Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway

Abstract

Serotonin (5-HT) and its specific transporter, SERT play important roles in pregnancy. Using placentas dissected from 18d gestational SERT-knock out (KO), peripheral 5-HT (TPH1)-KO, and wild-type (WT) mice, we explored the role of 5-HT and SERT in placental functions in detail. An abnormal thick band of fibrosis and necrosis under the giant cell layer in SERT-KO placentas appeared only moderately in TPH1-KO and minimally present in WT placentas. The majority of the changes were located at the junctional zone of the placentas in SERT. The etiology of these findings was tested with TUNEL assays. The placentas from SERT-KO and TPH1-KO showed 49- and 8-fold increase in TUNEL-positive cells without a concurrent change in the DNA repair or cell proliferation compared to WT placentas. While the proliferation rate in the embryos of TPH1-KO mice was 16-fold lower than the rate in gestational age matched embryos of WT or SERT-KO mice. These findings highlight an important role of continuous 5-HT signaling on trophoblast cell viability. SERT may contribute to protecting trophoblast cells against cell death via terminating the 5-HT signaling which changes cell death ratio in trophoblast as well as proliferation rate in embryos. However, the cell death in SERT-KO placentas is in caspase 3-independent pathway.

Keywords: placenta; serotonin; serotonin transporter.

Figure 1. Expression of SERT in E18 mouse placentas

The 4 μm sections of parafine embeded placentas dissected from E18 WT and SERT-KO mice were dewaxed, rehydrated and subjected to antigen retrieval. Sections were stained as described in the Methods with polyclonal SERT- and monoclonal TROP-1 antibodies (1:100 dilution) followed by Alexa549-conjugated donkey anti-goat and Alexa488 donkey anti-rabbit dye (1:250 dilution). Nuclei were counterstained with DAPI in blue (1:250 dilution). In placentas dissected from SERT-KO mouse only green TROP-1 and blue DAPI staining are found. In WT placentas red, SERT-staining appeared over TROP-1. Cells were analyzed with a Zeiss LSM510 laser confocal microscope. The lower panels are 10X the magnification of the area framed in white in the upper panels. The scale bars for the upper panels are in 1 μm and for the lower panels 10 μm indicate the magnification of the images. Figures show representative images from at least 2 separate experiments.

Figure 2

H&E stained E18 SERT-KO (A) and WT (B) placenta at 2X highlighting junctional zone showing necrotic area (indicated with an asterisk) in SERT-KO with minimal serous fluid (arrows) in WT placentas. H&E staining are the representative images of 10 slides per placenta, 6 placentas from each mouse, n=4. The insets at the right of the main figures are the 20X the magnification of the areas framed in white. Trichrome staining of placentas from E.18 d SERT-KO (C) and WT (D) mice. Photomicrographs of the junctional zone stained with trichrome. Royal blue color and arrows highlight connective tissue (collagen) within the necrosis in SERT-KO and WT at 10X magnification.

Figure 2

H&E stained E18 SERT-KO (A) and WT (B) placenta at 2X highlighting junctional zone showing necrotic area (indicated with an asterisk) in SERT-KO with minimal serous fluid (arrows) in WT placentas. H&E staining are the representative images of 10 slides per placenta, 6 placentas from each mouse, n=4. The insets at the right of the main figures are the 20X the magnification of the areas framed in white. Trichrome staining of placentas from E.18 d SERT-KO (C) and WT (D) mice. Photomicrographs of the junctional zone stained with trichrome. Royal blue color and arrows highlight connective tissue (collagen) within the necrosis in SERT-KO and WT at 10X magnification.

Figure 3. Tissue injury in placentas of E.18 d SERT- and TPH1-KO and WT mice placentas

(A) TUNEL assay performed in placentas of these mice. Note the uneven (patchy) TUNEL staining. The images in each panel are acquired at 200X magnification. (B) Quantification of the TUNEL. The apoptosis in placentas from SERT-KO mice were 65-fold higher than WT, 6-fold higher than the rates of placentas from TPH1-KO mice. (C) Quantification of the proliferation marker Ki67 mean intensity. The images in each panel are acquired at 400X magnification. No difference was seen on the proliferation rates of placentas from transgenic or WT mice. TUNEL assay and Ki67 staining were performed on 10 slides per placenta, 6 placentas from each mouse, n=4. Asterisks indicate statistical difference between WT and SERT-KO (*) mice P<0.05 assessed by one way ANOVA and a Bonferroni post hoc test. The scale bars represent 10 μm.

Figure 3. Tissue injury in placentas of E.18 d SERT- and TPH1-KO and WT mice placentas

(A) TUNEL assay performed in placentas of these mice. Note the uneven (patchy) TUNEL staining. The images in each panel are acquired at 200X magnification. (B) Quantification of the TUNEL. The apoptosis in placentas from SERT-KO mice were 65-fold higher than WT, 6-fold higher than the rates of placentas from TPH1-KO mice. (C) Quantification of the proliferation marker Ki67 mean intensity. The images in each panel are acquired at 400X magnification. No difference was seen on the proliferation rates of placentas from transgenic or WT mice. TUNEL assay and Ki67 staining were performed on 10 slides per placenta, 6 placentas from each mouse, n=4. Asterisks indicate statistical difference between WT and SERT-KO (*) mice P<0.05 assessed by one way ANOVA and a Bonferroni post hoc test. The scale bars represent 10 μm.

