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 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

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 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