Cyclosporine A improves adhesion and invasion of mouse preimplantation embryos via upregulating integrin β3 and matrix metalloproteinase-9

Abstract

Our previous study has demonstrated cyclosporin A (CsA) promotes the migration and invasiveness of human first-trimester trophoblast cells in vitro. Here, we further investigated the effect of CsA on the early implantation in vitro of mouse embryo. Female C57 mice were superovulated and mated, and then two-cell embryos were harvested from the oviducts and sequentially cultured in vitro in G1 and G2 media with 0, 0.1, 1.0 or 10 μM of CsA. Blastocyte formation, blastocyte cell number and apoptosis, embryo hatching were assessed in 4-6 dpc. The adhesion and stretching growth of hatched embryos in laminin coated dishes were evaluated from 5 dpc to 8 dpc, and the expressions of implantation serine proteinase 1 (ISP1), integrin (itg) β3 and matrix metalloproteinase (MMP)-9 were determined by real time PCR and immunofluorescence, respectively. We showed there was no significant difference in blastocyst formation rates, hatching rates, number of whole embryonic cells, apoptotic cells, and distribution of inner cell masses (ICMs) and trophoblasts (TB) between the CsA- and control-treated groups. Expression of ISP1 mRNA was unaffected on 5 dpc. After hatching, adhesion rate of 7 dpc significantly increased in 0.1 and 1.0 μM of CsA treatment, and embryo area of 8 dpc stretch growing on laminin were increased in 1.0 μM of CsA. The mRNA and protein expression of itgβ3 and MMP-9 on 7 dpc blastocyst were up-regulated. In conclusion, CsA in low dosage up-regulates itgβ3 and MMP-9 expression, and enhances embryonic adhesion and invasion, which is beneficial to the embryo implantation.

Keywords: Cyclosporine A; MMP; integrin; pre-implantation embryos.

Figures

Figure 1

Evaluation of total embryo cells, apoptosis cells and ICM cells by fluorescence microscopy. The blastocysts on 2 dpc were cultured in the media containing different concentrations of CsA. A: The blastocysts on 5 dpc were washed and cell number was determined by Hoechest 33258 staining. The data are from 176 blastocysts. B: The blastocysts on 5 dpc were washed and apoptosis was determined by using TUNEL and DAPI staining based on nuclear morphology and TUNEL assay. Data in B are from 98 embryos. The representative picture of apoptotic cells were showed in A with red arrows. (Bars: 50 μm).

Figure 2

The effect of CsA on the allocation and number of trophoblasts and ICM cells. The blastocysts on 5 dpc were washed and stained for OCT-4 and DAPI. The ICM cells were OCT-4-positive, the trophoblast cell numbers were the total cell number subtract ICM cell number. The data are from 160 blastocysts. TB: trophoblast, ICM: Inner cell masses. (Bars: 50 μm).

Figure 3

The effect of CsA on blastocyst hatching. The blastocysts on 4 dpc were cultured in the media containing different concentrations of CsA. The blastocysts on 6 dpc were harvested and the number of hatched blastocyst was counted as in Table 3. The total RNA was extracted to determine the mRNA level of ISP1 by using RT-PCR. The data were from 799 blastocysts.

Figure 4

The effect of CsA on the stretching growth of peri-implantation embryo on the laminin-coated dishes. The hatched embryos on 5 dpc were cultured on the laminin-precoated dishes in media containing different concentrations of CsA. On 8 dpc, the stretching area was measured at the back of the dish bottom. The representative pictures are showed in A: poor stretched embryo, B: mild stretched embryo and C: well stretched embryo. D: Embryonic C was measured by a red line for the border area. (Scale: 50 μm). E was from 124 embryos. *P

Figure 5

The regulation of CsA on itgβ3 expression of blastocysts. The hatched blastocysts on 5 dpc were cultured in media containing CsA on the laminin-coated dishes. On 7 dpc, itgβ3 mRNA level was determined by RT-PCR (A). The protein level of itgβ3 was detected by fluorescence microscopy (B). The rabbit-anti-mouse itgβ3 antibody and Alexa Fluor 488-congulated second antibody were used. Embryos were observed by confocal laser scanning microscope.

Figure 6

The regulation of CsA on MMP-9 expression of in blastocysts. The hatched blastocysts on 5 dpc were cultured in media containing CsA on the laminin-coated dishes. On 7 dpc, the adhesioned blastocysts were harvest, and MMP-9 mRNA was determined by RT-PCR (A), and MMP-9 protein level was determined by immunofluorescence (B). The rabbit-anti-mouse MMP-9 antibody and Alexa Fluor 488-congulated second antibody were used. Embryos were observed by confocal laser scanning microscope. **P