Decidual expression and regulation of fatty acid desaturase 3 during mouse decidualization

in Reproduction
Correspondence should be addressed to Z-M Yang; Email: zmyang@scau.edu.cn
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Decidualization is required for the successful establishment of pregnancy in rodents and primates. Fatty acid desaturase 3 (Fads3) belongs to the fatty acid desaturase family, which is a crucial enzyme for highly unsaturated fatty acid biosynthesis. However, the expression, regulation and function of Fads3 during early pregnancy in mice are still unknown. In this study, we examined Fads3 expression, regulation and function during mouse decidualization. The expression of Fads3 is detected in the subluminal stromal cells at implantation site on day 5 of pregnancy, but not at inter-implantation site and in day 5 pseudopregnant uteri. Compared to delayed implantation, Fads3 is strongly expressed after delayed implantation is activated by estrogen treatment. From days 6 to 8, Fads3 mRNA signals are significantly detected in the decidua. In ovariectomized mice, estrogen significantly stimulates Fads3 expression. However, estrogen has no effect on Fads3 expression in ovariectomized ERα-deficient mice, suggesting that estrogen regulation on Fads3 expression is ERα dependent. When ovariectomized mice were treated with progesterone, Fads3 expression is significantly increased by progesterone. Progesterone stimulation on Fads3 expression is also detected in cultured stromal cells, which is abrogated by RU486 treatment. These data indicate that progesterone upregulation on Fads3 expression is progesterone receptor-dependent. Fads3 knockdown by siRNA reduces in vitro decidualization of mouse stromal cells. Taken together, Fads3 may play an important role during mouse decidualization.

 

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    In situ hybridization of Fads1, Fads2 and Fads3 mRNA in mouse uteri during early pregnancy, showing the localization of Fads1, Fads2 and Fads3 mRNA in mouse uteri on days 1 (D1), 4 (D4), 5 (D5), 6 (D6) and 8 (D8). On each day of pregnancy, 3–5 mice were used. At least three implantation sites per mouse were used for in situ hybridization and a representative section was shown. Asterisk (*), embryo. Bar = 300 μm.

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    Expression of Fads3 mRNA during pseudopregnancy and under delayed implantation. (A) In situ hybridization of Fads3 expression at inter-implantation site on day 5 of pregnancy (D5 NI), on day 5 of pseudopregnancy and under delayed implantation and activated uteri. D5 NI, inter-implantation site on day 5; PD5, day 5 of pseudopregnancy; Asterisk (*), embryo. Bar = 300 μm. Three mice were used in each group. At least 3–5 sections from each mouse were used for in situ hybridization. A representative section was shown. (B) Real-time PCR analysis of Fads3 expression between inter-implantation and implantation on day 5. D5 IS, implantation site on day 5. Uteri from three mice were used for real-time PCR. Three independent repeats were performed.

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    Expression of Fads3 mRNA during artificial decidualization. (A) Real-time PCR analysis of Fads3 expression between inter-implantation and implantation on day 8. D8-NI, inter-implantation site on day 8; D8-IS, implantation site on day 8. (B) In situ hybridization of Fads3 expression under artificial decidualization. Bar = 300 μm. (C) Real-time PCR analysis of Fads3 expression under artificial decidualization. Uteri from three mice were used for real-time PCR. Three independent repeats were performed. At least 3–5 sections from each mouse were used for in situ hybridization. A representative section was shown.

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    Steroid hormones regulation on Fads3 mRNA expression. (A) In situ hybridization of Fads3 expression after ovariectomized mice were treated with sesame oil, estrogen and progesterone, respectively. (B) Real-time PCR of Fads3 mRNA expression after WT or ER KO ovariectomized mice were injected with estrogen. (C) Real-time PCR of Fads3 mRNA expression after WT ovariectomized mice were injected with progesterone. (D) Real-time PCR of Fads3 mRNA expression after stromal cells were treated with progesterone or progesterone plus RU486. Con, control; E, estrogen; P, progesterone; group P+RU486. Co-treatment with progesterone plus RU486. (E) Effects of JNK inhibitor on progesterone-induced Fads3 expression. Stromal cells were pretreated with JNK inhibitor for 1 h and then treated with progesterone. Three mice were used in each group. At least 3–5 sections from each mouse were used for in situ hybridization. A representative section was shown. All experiments were repeated at least three times.

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    Expression and function of Fads3 under in vitro decidualization. (A) Real-time PCR of Dtprp mRNA expression under in vitro decidualization from 12 h to 72 h. (B) Real-time PCR of Fads3 mRNA expression during in vitro decidualization. Fads3 expression was obviously upregulated compared to the control from 12 h to 72 h. (C) Fads3 mRNA expression after stromal cells were transfected with Fads3 siRNA and the nonspecific siRNA (NC) under in vitro decidualization for 48 h. (D) Western blot analysis showed that FADS3 protein level was significantly reduced by Fads3 siRNA. (E) Effects of Fads3 siRNA on Dtprp expression. (F) Effects of Fads3 siRNA on Prl3c1 expression. (G) Effects of Fads3 siRNA on Bmp8a expression. (H) Effects of Fads3 siRNA on Abp1 expression. Con, control; group E + P, Co-treatment with estrogen plus progesterone. Stromal cells from 3 to 6 mice were isolated, pooled and used for cell culture and treatments in triplicates. All experiments were repeated at least three times.

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    Effects of PGE2 on Fads3 action during in vitro decidualization. (A) PGE2 has no effects on Fads3 expression. (B) PGE2 has no stimulating effects on Dtprp, but can rescue the inhibitory effect of Fads3 siRNA on Dtprp level. (C) PGE2 has no stimulating effects on Dtprp, but can rescue the inhibitory effect of Fads3 siRNA on Prl3c1. Stromal cells from 3 to 6 mice were isolated, pooled and used for cell culture and treatments in triplicates. All experiments were repeated at least three times.

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