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. 2021 Apr 21;2(2):107-116.
doi: 10.1530/RAF-20-0035. eCollection 2021 Apr.

Nitric oxide positively affects endometrial receptivity via FAAH and NAPE-PLD in vitro

Sarah E Melford  1 , Anthony H Taylor  1 , Justin C Konje  1
Affiliations

Nitric oxide positively affects endometrial receptivity via FAAH and NAPE-PLD in vitro

Sarah E Melford et al. Reprod Fertil. .

Abstract

Objective: To determine if models of human 'receptive' and 'non-receptive endometrium' differ in their responses to nitric oxide (NO) supplementation by measuring the levels of the enzymes of the endocannabinoid system (ECS) (fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD)), which control the 'anandamide tone' essential for successful pregnancy.

Design: A study of FAAH and NAPE-PLD expression (using human endometrium) through the menstrual cycle and an in vitro using a model of 'receptive' (Ishikawa) and 'non-receptive' (HEC-1A) human endometrial cell lines treated with the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP).

Results: Immunoreactivity measured by optimised H-score for both FAAH and NAPE-PLD was reduced in secretory (receptive) endometrium compared to proliferative (non-receptive) endometrium (P = 0.0009 and <0.0001, respectively). FAAH and NAPE transcript levels were significantly higher in untreated Ishikawa cells than in HEC-1A cells (P = 0.0228 and 0.0001, respectively). Treatment of cultures with SNAP resulted in an increase in the amount of FAAH mRNA produced by Ishikawa cells and a decrease in NAPE-PLD mRNA. No effect of SNAP was observed in HEC-1A cells. Similarly, FAAH protein was significantly decreased in endometria representative of the receptive endometrium.

Conclusion: These data suggest that NO most likely affects the expression of ECS enzymes in the implantation site of a receptive endometrium; a phenomenon not seen in a non-receptive endometrium. These effects are most marked with FAAH expression, suggesting that FAAH may play the more critical role in ensuring the correct 'anandamide tone' for successful embryo implantation than NAPE-PLD.

Lay summary: Embryo implantation into the wall of the uterus is only successful when the inner wall of the uterus (the endometrium) is 'receptive', because if it is 'non-receptive', implantation will fail. Previous work showed that enzymes of the 'endocannabinoid system' are critical for implantation by maintaining the correct level of a fat called anandamide. This is by balancing its synthesis (by N-acylphosphatidylethanolamine specific phospholipase D, NAPE-PLD) and degradation (by fatty acid amide hydrolase, FAAH). Using immortalised cell lines as models of 'receptive' and 'non-receptive' human endometrium, we demonstrate a key stimulator of implantation, nitric oxide, has a positive effect on implantation by both increasing the mRNA levels of the degrading enzyme (FAAH) and decreasing the expression of the synthesising enzyme (NAPE-PLD). These effects are most marked with the degrading enzyme, suggesting that FAAH plays a more critical role than NAPE-PLD in ensuring the correct 'anandamide tone' for successful embryo implantation.

Keywords: N-acylphosphatidylethanolamine; endocannabinoid; endometrial receptivity; fatty acid amide hydrolase; nitric oxide.

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Conflict of interest statement