Figure 3. Tissue injury in placentas of E.18 d SERT- and TPH1-KO and WT mice placentas

(A) TUNEL assay performed in placentas of these mice. Note the uneven (patchy) TUNEL staining. The images in each panel are acquired at 200X magnification. (B) Quantification of the TUNEL. The apoptosis in placentas from SERT-KO mice were 65-fold higher than WT, 6-fold higher than the rates of placentas from TPH1-KO mice. (C) Quantification of the proliferation marker Ki67 mean intensity. The images in each panel are acquired at 400X magnification. No difference was seen on the proliferation rates of placentas from transgenic or WT mice. TUNEL assay and Ki67 staining were performed on 10 slides per placenta, 6 placentas from each mouse, n=4. Asterisks indicate statistical difference between WT and SERT-KO (*) mice P<0.05 assessed by one way ANOVA and a Bonferroni post hoc test. The scale bars represent 10 μm.

Figure 4. The location of the death cells in placentas of E.18 d SERT- and TPH1-KO and WT mice placentas

A. TUNEL assays were performed on maternal and fetus sides of the placentas from E.18 d SERT-KO, TPH1-KO and WT mice. The images in each panel are acquired at 200X magnification. The scale bars represent 10 μm. B. Quantification of the TUNEL assays showed that the highest level of the cell death occurred at the maternal side of the SERT-KO placentas was 65-fold higher than the apoptosis of the cells at the maternal side of the WT placentas. TUNEL assays were performed on 10 slides per placenta, 6 placentas from each mouse, n=4. Asterisks indicate statistical difference between WT and SERT-KO maternal (*) and fetal side (**) of placentas P<0.05 and P<0.001 assessed by one way ANOVA and a Bonferroni post hoc test.

Figure 4. The location of the death cells in placentas of E.18 d SERT- and TPH1-KO and WT mice placentas

A. TUNEL assays were performed on maternal and fetus sides of the placentas from E.18 d SERT-KO, TPH1-KO and WT mice. The images in each panel are acquired at 200X magnification. The scale bars represent 10 μm. B. Quantification of the TUNEL assays showed that the highest level of the cell death occurred at the maternal side of the SERT-KO placentas was 65-fold higher than the apoptosis of the cells at the maternal side of the WT placentas. TUNEL assays were performed on 10 slides per placenta, 6 placentas from each mouse, n=4. Asterisks indicate statistical difference between WT and SERT-KO maternal (*) and fetal side (**) of placentas P<0.05 and P<0.001 assessed by one way ANOVA and a Bonferroni post hoc test.

Figure 5. Ki67 staining of Embryonic heart from E.18 d WT and transgenic mice

(A) The embryonic tissues were stained with Ki67, proliferation marker and DAPI to orient the staining in nucleus. Merged image show the level of staining in each tissue. The images in each panel are acquired at 400X magnification. The scale bars represent 10 μm. (B) Quantification of the proliferation marker Ki67 mean intensity. The proliferation rates in embryos of TPH1-KO were 16-fold lower than the rates of WT. This difference was much less pronounced in between the proliferation rates of embryos from SERT-KO and WT which was only 1.3-fold less in SERT-KO embryos. Asterisks indicate statistical difference between WT and SERT-KO or TPH1-KO mice (**) P<0.01 or (***) P<0.001 assessed by one way ANOVA and a Bonferroni post hoc test. All assays were performed on 10 slides per embryo, 6 embryos from each mouse, n=4.

Figure 5. Ki67 staining of Embryonic heart from E.18 d WT and transgenic mice

(A) The embryonic tissues were stained with Ki67, proliferation marker and DAPI to orient the staining in nucleus. Merged image show the level of staining in each tissue. The images in each panel are acquired at 400X magnification. The scale bars represent 10 μm. (B) Quantification of the proliferation marker Ki67 mean intensity. The proliferation rates in embryos of TPH1-KO were 16-fold lower than the rates of WT. This difference was much less pronounced in between the proliferation rates of embryos from SERT-KO and WT which was only 1.3-fold less in SERT-KO embryos. Asterisks indicate statistical difference between WT and SERT-KO or TPH1-KO mice (**) P<0.01 or (***) P<0.001 assessed by one way ANOVA and a Bonferroni post hoc test. All assays were performed on 10 slides per embryo, 6 embryos from each mouse, n=4.

Figure 6. Caspase3 staining of the placentas from SERT-KO mice

(A) E.18d placentas from WT and SERT-KO mice were first analyzed in TUNEL assays and the same field was evaluated for caspase3 staining (n=3). Only one of the three SERT-KO placentas gave a negligible levels of Caspase3 staining suggest that in the absence of SERT the placental damage occurs in caspase3-independent signaling pathway. The images in each panel are acquired at 400X magnification. The scale bars represent 10 μm. (B) Placental cells prepared from WT and SERT-KO (E.18d) (Yi et al., 2014) were analyzed in 5-12% gradient SDS. The WB analysis were performed with antibodies against Bax or Bcl2 or Caspase-3. The results of Western blot analysis are the summaries of combined data from four densitometric scans of the protein bands.