Associations between testicular development and fetal size in the pig

BACKGROUND: Impaired reproductive performance is the largest contributing factor for the removal of boars from commercial systems. Intrauterine growth restricted piglets represent 25% of the total number of piglets born and have impaired reproductive performance. This study aimed to improve the understanding of temporal changes in testicular gene expression during testes development in fetuses of different size. The lightest and closest to mean litter weight (CTMLW) male Large White × Landrace littermates were collected at gestational days (GD) 45, 60 and 90 (n = 5–6 litters/GD). RESULTS: Testes weight and testes weight as a percentage of fetal weight were not associated with fetal size at GD60 or 90. Fetal plasma testosterone was not associated with fetal size at GD90. There was no association between fetal size and seminiferous tubule area and number, number of germ or Sertoli cells per tubule. The lightest fetuses tended to have wider seminiferous tubules compared to the CTMLW fetuses at GD90 (P = 0.077). The testicular expression of KI67 (P ≤ 0.01) and BAX:BCL2 ratio (P = 0.058) mRNAs decreased as gestation progressed. Greater SPP1 mRNA expression was observed at GD60 when compared with GD45 and 90 (P ≤ 0.05). Lower expression of DMRT1 and SPP1 (P < 0.01) mRNAs was observed in testes associated with the lightest fetuses compared to the CTMLW fetuses at GD90. CONCLUSIONS: These findings provide novel insights into the expression profiles of genes associated with testicular development and function. Further, these data suggest that programming of reproductive potential in IUGR boars occurs late in gestation, providing a platform for further mechanistic investigation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40104-022-00678-3

KLB dysregulation mediates disrupted muscle development in intrauterine growth restriction

ABSTRACT: Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified β‐Klotho (KLB), a fibroblast growth factor 21 (FGF21) co‐receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally‐occurring disease model, we performed transcriptome‐wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal‐weight (NW) littermates. We found that, alongside large‐scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose‐dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill‐health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large‐scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co‐receptor, β‐Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals

Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the “Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)” to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs

Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the "Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)" to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs

Mechanisms involved in the adaptation of Escherichia coli O157:H7 to the host intestinal microenvironment

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: MaCateer, Sean P.. University of Edinburgh; Reino UnidoFil: Montañez Culma, Leidy Johanna. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Cortés Araya, Yennifer. University of Edinburgh; Reino UnidoFil: Tree, Jai. University of Edinburgh; Reino UnidoFil: Bernal, Alan Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fuentes, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fitzgerald, Stephen. University of Edinburgh; Reino UnidoFil: Pineda, Gonzalo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gally, David. University of Edinburgh; Reino UnidoFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Effects of intrauterine growth restriction (IUGR) on porcine muscle development

Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans. Preferential allocation of available resources to vital tissues in the IUGR foetus occurs at the expense of skeletal muscle, resulting in reduced tissue growth and metabolic adaptations that increase the risk of premature muscle loss and metabolic disease later in life. Several animal models have been used to study the effects of IUGR on skeletal muscle. However, the developmental mechanisms driving impaired myogenesis in IUGR have only been partly elucidated. IUGR occurs spontaneously in the pig, generating severe consequences on muscle development, providing a convenient, physiologically-relevant large animal model for studying developmental aspects of IUGR. The studies in this thesis aimed to identify genome-wide transcriptional signatures associated with the adaptive response of foetal skeletal muscle to IUGR in the pig. Another aim was to establish a suitable in vitro system and to use this to functionally target identified candidate genes to elucidate their involvement in the IUGR muscle phenotype. Using RNA-sequencing of muscle from IUGR and normal-weight (NW) pig littermates at a late foetal stage (Day 90 of gestation), a total of 1031 genes were identified to be differentially expressed (DE). Those DE transcripts mapped to multiple biological pathways involved in Development, Tissue injury (Inflammation, Coagulation and Anti-oxidation/Detoxification) and Metabolism (Glucose metabolism, Lipid biosynthesis/transport, and Amino acid degradation). To establish an in vitro system for investigating the involvement of some of the consequences identified in IUGR, progenitor muscle cells (PMCs) from IUGR and normal weight (NW) littermates were obtained and were differentiated under established myogenic or adipogenic conditions, followed by analyses of lineage markers by immunochemistry and RT-qPCR. IUGR PMCs displayed reduced myogenic capacity and an increased ability to undergo adipogenic differentiation compared to PMCs from NW littermates. These results demonstrated that PMCs are developmentally programmed in utero by IUGR. At the same time, they provided a powerful tool that was later used to elucidate some of the mechanisms involved in the IUGR phenotype. Among the up-regulated transcripts identified in IUGR muscle was the global metabolic regulator, KLB, a co-receptor for FGF21. In addition, FGF21 levels in plasma were increased in IUGR relative to NW littermates. Functional targeting of KLB was performed by determining myogenesis and mTOR signalling responses to transfection with KLB siRNA and treatment with the KLB ligand, FGF21 (0-100 ng/ml), using porcine PMCs as well as PMCs isolated from human foetuses. Transfection of PMCs with KLB siRNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and human PMCs. This showed that the effects of KLB on muscle growth occur through inhibition of mTOR signalling and may involve both direct and indirect mechanisms. Importantly, these results indicate that the effects of KLB on muscle cells are conserved in pigs and humans. Finally, some of the identified DE transcripts were analysed on foetal muscle samples from IUGR and NW littermates at gestational days (GD) 45, 60 and 90. RT-qPCR analysis showed that, in general, altered expression of developmental transcripts in IUGR foetal muscle started on GD45, whereas for tissue injury and metabolic transcripts, changes were observed starting on GD60 and GD90, respectively. These results indicate that different functional gene categories are sequentially affected in muscle by IUGR during foetal development and that interventions at the sow level aimed at preventing or ameliorating the IUGR phenotype in offspring should be implemented early during gestation. Altogether, this thesis provides new insight into the molecular mechanisms underlying the effects of IUGR on muscle development. The results may aid the development of novel strategies to ameliorate the effects of IUGR in the pig industry