34 research outputs found
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 ā Integrin Subunits, Secreted Phosphoprotein 1
(SPP1) and Fibronectin (FN)
Integrin receptors exist as heterodimers consisting of an Ī± and a Ī² 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) ITGĪ±2, ITGĪ±V, ITGĪ²1, ITGĪ²3, ITGĪ²5, ITGĪ²6 and ITGĪ²8, 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 ITGĪ±2
expression compared to those supplying the CTMLW foetuses (P=0.07).
Endometrial samples supplying the lightest foetuses had decreased
expression of ITGĪ²1 and SPP1 at GD45 and 60 respectively (Pā¤0.05).
Placental samples associated with female foetuses had decreased expression
of ITGĪ²6 and FN (Pā¤0.05) compared to those supplying male foetuses at
GD45 and 90 respectively. FN and ITGĪ²3expression was increased in
endometrial samples supplying female foetuses compared to their male
littermates at GD30 and 60 respectively (Pā¤0.05). In contrast, SPP1
expression was decreased in endometrial samples supplying female foetuses
compared to their male littermates at GD60 (Pā¤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 ā 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 ā 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 (Pā¤0.001). At GD60 and 90 foetal heart rate decreased
with increasing duration of sedation (Pā¤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
(Pā¤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 ā 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 (Pā¤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 (Pā¤0.05), total number of
BV (Pā¤0.05) and mean number of BV (Pā¤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 F2Ī± 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 ā 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 HPLCāMS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (nā=ā5ā11 gilts/GD). Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (Pā<ā0.05). 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 (Pā=ā0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (Pā<ā0.05) 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 (Pā<ā0.05). 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 Ć Landrace littermates were collected at gestational days (GD) 45, 60 and 90 (nĀ =ā5ā6 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 (PĀ =ā0.077). The testicular expression of KI67 (PĀ ā¤ā0.01) and BAX:BCL2 ratio (PĀ =ā0.058) mRNAs decreased as gestation progressed. Greater SPP1 mRNA expression was observed at GD60 when compared with GD45 and 90 (PĀ ā¤ā0.05). Lower expression of DMRT1 and SPP1 (PĀ <ā0.01) 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
Intrauterine growth restriction: screening and outcomes
Intrauterine growth restriction (IUGR) is a condition in which the fetus does notachieve its full growth potential, resulting in low birthweight infants. IUGR represents oneof the leading causes of infant mortality worldwide and has been suggested to predisposenewborns to a variety of diseases. This condition does not only impact human pregnanciesand is a significant problem in livestock species. Thus, there is a clear need to improve theunderstanding of the mechanisms regulating fetal growth and to allow the development oftherapeutic strategies. The articles in this Research Topic āIntrauterine growth restriction:screening and outcomesā describe some of the latest studies aimed to improve theunderstanding of the mechanisms regulating fetal growth and development
Associations between Fetal Size, Sex and both Proliferation and Apoptosis at the Porcine Feto-Maternal Interface
Differentiation potential of Mesenchymal Stem/Stromal cells is altered by Intrauterine Growth Restriction
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 Ī²āKlotho (KLB), a fibroblast growth factor 21 (FGF21) coāreceptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturallyāoccurring disease model, we performed transcriptomeāwide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normalāweight (NW) littermates. We found that, alongside largeāscale 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 doseādependent 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 illāhealth in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with largeāscale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) coāreceptor, Ī²āKlotho (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 āImproved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)ā 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