Decidualized Endometrial Stromal Cells Promote Mitochondrial Beta-Oxidation to Produce the Octanoic Acid Required for Implantation

Abstract

Decidualization denotes the morphological and biological differentiating process of human endometrial stromal cells (HESCs). Fatty acid pathways are critical for endometrial decidualization. However, the participation of fatty acids as an energy source and their role in endometrial decidualization have received little attention. To identify fatty acids and clarify their role in decidualization, we comprehensively evaluated free fatty acid profiles using liquid chromatography/Fourier transform mass spectrometry (LC/FT-MS). LC/FT-MS analysis detected 26 kinds of fatty acids in the culture medium of decidualized or un-decidualized HESCs. Only the production of octanoic acid, which is an essential energy source for embryonic development, was increased upon decidualization. The expressions of genes related to octanoic acid metabolism including ACADL, ACADM, and ACADS; genes encoding proteins catalyzing the first step of mitochondrial fatty acid beta-oxidation; and ACSL5 and ACSM5; genes encoding fatty acid synthesis proteins were significantly altered upon decidualization. These results suggest that decidualization promotes lipid metabolism, implying that decidualized HESCs require energy metabolism of the mitochondria in embryo implantation.

Keywords: decidualization; embryo implantation; mitochondrial beta-oxidation; octanoic acid.

Figure 1

Effects of decidualizing stimuli using 8-br-cAMP and MPA in HESCs. (a) Morphological changes in HESCs. Undifferentiated primary HESCs exhibit a fibroblastic spindle-shaped morphology (CTL). Primary HESCs, upon decidualization by 8-br-cAMP and MPA, show abundant cytoplasm and larger nuclei, which are the typical morphology of decidualized cells. (b) Primary HESCs are stimulated in the absence (CTL) or presence of 8-br-cAMP and MPA. IGFBP1 and PRL mRNA expressions are measured using RT-qPCR. Results were normalized to GAPDH and compaired with CTL was set as 1. Statistical analysis performed by two-tailed Student’s t-tests. Data are shown as the mean ± SEM (standard error of the mean, n = 3). ** p < 0.01, *** p < 0.001.

Figure 2

Evaluation of 26 kinds of medium-chain fatty acids and long-chain fatty acids in the culture medium of decidualized HESCs analyzed using LC/FT-MS. All kinds of fatty acids but octanoic acid did not increase upon decidualization in the culture medium. Statistical analysis of un-decidualized (CTL) and decidualized (cAMP+MPA) are performed by two-tailed Student’s t-tests. Data are shown as the mean ± SEM (standard error of the mean, n = 3–4). * p < 0.05, ** p < 0.01, *** p < 0.001. NS; No significant difference. “Medium” refers to the culture medium before use.

Figure 3

Evaluation of the fatty acid beta-oxidation activity by palmitate with the radiolabel at position C-1. Primary HESCs are stimulated in the absence (CTL) or presence of 8-br-cAMP and MPA, and the incomplete oxidation of [1–14C]-labeled capric acids (C10:0) (a) or [1–14C]-labeled palmitic acid (C16:0) (b) is measured as the fatty acid oxidation rate. Statistical analysis performed by two-tailed Student’s t-tests. Data are shown as the mean ± SEM (standard error of the mean, n = 6). * p < 0.05, *** p < 0.001.

Figure 4

Expression profiles of the genes associated with the beta-oxidation process. Primary HESCs are stimulated in the absence (CTL) or presence of 8-br-cAMP and MPA. The expression of each gene mRNA are measured using RT-qPCR and normalized to GAPDH. The diagram in the middle shows the metabolic pathways of fatty acids. Expressions of long chain (green), medium-chain (red), and short chain (blue) fatty acid metabolism-related genes are analyzed using RT-PCR. Statistical analysis performed by two-tailed Student’s t-tests. Data are shown as the mean ± SEM (standard error of the mean, n = 3-6). * p < 0.05, ** p < 0.01, *** p < 0.001, NS; No significant difference.

Figure 5

Effects of octanoic acid addition in culture medium for HTR-8/SVneo cell invasion. (a) HTR-8/SVneo cells invaded by incubation with control (10% FBS) or octanoic acid (200 ng/mL in 10% FBS). (b) Quantification of the average number of invaded HTR-8/SVneo cells (mean ± SEM, n = 3, p = 0.1; Mann–Whitney U test). Each experiment is performed in triplicate (n = 3). The black dots represent the average of five fields for each experiment.