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. 2021 Jun 1;162(6):bqab054.
doi: 10.1210/endocr/bqab054.

Endometrium On-a-Chip Reveals Insulin- and Glucose-induced Alterations in the Transcriptome and Proteomic Secretome

Tiago H C De Bem  1   2 , Haidee Tinning  1 , Elton J R Vasconcelos  3 , Dapeng Wang  3 , Niamh Forde  1   3
Affiliations

Endometrium On-a-Chip Reveals Insulin- and Glucose-induced Alterations in the Transcriptome and Proteomic Secretome

Tiago H C De Bem et al. Endocrinology. .

Abstract

The molecular interactions between the maternal environment and the developing embryo are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multicellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of nonpregnant cows in the early luteal phase (Days 4-7) were seeded in the upper chamber of the device (epithelial cells; 4-6 × 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 × 104 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0, or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) was performed at a flow rate of 1 µL/minute for 72 hours. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro-derived uterine luminal fluid were determined by RNA-sequencing and tandem mass tagging mass spectrometry, respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (P < .05), with a dose-dependent quantitative change in the protein secretome (1 and 23 proteins). Altering insulin concentrations resulted in limited transcriptional changes including transcripts for insulin-like binding proteins that were cell specific but altered the quantitative secretion of 196 proteins. These findings highlight 1 potential mechanism by which changes to maternal glucose and insulin alter uterine function.

Keywords: Endometrium on-a-chip; bovine; cattle; microfluidics; uterine luminal fluid; uterus.

