Investigating the porcine feto-maternal interface throughout gestation: associations with foetuses of different size and sex

Background Inadequate foetal growth cannot be remedied postnatally, leading to severe consequences for neonatal and adult development. Furthermore, sexual dimorphism in placental development has been suggested in humans although this remains poorly investigated in the pig. Hypotheses Intrauterine Growth Restriction (IUGR) occurs due to aberrant conceptus attachment, which leads to alterations in angiogenesis and vascularity of the feto-maternal interface. Altered gene expression and vascularity will be observed at the feto-maternal interface in male foetuses compared to female foetuses. Increased apoptosis and decreased proliferation will be observed in the feto-maternal interface associated with the lightest foetuses compared to the closest to mean litter weight (CTMLW) foetuses. Aims This thesis aimed to investigate the association between foetal size and sex and: integrin signalling; apoptotic and proliferation pathways; umbilical arterial (UA) blood flow; and angiogenesis and vascularity at the feto-maternal interface. This was performed by the collection of placental and endometrial samples associated with conceptuses or foetuses of different size (lightest and CTMLW) and sex at gestational day (GD) 18, 30, 45, 60 and 90. Results – Integrin Subunits, Secreted Phosphoprotein 1 (SPP1) and Fibronectin (FN) Integrin receptors exist as heterodimers consisting of an α and a β subunit at the porcine feto-maternal interface. They bind to a number of ligands including secreted phosphoprotein 1 (SPP1) and fibronectin (FN), and play a central role in the establishment of pregnancy. The mRNA expression of the integrin subunits (ITG) ITGα2, ITGαV, ITGβ1, ITGβ3, ITGβ5, ITGβ6 and ITGβ8, and the ligands SPP1 and FN was quantified by qPCR in placental and endometrial samples supplying foetuses of different size and sex throughout gestation. Temporal changes in mRNA expression were observed in both tissues. At GD45, placental samples supplying the lightest foetuses had increased ITGα2 expression compared to those supplying the CTMLW foetuses (P=0.07). Endometrial samples supplying the lightest foetuses had decreased expression of ITGβ1 and SPP1 at GD45 and 60 respectively (P≤0.05). Placental samples associated with female foetuses had decreased expression of ITGβ6 and FN (P≤0.05) compared to those supplying male foetuses at GD45 and 90 respectively. FN and ITGβ3expression was increased in endometrial samples supplying female foetuses compared to their male littermates at GD30 and 60 respectively (P≤0.05). In contrast, SPP1 expression was decreased in endometrial samples supplying female foetuses compared to their male littermates at GD60 (P≤0.05). SPP1 protein staining per uterine gland was assessed throughout gestation by immunofluorescence. Whilst temporal changes in staining were observed, with a similar profile to the mRNA expression data, no associations between foetal size or sex, and uterine gland staining were observed. Results – Apoptosis and Proliferation The mRNA expression of candidate genes involved in apoptosis or proliferation (Bax, Bcl2, P53 and Ki67) was quantified by qPCR. Temporal changes were observed in both tissues. Placentas associated with the lightest foetuses had decreased P53 and Ki67 expression compared to the CTMLW foetuses at GD45. However, at GD60, P53 expression was increased in placentas supplying the lightest foetuses compared to CTMLW foetuses. Intriguingly, endometrial P53 expression was increased in samples associated with the lightest foetuses compared to the CTMLW foetuses at GD45. A trend towards females having decreased placental Bax expression was observed at GD45 (P=0.06). At GD60 the expression of Bcl2, P53 and Ki67 was decreased in endometrial samples associated with females compared to their male littermates. At GD30, Bax expression was increased in endometrial samples associated with female foetuses compared to their male littermates. TUNEL staining revealed an increased prevalence of apoptotic cells in placentas at GD60 compared to GD45 however, no association between foetal size or sex, and apoptotic cell number was observed. Results – Doppler Ultrasound Human IUGR is diagnosed prenatally by alterations in UA blood flow, detected by Doppler ultrasound. Doppler ultrasound was used under moderate sedation over a 30-minute period to monitor umbilical arterial (UA) blood flow in the right uterine horn of Large White X Landrace gilts at GD30, 45, 60 and 90. Gilts were scanned prior to euthanasia to examine relationships between litter size, sex ratio and five UA parameters of interest. In GD90 gilts where scans were obtained from all foetuses in the scanned horn, relationships between individual foetal weight and sex were examined. A subset of the gilts were sedated, scanned and recovered (SSR) earlier in gestation to assess the influence of sedation on later foetal development by comparing with control litters that had not been sedated previously. Temporal changes