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Figure 1
Figure 1
Immunohistochemical staining and histomorphometric analyses of NAPE-PLD and FAAH protein expression through the menstrual cycle. Endometrial biopsies taken from the menstrual (Men, n  = 6), early- (EP, n  = 8), mid- (MP, n  = 8) and late-proliferative (LP, n  = 8) phases and the early- (ES, n  = 8), mid- (MS, n  = 8) and late-secretory (LS, n  = 8) phases of the menstrual cycle were subjected to immunohistochemistry with FAAH and NAPE-PLD specific (Panel A) antibodies. After image capture, the intensity of brown DAB staining was determined histomorphometrically (Panel B) for the endometrial glandular epithelial cells (left panels) and endometrial stromal cells (right panels) as separate measurements for each enzyme. The data are presented as the mean ± s.d. histoscore (H-score). FAAH immunoreactivity was reduced in both the glandular epithelial and stromal cells of receptive (open bars) endometria (MS; ***P = 0.0009; ****P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test) when compared to the H-score of the non-receptive (solid bars) endometria (MP). NAPE-PLD immunoreactivity was also reduced in the glandular epithelial and stromal cell compartments during the receptive phase of the menstrual cycle (ES vs MP ****P < 0.0001; MS vs MP ****P < 0.0001).
Figure 2
Figure 2
Histomorphometric analyses of NAPE-PLD and FAAH protein expression in receptive and non-receptive endometria. Endometrial biopsies were taken from the early and middle secretory phases of the menstrual cycle (ES and MS = receptive), and biopsies from the proliferative and late secretory phase of the menstrual cycle (EP, MP, LP and LS = non-receptive) were subjected to immunohistochemistry (see Fig. 1) with FAAH (upper panels) and NAPE-PLD-specific (lower panels) antibodies. After image capture, the intensity of brown DAB staining was determined histomorphometrically for the endometrial glandular epithelial cells (left panels) and endometrial stromal cells (right panels). The data are presented as the mean ± s.d. histoscore (H-score). Both FAAH and NAPE-PLD immunoreactivity were reduced in the glandular epithelial cells of receptive endometria (n = 16; ****P < 0.0001; Student’s unpaired t-test with Welch correction for unequal variances) when compared to that of the non-receptive endometria (n = 38). There was no change in the amount of FAAH expressed in the stroma. By contrast, there was a significant reduction in NAPE-PLD expression in the stroma in the receptive endometria when compared to the non-receptive endometria (****P < 0.0001).
Figure 3
Figure 3
A comparison of the relative levels of FAAH (upper panel) and NAPE-PLD (lower panel) mRNA in untreated Ishikawa and HEC-1A cells. Ishikawa and HEC-1A cells were cultured for 48 h and total cellular RNA prepared as described in the 'Materials and Method' section. The amounts of FAAH and NAPE-PLD mRNA generated from RT and by using gene-specific TaqMan based PCR were normalised to mRNA levels of the reference gene GAPDH to provide a relative mRNA level. The data are presented as the mean ± s.d. of FAAH or NAPE-PLD mRNA levels relative to the amounts of GAPDH. Both sets of data showed a significant lower amount of transcripts for the enzymes in the HEC-1A cells (FAAH ****P  < 0.0001, NAPE-PLD *P = 0.0228; Student’s unpaired t-test) when compared to the levels found in Ishikawa cells.
Figure 4
Figure 4
The effect of SNAP on FAAH and NAPE-PLD mRNA expression in receptive and non-receptive endometrial cell lines. Ishikawa cells (upper panel) and HEC-1A cells (lower panel) were exposed to the indicated concentrations of SNAP for 48 h. The amount of FAAH and NAPE-PLD mRNA present was determined after RT and by using gene-specific TaqMan based PCR (see Materials & Methods section). For each sample, FAAH and NAPE-PLD amplicon levels were then normalised against those for the reference gene GAPDH. The data are presented as the mean ± s.d. relative amounts of FAAH (n = 6) or NAPE-PLD (n = 6) with the error bars not shown when encompassed by the bar. One-way ANOVA (P < 0.0001) revealed that FAAH transcript levels in Ishikawa cells treated with 2000 µM SNAP was significantly higher than 0, 50, 100 and 500 µM SNAP (***P < 0.001 for 0 and 50 µM, **P < 0.01 for 100 and 500 µM SNAP; Tukey’s multiple comparison test). By contrast, SNAP had no significant effect on FAAH transcript levels in HEC-1A cells (P > 0.05, Tukey’s multiple comparison test). Similarly, in Ishikawa cells, 500, 1000 and 2000 µM SNAP caused a significant decrease in NAPE-PLD transcript levels (**P < 0.01 for 500 and 1000 µM, *P < 0.05 for 2000 µM; Tukey’s multiple comparison test) when compared to the untreated control and 50 μM SNAP. Similar to the data for FAAH expression, SNAP had no effect on NAPE-PLD transcript levels in HEC-1A cells (P > 0.05).
Figure 5
Figure 5
The effect of the NO inducer SNAP on the ratio of FAAH to NAPE-PLD transcript levels in Ishikawa and HEC-1A cells. The data are presented as a single plot for each FAAH:NAPE-PLD ratio for Ishikawa (upper panel) and HEC-1A cells (lower panel) treated for 48 h with the indicated concentrations of SNAP. Linear regression analysis (Pearson’s correlation) revealed a strong correlation between SNAP concentrations and the FAAH:NAPE-PLD ratio in Ishikawa cells (r2 = 0.9844, P  < 0.0001), whereas there was no correlation between the FAAH:NAPE-PLD ratio and SNAP concentrations in HEC-1A cells (r2 = 0.1295, P = 0.4835).
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References

    1. Aban CE, Accialini PL, Etcheverry T, Leguizamon GF, Martinez NA, Farina MG.2018. Crosstalk between nitric oxide and endocannabinoid signaling pathways in normal and pathological placentation. Frontiers in Physiology 9 1699. (10.3389/fphys.2018.01699) - DOI - PMC - PubMed
    1. Ayakannu T, Taylor AH, Bari M, Mastrangelo N, Maccarrone M, Konje JC.2019. Expression and function of the endocannabinoid modulating enzymes fatty acid amide hydrolase and N-acylphosphatidylethanolamine-specific phospholipase D in endometrial carcinoma. Frontiers in Oncology 9 1363. (10.3389/fonc.2019.01363) - DOI - PMC - PubMed
    1. Bambang KN, Karasu T, Gebeh A, Taylor AH, Marczylo TH, Lam P, Willets JM, Konje JC.2010. From fertilisation to implantation in mammalian pregnancy – modulation of early human reproduction by the endocannabinoid system. Pharmaceuticals 3 2910–2929. (10.3390/ph3092910) - DOI - PMC - PubMed
    1. Castelbaum AJ, Ying L, Somkuti SG, Sun J, Ilesanmi AO, Lessey BA.1997. Characterization of integrin expression in a well differentiated endometrial adenocarcinoma cell line (Ishikawa). Journal of Clinical Endocrinology and Metabolism 82 136–142. (10.1210/jcem.82.1.3658) - DOI - PubMed
    1. Chwalisz K, Garfield RE.2000. Role of nitric oxide in implantation and menstruation. Human Reproduction 15 (Supplement 3) 96–111. (10.1093/humrep/15.suppl_3.96) - DOI - PubMed

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