Jianfei Gong

Spermatogenesis is a complex, tightly regulated biological process crucial for male fertility in mammals. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) play essential functions in regulating gene expression during spermatogenesis, and their specific roles in Holstein bull testicular development remain unclear. The study analyzed testicular tissues from young and mature Holstein bulls, unveiling the post-sexual maturation transcriptome landscape with a focus on spermatogenesis-associated miRNAs and mRNA profiles. The cDNA libraries from the testicular tissues of three young Holstein bulls (X1, X2, and X3; group YB) and three mature Holstein bulls (D5, D6, and D7; group AB) were established for RNA-sequencing analysis using Illumina high-throughput sequencing technology, followed by bioinformatics analysis. A total of 1114 miRNAs were identified in the X vs. D groups: 683 known miRNAs and 431 novel miRNAs. Notably, 221 known miRNAs exhibit differential expression between the X and D groups, with 118 up-regulated and 103 down-regulated genes. Moreover, a total of 237 previously unidentified novel miRNAs were also observed to exhibit differential expression between X and D, with 77 up-regulated and 160 down-regulated miRNA genes. These miRNA genes were linked to Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to male reproduction and spermatogenesis. The DE analysis of miRNA genes identified the top five up-regulated miRNAs (bta-miR-127, bta-miR-495, bta-miR-493, bta-miR-432, and bta-miR-3956) and down-regulated miRNAs (bta-miR-34c, bta-miR-34b, bta-miR-449a, bta-miR-449b, and 23_28233-5p (hsa-miR-6774-3p)) between the young bull and adult bull groups. The miRNA-mRNA network analysis also identified 17_16057-5p, X_45311-3p (cpo-miR-12072-3p), bta-miR-34c, bta-miR-375, 17_15431-5p (eca-miR-8912), 10_2547-3p (isc-miR-5313), bta-miR-449b, and mRNA genes such as BCL2L11, ADAMTSI1, MED1, and PRDM1; these genes were associated with reproductive system development. The mRNA genes (STRB and BCL2L11) and miRNA genes (bta-miR-134, bta-let-7e, bta-miR-181b, and bta-miR-214) were also found to have a significant correlation with the spermatogenic process. Thus, the comprehensive analysis of miRNA and mRNA enhances our knowledge of the regulatory networks involved in testicular development and identifies potential targets for further investigation and manipulation to improve cattle reproductive efficiency and genetic selection.

The Polled Celtic (Pc) mutation locus is a genetically simple single mutation that is the best choice for breeding polled cattle using gene editing. However, the mechanism of the Pc locus for regulating horn development is unclear, so we used gene editing, somatic cell nuclear transfer and embryo transfer to obtain polled Holstein fetal bovine (gestation time 90 days) with a homozygous Pc insertion (gene-edited Holstein fetal bovine, EH) and the wild-type 90 days Holstein fetal bovine (WH) as controls. The hematoxylin-eosin (HE) staining results showed that, compared to the WH, the EH horn buds had no white keratinized projections or vacuolated keratinocytes and no thick nerve bundles under the dermal tissue. Furthermore, DNA sequencing results showed that the Pc locus was homozygously inserted into the fetal bovine genome. A total of 791 differentially expressed genes were identified by transcriptome sequencing analysis. Enrichment analysis and protein interaction analysis results of differentially expressed genes showed that abundant gene changes after Pc insertion were associated with the adhesion molecule regulation, actin expression, cytoskeletal deformation and keratin expression and keratinization. It was also noted that the results contained several genes that had been reported to be associated with the development of horn traits, such as RXFP2 and TWIST1. This study identified these changes for the first time and summarized them. The results suggested that the Pc mutant locus may inhibit neural crest cell EMT generation and keratin expression, leading to failures in neural crest cell migration and keratinization of the horn bud tissue, regulating the production of the polled phenotype.

Previous studies reported the physical, transcriptome, and metabolome changes in in vitro acute heat-stressed (38 °C versus 43 °C for 2 h) bovine granulosa cells. Granulosa cells exhibited transient proliferation senescence, oxidative stress, an increased rate of apoptosis, and a decline in steroidogenic activity. In this study, we performed a joint integration and network analysis of metabolomic and transcriptomic data to further narrow down and elucidate the role of differentially expressed genes, important metabolites, and relevant cellular and metabolic pathways in acute heat-stressed granulosa cells. Among the significant (raw p-value < 0.05) metabolic pathways where metabolites and genes converged, this study found vitamin B6 metabolism, glycine, serine and threonine metabolism, phenylalanine metabolism, arginine biosynthesis, tryptophan metabolism, arginine and proline metabolism, histidine metabolism, and glyoxylate and dicarboxylate metabolism. Important significant convergent biological pathways included ABC transporters and protein digestion and absorption, while functional signaling pathways included cAMP, mTOR, and AMPK signaling pathways together with the ovarian steroidogenesis pathway. Among the cancer pathways, the most important pathway was the central carbon metabolism in cancer. Through multiple analysis queries, progesterone, serotonin, citric acid, pyridoxal, L-lysine, succinic acid, L-glutamine, L-leucine, L-threonine, L-tyrosine, vitamin B6, choline, and CYP1B1, MAOB, VEGFA, WNT11, AOX1, ADCY2, ICAM1, PYGM, SLC2A4, SLC16A3, HSD11B2, and NOS2 appeared to be important enriched metabolites and genes, respectively. These genes, metabolites, and metabolic, cellular, and cell signaling pathways comprehensively elucidate the mechanisms underlying the intricate fight between death and survival in acute heat-stressed bovine granulosa cells and essentially help further our understanding (and will help the future quest) of research in this direction.

Backfat deposition is strongly related to carcass traits, growth rate, feed conversion rate, and reproductive performance in pig production. To understand the molecular mechanisms underlying porcine backfat thickness phenotypes, transcriptome and miRNA profiling of backfat from high-backfat thickness and low-backfat thickness pigs were performed by RNA sequencing. Twenty genes encoding for miRNAs and 126 genes encoding for protein-coding genes were found to be differentially expressed between the two libraries. After integrative analysis of DEMs targets and DEGs, a total of 33 mRNA‒miRNA interaction pairs were identified, and the regulatory networks of these pairs were determined. Among these genes, five (AQP9, DKK3, GLYCTK, GLIPR1, and DUSP2) related to fat deposition were found to be strong candidate genes, and mir-31-5p/AQP9 and mir-31-5p/GLIPR1 may play important roles in fat deposition. Additionally, potential adipogenesis-related genes and miRNAs were identified. These findings improve the current understanding of the molecular genetic mechanisms of subcutaneous fat deposition in pigs and provide a foundation for further studies.