were observed in all UA parameters (P≤0.001). At GD60 and 90 foetal heart rate decreased with increasing duration of sedation (P≤0.001). Sex ratio and foetal weight were associated with UA blood flow whereas litter size and foetal sex were not. SSR at GD30 and 45 was associated with decreased foetal weight at GD60 (P≤0.001) and 90 (P=0.06), respectively, when compared to controls. These results suggest that maternal sedation during gestation has a significant effect on foetal development, the mechanisms of which warrant further investigation. Results – Angiogenesis and Vascularity Angiogenesis is essential for foetal and placental development and has been shown to be inadequately regulated in instances of complicated pregnancies. Immunohistochemistry on placental (GD45, 60 and 90) and endometrial (GD18, 30, 45, 60 and 90) samples for the endothelial cell marker Platelet and Endothelial Cell Adhesion Molecule 1 (CD31) was performed to assess placental and endometrial vascularity. At GD60, the percentage staining in the chorioallantoic membrane (CAM) of placentas supplying the lightest foetuses was increased compared to those supplying the CTMLW foetuses (P=0.06). The direction of this relationship switched at GD90, with placentas supplying the lightest foetuses having decreased percentage CD31 staining in the CAM compared to those supplying the CTMLW foetuses (P≤0.05). No association between foetal sex and placental vascularity was observed. At GD60, a trend for both the total (P=0.07) and mean number of blood vessels (BV) (P=0.07) to be decreased in endometrial samples supplying the lightest foetuses compared to the CTMLW foetuses was observed. Similarly, a trend towards a decrease in the mean number of uterine glands in endometrial samples supplying the lightest compared to the CTMLW foetuses was observed at GD90 (P=0.08). The total number of uterine glands (P≤0.05), total number of BV (P≤0.05) and mean number of BV (P≤0.05) were increased in endometrial samples supplying female foetuses compared to male foetuses at GD45. The mRNA expression of candidate genes which are quantitative trait loci (QTL) associated or have a central role in angiogenesis and foetal development, including: Uteroferrin (ACP5), CD31, Hypoxia Inducible Factor 1 Alpha Subunit (HIF1A), Heparanase (HPSE), Prostaglandin F2α Receptor (PTGFR) and Vascular Endothelial Growth Factor A (VEGFA) were investigated by qPCR. Temporal changes in expression were observed in both tissues. ACP5 expression was increased (GD60), whilst HIF1A (GD90) were decreased in placentas supplying the lightest foetuses compared to the CTMLW foetuses. CD31 expression was decreased at GD45, and increased at GD60, in placental samples supplying the lightest foetuses compared to the CTMLW foetuses. Decreased expression of CD31 (GD60), HPSE and VEGFA (GD90), alongside increased HIF1A (GD45) expression were observed in endometrial samples supplying the lightest foetuses compared to the CTMLW foetuses. Decreased expression of CD31, PTGFR and VEGFA was observed in endometrial samples associated with male compared to female foetuses at GD30. At GD60, the direction of these differences in endometrial expression was reversed, with increased expression of ACP5, CD31 and VEGFA in endometrial samples associated with male foetuses compared to their female littermates. Results – In vitro Angiogenic Potential of Placental and Endometrial Samples Matrigel branching assays were used to assess the angiogenic potential of GD45 and 60 placental and endometrial conditioned media on endothelial cell branching in vitro. Conditioned media from GD45 placental and endometrial samples increased endothelial cell branching in vitro compared to treatment with media from GD60 samples. Endothelial cell branching was impaired in response to treatment with conditioned media from placental and endometrial samples associated with the lightest foetuses compared to the CTMLW foetuses at both GD. At GD60, conditioned media from female placentas had increased endothelial cell branching compared to those supplying males. Interestingly, the inverse of this was observed in the endometrium, with samples supplying females having a decreased ability to induce endothelial cell branching compared with males. Conclusion This thesis has presented novel findings of associations between foetal size and sex, and placental and endometrial integrin signalling, apoptosis and proliferation, and angiogenesis and vascularity. Currently, this is the first suggestion in the literature that foetal size, and more intriguingly foetal sex, may have a strong influence on the activity of the endometrium. The mechanisms behind these findings warrant further investigation. Switches in the direction of differences at the feto-maternal interface between foetuses of different size were observed throughout gestation, notably between GD45 and 60, highlighting the dynamic nature of the feto-maternal interface and suggesting this as a potential window that could be manipulated by the industry to attempt to rescue the postnatal phenotype of IUGR piglets

