Effects of fetal sex and genetics on the bovine placenta: from baseline data to fetal programming and heterosis

The placenta is a major determinant of fetal growth and central to fetal programming effects that impact postnatal performance and health. Most reports in human and animals on prenatal growth and programming are based on studies of term placentae and/or birth weight, but information at critical time points of development on (i) fundamental gross morphological and histomorphological characteristics and developmental changes of the placenta that could impact embryo/fetal growth, (ii) influence of sex-specific placental and umbilical cord phenotype on sex differences in fetal growth, (iii) contribution of placenta and umbilical cord in mediating effects of genetics and epigenetics on heterotic fetal growth, and (iv) differences in placental expression of insulin-like growth factor (IGF) system components between sexes and fetal genetic groups, are generally lacking. This information is highly relevant for both animal production and human health, and the present study used cattle, a major livestock species and valuable biomedical animal model, to address research questions and close gaps in knowledge. Purebred and reciprocal cross Bos taurus taurus (Angus, A) and Bos taurus indicus (Brahman, B) concepti recovered at the late embryo (Day 48, n=60) and early accelerated fetal growth (Day 153, n=73) stages (term, 277-291) were used to examine effects of fetal sex and genetics on a broad range of conceptus traits. Data analyzed using general linear models included gross- and histomorphological parameters of the placenta, umbilical cord traits and fetal fluid volume as well as clinical-chemical parameters, circulating IGFs in cord blood, and tissue-specific transcript abundances of IGF system components. The main findings include (i) the significant contribution of convex but not flat placentomes to embryo-fetal growth as revealed by the exclusive positive relationships between number of convex placentomes and embryo-fetal weights and the higher number and average weight of convex placentomes in placentae of Bpaternal×Amaternal and A×A concepti, which ultimately show the highest birth weights; (ii) fetal sex effects on placental and umbilical cord phenotype which mediate sex-specific fetal growth, where normal female fetuses display hallmarks of intrauterine growth restriction that provide a mechanism for sexdifferences in susceptibility to non-communicable diseases; (iii) heterosis of B×A hybrids in utero that is characterized by polar overdominance of paternal B genome on umbilical cord phenotype and complements the superior maternal A genome effects on placenta, a clear indication of heterosis due to high capacity for nutrient extraction and high capacity for nutrient supply; and (iv) differences in circulating IGF2 and transcript abundance of IGF2 in fetal brain and heart between sexes, as well as differences in expression of IGF2, IGF2R, H19, AIRN in placenta between fetal genetic groups demonstrate the important role of IGF2 for fetal growth at mid-gestation. In conclusion, the results of the present study support the hypothesis that differences in placental and umbilical cord phenotypes between males and females and between purebreds and reciprocal cross hybrids determine variation in intrauterine growth at midgestation and contribute to programming of postnatal performance and health. Future studies to be explored include analyses of growth factors associated with development of the placental vasculature and umbilical cord, and involvement of additional imprinted gene clusters in prenatal growth and development.Thesis (Ph.D.) -- University of Adelaide, School of Animal & Veterinary Sciences, 201

Asymmetric growth-limiting development of the female conceptus

IntroductionSex differences in prenatal growth may contribute to sex-dependent programming effects on postnatal phenotype. MethodsWe integrated for the first time phenotypic, histomorphological, clinico-chemical, endocrine and gene expression analyses in a single species, the bovine conceptus at mid-gestation. ResultsWe demonstrate that by mid-gestation, before the onset of accelerated growth, the female conceptus displays asymmetric lower growth compared to males. Female fetuses were smaller with lower ponderal index and organ weights than males. However, their brain:body weight, brain:liver weight and heart:body weight ratios were higher than in males, indicating brain and heart ‘sparing’. The female placenta weighed less and had lower volumes of trophoblast and fetal connective tissue than the male placenta. Female umbilical cord vessel diameters were smaller, and female-specific relationships of body weight and brain:liver weight ratios with cord vessel diameters indicated that the umbilico-placental vascular system creates a growth-limiting environment where blood flow is redistributed to protect brain and heart growth. Clinico-chemical indicators of liver perfusion support this female-specific growth-limiting phenotype, while lower insulin-like growth factor 2 (IGF2) gene expression in brain and heart, and lower circulating IGF2, implicate female-specific modulation of key endocrine mediators by nutrient supply. ConclusionThis mode of female development may increase resilience to environmental perturbations in utero and contribute to sex-bias in programming outcomes including susceptibility to non-communicable diseases

Tissue-specific expression profiles of IGF system binding proteins in bovine pre- and postnatal developmental stages.

<p>Abundances of transcripts for <i>IGFBP1</i>, <i>IGFBP2</i>, <i>IGFBP3</i>, <i>IGFBP4</i>, <i>IGFBP5</i>, <i>IGFBP6</i>, <i>IGFBP7</i> and <i>IGFBP8</i> were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants.

