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. 2016 Sep 26:6:33811.
doi: 10.1038/srep33811.

Compartmentalized gene expression profiling of receptive endometrium reveals progesterone regulated ENPP3 is differentially expressed and secreted in glycosylated form

Nageswara Rao Boggavarapu  1 , Sujata Lalitkumar  1 , Vijay Joshua  1 , Sergo Kasvandik  2   3   4 , Andres Salumets  2   3   4 , Parameswaran Grace Lalitkumar  1 , Kristina Gemzell-Danielsson  1
Affiliations

Compartmentalized gene expression profiling of receptive endometrium reveals progesterone regulated ENPP3 is differentially expressed and secreted in glycosylated form

Nageswara Rao Boggavarapu et al. Sci Rep. .

Abstract

The complexity of endometrial receptivity at the molecular level needs to be explored in detail to improve the management of infertility. Here, differential expression of transcriptomes in receptive endometrial glands and stroma revealed Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a progesterone regulated factor and confirmed by various methods, both at mRNA and protein level. The involvement of ENPP3 in embryo attachment was tested in an in vitro model for human embryo implantation. Interestingly, there was high expression of ENPP3 mRNA in stroma but not protein. Presence of N-glycosylated ENPP3 in receptive phase uterine fluid in women confirms its regulation by progesterone and makes it possible to use in a non-invasive test of endometrial receptivity.

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Figures

Figure 1
Figure 1. Heatmaps for hierarchical clustering of significant genes.
Genes regulated by progesterone in endometrial glandular (G, left panel) and stromal (S, right panel) compartments shown by heatmaps. Gene expression was studied in the receptive endometrium (C) in a non-treated cycle and non-receptive endometrium (T) with the treatment of progesterone receptor antagonist mifepristone. Each woman in the study acted as their own control.
Figure 2
Figure 2. Progesterone regulated genes in endometrial stroma.
Tukey plots of significant genes of stromal compartment as analyzed by real time PCR. Progesterone inhibition upregulated CPM and SFRP4 and down regulated MT1G, MT2A and ENPP3.
Figure 3
Figure 3. Tukey plot for progesterone regulated genes in endometrial glands.
Endometrial epithelial compartment showed down regulation of MT2A, MT1and ENPP3 with the inhibition of P by mifepristone. Gene expression for SFRP4 and UBE2E2 was significantly upregulated with the inhibition of progesterone.
Figure 4
Figure 4. Regulation of ENPP3 by progesterone.
The expression of ENPP3 was observed very specifically in the apical border of glands in P dominant mid-luteal phase as studied by immunohistochemistry (A,C,D) in women without mifepristone treatment (control). Downregulation of ENPP3 was observed in endometrial glands with the suppression of progesterone action by mifepristone treatment (B,C). In endometrium of women without mifepristone treatment, high level of ENPP3 was observed during P upregulated secretory phase, on comparing with proliferative phase of the menstrual cycle. Scale bar indicates 50 μm.
Figure 5
Figure 5. Expression of ENPP3 in endometrium and uterine fluid.
Western blot analysis of ENPP3 in receptive phase endometrial tissue lysates (A,B) and uterine fluid (C,D) by Wes showed good expression levels of ENPP3 in control samples (lanes 2–7). Antiprogestin treatment (A,C): lanes 8–13) showed no detectable levels of ENPP3, both in endometrial tissue and uterine fluid, confirming the regulation of ENPP3 protein by progesterone. (B,D) show semiquantitative analysis of immunodetectable ENPP3 by Wes, expressed in log2 AUC (area under the curve). Lane 1: protein molecular weight marker.
Figure 6
Figure 6. N-glycosylated ENPP3 in human endometrium.
Deglycosylation of uterine fluid showed a shift of band from 165 kD to a single band at 110 kD, meaning that ENPP3 in the uterine fluid is present in its glycosylated form. The glycosylated (lanes 2–7) form of ENPP3 showed a band at 165 kD and the deglycosylated (lanes 8–13) form had a single band at 110 kD.
Figure 7
Figure 7. Embryo attachment and ENPP3 expression.
Human embryo attached to the in vitro three-dimensional cell culture model with progesterone exposure (A). None of the 8 embryos in the anti-progestin treated group, where ENPP3 was significantly down regulated had attached. In the control group, 7 out of 10 blastocysts attached (B) to the construct, with very good expression of ENPP3 in the three-dimensional endometrial cell culture system.
Figure 8
Figure 8. Expression of ENPP3 in the uterine fluid of receptive and non-receptive phase.
Tukey plot for the expression of ENPP3 quantified by nano-ESI-LC/MS in uterine fluids from early secretory phase (LH + 2) and mid-secretory phase (LH + 8) of the same set of women. A significant upregulation of ENPP3 (p = 0.0032) was observed in P dominant, receptive phase (LH + 8) on comparison with non-receptive phase (LH + 2).
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