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Figures

Figure 1.
Figure 1.
Flow cytometry validation of bovine endometrial (A) stromal enriched or (B) epithelial enriched cell types. Primary bovine endometrial cells were isolated as described and cultured to confluency. Cells were lifted using trypsin (0.025%) in PBS, washed in PBS, and 1 × 106 cells from each cell type fixed and permeabilized using the FIX & PERM kit as per the manufacturers protocol (ThermoFisher Scientific). (A) Antikeratin 18 rabbit IgG (1 μg/mL) (SAB5500126) was added to the epithelial-enriched cell fraction, incubated for 15 minutes, washed with PBS, and incubated with anti-rabbit IgG with 680 nm flurophore (1 μg/mL) (SAB4600395) for 15 minutes. After a final wash cells were immediately analyzed on a CytoFLEX S (Beckman Coulter) (640-nm laser). (B) Antivimentin mouse IgG (1 μg/mL) (SAB4200761) was added to the stromal enriched cell fraction, incubated for 15 minutes, washed in PBS, and incubated with antimouse IgG with 488 nm flurophore (1 μg/mL) (SAB4600029) for 15 minutes. After a final wash cells were immediately analyzed on a CytoFLEX S (488 nm laser). Appropriate gating was used to exclude cell debris and clumps of cells, and control samples including no antibody, primary only, and secondary only were included. The % of positively stained cells were determined by gating the area to the right of the unstained peak.
Figure 2.
Figure 2.
(A) Schematic diagram of endometrium-on-a-chip microfluidic device and experimental design used to mimic the physiological extremes of glucose and insulin. Representative images of (B) epithelial cells seeded into the upper chamber, (C) cells attached on and under the porous membrane, and (D) stromal cells seeded into the lower chamber. The rate of flow for both experiments was performed at 1 μL/min per 72 hours.
Figure 3.
Figure 3.
(A) Principal component analysis (PCA) plot of overall transcriptional profile determined via RNA sequencing of bovine endometrial epithelial and stromal cells exposed to either 5 mM or 50 mM of glucose for 72 hours under flow (n = 3 biological replicates). Epithelial (left hand side) and stromal cells (right hand side) clustered into 2 distinct populations. (B) Heatmap showing the transcript expression for individual samples with lower levels (blue) and those with higher expression shown in red. Samples from epithelial cells (n = 6) and samples from stromal cells (n = 6) are arranged from left to the right.
Figure 4.
Figure 4.
Gene ontology overrepresented Biological Process, Cellular Component, and Molecular Function from DEGs in (A) epithelial (n = 21) and (B) stromal cells (n = 191) following exposure to different concentrations of glucose in vitro. The transcript accession numbers (Ensembl Transcript ID) were inputted into DAVID—Functional Annotation Tool (DAVID Bioinformatics Resources 6.7, NIAID/NIH—https://david.ncifcrf.gov/summary.jsp) and those that were significantly overrepresented in the list of DEGs are presented above for biological processes (blue bars), cellular component (orange bars) and molecular function (plum bars) with associated enrichment score.
Figure 5.
Figure 5.
(A) Principal component analysis (PCA) plot shows the distribution of in vitro-derived ULF following tandem mass tag (TMT) mass spectrometry analysis of the proteomic content following exposure to: 0.5 mM (green circle), 5.0 mM (yellow circle), or 50 mM (red circle) concentrations of glucose for 72 hour (n = 3 biological replicates). (B) Venn diagram analysis of overlap and unique proteins that are differentially abundance following exposure to different concentrations of glucose. Fold change differences in protein abundances between groups was determined using paired t-tests and were considered significant when P < .05.
Figure 6.
Figure 6.
(A) Principal component analysis (PCA) plot of overall transcriptional profile determined via RNA sequencing of bovine endometrial epithelial and stromal cells exposed to either vehicle control, 1 ng/mL, or 10 ng/mL of Insulin for 72 hour under flow (n = 3 biological replicates). Epithelial (left hand side) and stromal cells (right hand side) clustered into 2 distinct populations. (B) Heatmap showing the transcript expression for individual samples with lower levels (blue) and those with higher expression shown in red. Samples from epithelial cells (n = 9) and samples from stromal cells (n = 9) are arranged from left to the right.
Figure 7.
Figure 7.
(A) Principal component analysis (PCA) plot shows the distribution of in vitro–derived ULF following tandem mass tag (TMT)-mass spectrometry analysis of the proteomic content following exposure to: vehicle control (green circle), 1 ng/mL Insulin (yellow circle), or 10 ng/mL Insulin (red circle) for 72 hours (n = 3 biological replicates). (B) Venn diagram analysis of overlap and unique proteins that are differentially abundant following exposure to different concentrations of insulin. Fold change differences in protein abundances between groups was determined using paired t-tests and were considered significant when P < .05.
Figure 8.
Figure 8.
Network interaction between the differentially abundant proteins and their signaling pathways (P < .05) from in vitro–derived ULF from endometrium on-a-chip exposed to insulin for 72 hours. (A) GO pathways associated with proteins altered in abundance following treatment with 1.0 ng/mL of insulin compared to vehicle control. (B) GO pathways associated with proteins altered in abundance following treatment with 10.0 ng/mL of insulin was compared to vehicle control. The color circle represents an individual signaling pathway and the proteins around it are proteins identified as altered in our treatments associated with signaling pathway(s) that are either upregulated (red) or downregulated (blue) following insulin treatment. The color scale represents the fold enrichment that reflects the involvement of the proteins in each signaling pathway.
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References

    1. Rizos D, Clemente M, Bermejo-Alvarez P, de La Fuente J, Lonergan P, Gutiérrez-Adán A. Consequences of in vitro culture conditions on embryo development and quality. Reprod Domest Anim. 2008;43(Suppl 4):44-50. - PubMed
    1. Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 2002;61(2):234-248. - PubMed
    1. Maillo V, Lopera-Vasquez R, Hamdi M, Gutierrez-Adan A, Lonergan P, Rizos D. Maternal-embryo interaction in the bovine oviduct: Evidence from in vivo and in vitro studies. Theriogenology. 2016;86(1):443-450. - PubMed
    1. de Ávila ACFCM, da Silveira JC. Role of extracellular vesicles during oocyte maturation and early embryo development. Reprod Fertil Dev. 2019;32(2):56-64. - PubMed
    1. Nguyen HP, Simpson RJ, Salamonsen LA, Greening DW. Extracellular vesicles in the intrauterine environment: challenges and potential functions. Biol Reprod. 2016;95(5):109. - PMC - PubMed

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