞中的定位。结果表明,BMI DAZL cDNA全长2 985 bp（KU705632）,包含888 bp的CDS区,编码295个氨基酸（AOC89050）;该基因位于猪13号染色体,含11个外显子。qPCR结果显示,DAZL mRNA在睾丸中特异高表达。生物信息学分析表明,猪DAZL蛋白含有哺乳动物RMP和DAZ同源区,无规则卷曲在二级结构中超过50%。进化分析表明,BMI DAZL氨基酸序列高度保守,与牛科的亲缘关系最为接近。pEGFP-C1-DAZL转染ST细胞后的荧光共定位结果显示,DAZL蛋白主要分布在细胞核中。本研究分别从DNA、mRNA和蛋白质层面阐明了BMI DAZL基因的序列特征、表达、蛋白质结构和定位,为进一步研究DAZL在BMI精子发生方面的功能奠定基础。

The goat PRNT gene was initially identified as a testis-specific gene with a role in spermatogenesis. In this study, we used quantitative real-time PCR (qPCR) to first determine the mRNA expression profile of this gene in different goat tissues. Surprisingly, we found that PRNT was expressed not only in the testis but also in nine other tissues in goats. Moreover, PRNT was weakly expressed in the testis, while its expression was strongest in the ovary. These results, combined with those of other studies, led us to hypothesize that the goat PRNT gene has a role in both male and female reproduction. We further used direct DNA sequencing to detect potential SNPs within this gene in Shaanbei whit cashmere (SBWC) rams and ewes, and identified three SNPs within the PRNT gene, namely, c.-58C > T, c.71A > G (p.Alanine24Valine), and c.102C > T (synonymous). In rams, c.-58C > T and c.102C > T were strongly linked with each other (D' = 1.000, r(2) = 0.504), whereas no significant association (P > 0.05) was found between the three SNPs and semen quality, which was consistent with the low expression of the PRNT gene in the testis. Interestingly, in ewes (n = 502), c.-58C > T and c.71A > G were also strongly linked with each other (D' = 0.973, r(2) = 0.537). Additionally, the c.71A > G locus, especially the AA genotype, had a significant influence on litter size (P = 0.006), consistent with the high PRNT expression in the ovary. Combined, the results of the expression profiling and analysis of the association between the SNPs and reproductive traits showed that two strongly linked nucleotide sequence variants within PRNT were significantly associated with goat litter size. These findings provide potential DNA markers for use in the marker-assisted selection (MAS) of goats with high-fertility traits. (C) 2021 Elsevier Inc. All rights reserved.