<p>The exon-intron structure of bovine insulin/insulin-like growth factor 2 (<i>INS/IGF2</i>, GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters (<i>IGF2</i>-P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human <i>IGF2</i>-P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine <i>IGF2</i>-P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 (<i>IGF2</i>-P1e2) and exon 3 (<i>IGF2</i>-P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for <i>IGF2</i>-P1e2) and exons 3 and 8 (for <i>IGF2</i>-P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since <i>IGF2</i>-transcript P1 exon 2 is part of the first exonic region of transcript <i>IGF2</i>-P0, and exon 3 is present in both <i>IGF2-</i>P0 and <i>IGF2-</i>P1 transcripts, the <i>IGF2</i>-P1e2 and -P1e3 amplicons could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from <i>IGF2-</i>P2 promoter which comprise leading exon 4 (<i>IGF2</i>-P2e4) or leading exons 4 and 5 (<i>IGF2</i>-P2e5) as well as the protein coding exons. The forward primer for <i>IGF2</i>-P2e4 was designed to span the junction of exons 4 and 8, and for <i>IGF2</i>-P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify <i>IGF2</i>-P3 and <i>IGF2</i>-P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200466#pone.0200466.s003" target="_blank">S3 Table</a></b>.</p

Tissue-specific expression profiles of IGF system receptors in bovine pre- and postnatal developmental stages.

<p>Abundances of global <i>IR</i> transcript and splice variants <i>IR</i>-A and <i>IR</i>-B, <i>IGF1R</i> and <i>IGF2R</i> were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Tissue-specific expression profiles of IGF system ligands in bovine pre- and postnatal developmental stages.

<p>Abundances of global <i>IGF1</i> transcript and splice variants <i>IGF1</i> class 1 and 2, global <i>IGF2</i> transcript and promoter and splice variant-specific <i>IGF2</i>-P0, <i>IGF2</i>-P1e2, <i>IGF2</i>-P1e3, <i>IGF2</i>-P2e4, <i>IGF2</i>-P2e5, <i>IGF2</i>-P3 and <i>IGF2</i>-P4 transcript were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Tissue-specific expression profiles of long non-coding RNAs associated with the IGF system in bovine pre- and postnatal developmental stages.

<p>Abundances of <i>H19</i> and <i>AIRN</i> transcript were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Relative contribution of promoter and splice variant-specific <i>IGF2</i> transcripts to global <i>IGF2</i> transcript abundance in fetal tissues and placenta.

<p><i>IGF2</i>-P0, <i>IGF2</i>-P3 and <i>IGF2</i>-P4 are percent transcript abundance derived from P0, P3 and P4 promoters, respectively. Splice variants of promoter P2 transcript are <i>IGF2</i>-P2e4 with untranslated leader exon 4 and <i>IGF2</i>-P2e5 with untranslated leader exons 4 and 5. Estimated means are from 73 fetal cDNA samples per tissue and 95% confidence intervals are detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200466#pone.0200466.s009" target="_blank">S9 Table</a></b>. Transcript abundances were calculated by the standard curve method, normalized with reference genes and expressed in relative units. The relative contribution of each promoter-specific transcript to global <i>IGF2</i> transcript abundance was calculated by Johnson’s Relative Weight procedure.</p

Atlas of tissue- and developmental stage specific gene expression for the bovine insulin-like growth factor (IGF) system

The insulin-like growth factor (IGF) axis is fundamental for mammalian growth and development. However, no comprehensive reference data on gene expression across tissues and pre- and postnatal developmental stages are available for any given species. Here we provide systematic promoter- and splice variant specific information on expression of IGF system components in embryonic (Day 48), fetal (Day 153), term (Day 277, placenta) and juvenile (Day 365–396) tissues of domestic cow, a major agricultural species and biomedical model. Analysis of spatiotemporal changes in expression of IGF1, IGF2, IGF1R, IGF2R, IGFBP1-8 and IR genes, as well as lncRNAs H19 and AIRN, by qPCR, indicated an overall increase in expression from embryo to fetal stage, and decrease in expression from fetal to juvenile stage. The stronger decrease in expression of lncRNAs (average ―16-fold) and ligands (average ―12.1-fold) compared to receptors (average ―5.7-fold) and binding proteins (average ―4.3-fold) is consistent with known functions of IGF peptides and supports important roles of lncRNAs in prenatal development. Pronounced overall reduction in postnatal expression of IGF system components in lung (―12.9-fold) and kidney (―13.2-fold) are signatures of major changes in organ function while more similar hepatic expression levels (―2.2-fold) are evidence of the endocrine rather than autocrine/paracrine role of IGFs in postnatal growth regulation. Despite its rapid growth, placenta displayed a more stable expression pattern than other organs during prenatal development. Quantitative analyses of contributions of promoters P0-P4 to global IGF2 transcript in fetal tissues revealed that P4 accounted for the bulk of transcript in all tissues but skeletal muscle. Demonstration of IGF2 expression in fetal muscle and postnatal liver from a promoter orthologous to mouse and human promoter P0 provides further evidence for an evolutionary and developmental shift from placenta-specific P0-expression in rodents and suggests that some aspects of bovine IGF expression may be closer to human than mouse