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species. However, the temporal changes in maternal vitamin D status throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated. The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (n = 5–11 gilts/GD). Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (P < 0.05). The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight, ovulation rate, mean litter weight, number of live foetuses, percentage prenatal survival, and sex ratio of the litter was assessed. Maternal 25(OH)D (P = 0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (P < 0.05) concentrations were inversely associated with gilt weight. Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90 (P < 0.05). This study has provided novel insights into temporal changes in maternal vitamin D status throughout gestation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight, percentage prenatal survival and percentage of male foetuses in the litter. Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40104-022-00760-w

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

Accessory Genome Dynamics and Structural Variation of Shigella from Persistent Infections

Shigellosis is a diarrheal disease caused mainly by Shigella flexneri and Shigella sonnei Infection is thought to be largely self-limiting, with short- to medium-term and serotype-specific immunity provided following clearance. However, cases of men who have sex with men (MSM)-associated shigellosis have been reported where Shigella of the same serotype were serially sampled from individuals between 1 and 1,862 days apart, possibly due to persistent carriage or reinfection with the same serotype. Here, we investigate the accessory genome dynamics of MSM-associated S. flexneri and S. sonnei isolates serially sampled from individual patients at various days apart to shed light on the adaptation of these important pathogens during infection. We find that pairs likely associated with persistent infection/carriage and with a smaller single nucleotide polymorphism (SNP) distance, demonstrated significantly less variation in accessory genome content than pairs likely associated with reinfection, and with a greater SNP distance. We observed antimicrobial resistance acquisition during Shigella carriage, including the gain of an extended-spectrum beta-lactamase gene during carriage. Finally, we explored large chromosomal structural variations and rearrangements in seven (five chronic and two reinfection associated) pairs of S. flexneri 3a isolates from an MSM-associated epidemic sublineage, which revealed variations at several common regions across isolate pairs, mediated by insertion sequence elements and comprising a distinct predicted functional profile. This study provides insight on the variation of accessory genome dynamics and large structural genomic changes in Shigella during persistent infection/carriage. In addition, we have also created a complete reference genome and biobanked isolate of the globally important pathogen, S. flexneri 3a.IMPORTANCE Shigella spp. are Gram-negative bacteria that are the etiological agent of shigellosis, the second most common cause of diarrheal illness among children under the age of five in low-income countries. In high-income countries, shigellosis is also a sexually transmissible disease among men who have sex with men. Within the latter setting, we have captured prolonged and/or recurrent infection with shigellae of the same serotype, challenging the belief that Shigella infection is short lived and providing an early opportunity to study the evolution of the pathogen over the course of infection. Using this recently emerged transmission scenario, we comprehensively characterize the genomic changes that occur over the course of individual infection with Shigella and uncover a distinct functional profile of variable genomic regions, findings that have relevance for other Enterobacteriaceae