Changes in global gene expression in rat myometrium in transition from late pregnancy to parturition

Abstract

The process of parturition involves the complex interplay of factors that change the excitability and contractile activity of the uterus. We have compared the relative gene expression profile of myometrium from rats before parturition (21 days pregnant) and during delivery, using high-density DNA microarray. Of 8,740 sequences available in the array, a total of 3,782 were detected as present. From the sequences that were significantly altered, 59 genes were upregulated and 82 genes were downregulated. We were able to detect changes in genes described to have altered expression level at term, including connexin 43 and 26, cyclooxygenase 2, and oxytocin receptor, as well as novel genes that have been not previously associated with parturition. Quantitative real-time PCR on selected genes further confirmed the microarray data. Here we report for the first time that aquaporin5 (AQP5), a member of the aquaporin water channel family, was dramatically downregulated during parturition (approximately 100-fold by microarray and approximately 50-fold by real-time PCR). The emerging profile highlights biochemical cascades occurring in a period of approximately 36 h that trigger parturition and the initiation of myometrium reverse remodeling postpartum. The microarray analysis uncovered genes that were previously suspected to play a role in parturition. This regulation involves genes from immune/inflammatory response, steroid/lipid metabolism, calcium homeostasis, cell volume regulation, cell signaling, cell division, and tissue remodeling, suggesting the presence of multiple and redundant mechanisms altered in the process of birth.

Fig. 1.

Assessment of total RNA quality and criteria for quantitative real-time PCR (QRT-PCR) quantification. A: agarose gel showing typical high 28S-to-18S ratio (∼3.5) indicating good RNA quality from late-pregnancy (LP) and during-labor (DL) myometrium. B: single PCR product of ∼500 nt obtained with primers spanning a 200-bp intronic region of β-actin gene, indicating no detectable genomic DNA contamination in cDNAs from both LP and DL myometrium. C: fluorescence intensity vs. cycle number plots of QRT-PCR reactions for Fibulin 5 in LP in the presence (+RT) and absence (−RT) of reverse transcriptase and when water was used instead of RNA. D: melting curve [first derivative of fluorescence (dF/dt) vs. temperature (T)] of QRT-PCR product showing a single peak in the presence of RT. E: single QRT-PCR product of the expected molecular mass of ∼180 nt was obtained only in the presence of RT at the end of the QRT-PCR experiment. No product was detected without RT.

Fig. 2.

Scatter plot of gene expression in rat uterine smooth muscle from LP vs. DL. Expression index (EI) data are the average of 3 microarrays per condition. Central thin gray diagonal line shows identical expression; parallel thick black and blue lines represent 2-fold and 10-fold deviation, respectively. Small gray dots represent sequences with no significant changes, red dots sequences significantly upregulated sequences, and blue dots downregulated sequences (P < 0.05). Insets: probe pair sets from LP and DL rats for β-actin (V01217), CX43 (NM_012567), and aquaporin-5 (AQP5; U16245) in rat myometrium. Intensity level of each cell reflects the degree of hybridization, which correlates with the amount of the corresponding transcripts in the original mRNA.

Fig. 3.

Validation of microarray data with QRT-PCR. Relative transcript levels were measured in the linear range of the fluorescence vs. cycle curve as in Fig. 1C. A: Fc-γ. B: Signal peptidase. C: Deiodinase iodothyronine type III. D: Retinaldehyde dehydrogenase. E: Fibulin 5. F: AQP5. G: Sodium-myo-inositol cotransporter (SMIT). H: P-glycoprotein/multidrug resistance1 (MDR). I: transforming growth factor-β3 (TGF-β3). J: glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mean values were normalized to LP from 3–5 independent RNA isolations in each LP and DL group. Values were FC-γ 1 ± 0.11 (n = 5, LP), 0.49 ± 0.04 (n = 5, DL); Signal peptidase 1 ± 0.04 (LP), 0.75 ± 0.05 (DL); Deiodinase iodothyronine type III 1 ± 0.23 (n = 5, LP), 0.33 ± 0.06 (n = 5, DL); Retinaldehyde dehydrogenase 1 ± 0.14 (n = 5, LP), 2.11 ± 0.05 (n = 5, DL); Fibulin 5 1 ± 0.03 (n = 5, LP), 1.6 ± 0.08 (n = 5, DL); AQP5, 1 ± 0.10 (n = 3, LP), 0.022 ± 0.008 (n = 3, DL); SMIT 1 ± 0.06 (n = 3, LP), 0.26 ± 0.03 (n = 3, DL); MDR 1 ± 0.05 (n = 3, LP), 0.008 ± 0.003 (n = 3, DL); TGF-βIII 1 ± 0.08 (n = 3, LP), 7.43 ± 0.16 (n = 3, DL). GAPDH transcripts were practically identical in LP (1 ± 0.10, n = 8) and DL (1.04 ± 0.09, n = 8) rats.

Fig. 4.

Genes differentially expressed in LP and DL. Ten microarrays were analyzed: 5 LP (arrays 1, 2, 3 and 2 replicates of array 1) and 5 DL (arrays 1, 2, 3 and 2 replicates of array 1) (NIH GEO Database accession no. GSE12799). Genes are grouped by their biological function. Color columns show hierarchical cluster analysis of LP and DL in 3 single arrays for each. Color indicates expression level relative to the mean (white): low is blue, and high is red. Top: relationship between the arrays. Each row displays individual genes, with their gene description, GenBank accession no., fold change, and significance (P value > 0.05). Fold changes are the average of 3 arrays from LP and DL animals.