*2.4. Western Blot Analysis of the Implantation-Related Genes Reveals Alterations Between Fractalkine Treated Mono- and Co-Cultured JEG-3 Cells*

After the mRNA expression analysis, it was unravelled that FKN influenced the expression of genes that are implicated in the implantation process. We also examined if these alterations appeared at protein level, suggesting that FKN regulates implantation by controlling proliferation, differentiation and invasion related proteins of trophoblast cells.

In the monocultures, PR and CX3CR1 protein levels showed delays between the elevation of mRNA and protein expressions at F10 treatment, suggesting that the lower FKN concentration has a slower effect on the JEG-3 cells. The effect of F20 lasted longer as the mRNA levels decreased at 48 h but the protein levels were still higher compared to the control cells (Figure 3A,B and Figure 4A).

Activin receptor, MMP2, MMP9 and SRC-1 protein levels correlated with the changes of the mRNA expression levels (Figure 3C–F and Figure 4A), but their expressions varied by time and by FKN concentration. Activin receptor was upregulated by both FKN concentrations at 24 h, and then, at 48 h, the protein level was significantly reduced (Figure 4A,E).

MMP2 protein level raised at 6 h and 24 h and then, at 48 h, the protein level significantly decreased (Figure 4A,F). In case of MMP9, significant elevation was observed at only 24 h (Figure 4A,G). The same alteration was found in case of SRC-1 protein at 24 h, and then F10 decreased SRC-1 level at 48 h (Figure 4A,H). These results show that in the monocultured JEG-3 cells, F20 was more effective in upregulating the protein levels.

In the case of the co-cultured JEG-3 cells, the correlation between mRNA expression and protein level is not as straightforward as for monoculture suggesting that the interaction between the two cell types influences the effect of FKN and the translational rate of the examined proteins.

FKN treatments increased the protein level of PR at 6 h and 48 h, while at 24 h, F5 still increased the expression. On the other hand F10 and F20 had an opposite effect, significantly decreasing the level of PR, showing that the effect of FKN was concentration dependent (Figure 4B,I). SRC-1, a progesterone co-receptor, was elevated by F20 at 6 h, while F10 and F20 treatments reduced SRC-1 protein level at 24 h, and elevated it at 48 h (Figure 4B,N). These changes were parallel to the alterations found at PR. In the case of CX3CR1, the strongest effect was mediated by F10 which significantly reduced CX3CR1 expression at 24 h and 48 h (Figure 4B,J). The activin receptor was downregulated by F10 and F20 at 6 h and 24 h, but it was significantly elevated at 48 h by all three FKN concentrations (Figure 4B,K).

F5 reduced MMP2 protein level at 6 h and increased it at 48 h (Figure 4B,L). F10 and F20 decreased MMP2 level at 24 h and had a reverse effect at 48 h (Figure 4B,L). Interestingly, MMP9 protein level was elevated during the whole experiment by each FKN concentration (Figure 4B,M). This suggests that MMP9 had a specific role in the invasion and may work together with MMP2 at 48 h. decreased MMP2 level at 24 h and had a reverse effect at 48 h (Figure 4B,L). Interestingly, MMP9 protein level was elevated during the whole experiment by each FKN concentration (Figure 4B,M). This suggests that MMP9 had a specific role in the invasion and may work together with MMP2 at

F5 reduced MMP2 protein level at 6 h and increased it at 48 h (Figure 4B,L). F10 and F20

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 7 of 19

concentration. Activin receptor was upregulated by both FKN concentrations at 24 h, and then, at 48

MMP2 protein level raised at 6 h and 24 h and then, at 48 h, the protein level significantly decreased (Figure 4A,F). In case of MMP9, significant elevation was observed at only 24 h (Figure 4A,G). The same alteration was found in case of SRC-1 protein at 24 h, and then F10 decreased SRC-1 level at 48 h (Figure 4A,H). These results show that in the monocultured JEG-3 cells, F20 was more

In the case of the co-cultured JEG-3 cells, the correlation between mRNA expression and protein level is not as straightforward as for monoculture suggesting that the interaction between the two

FKN treatments increased the protein level of PR at 6 h and 48 h, while at 24 h, F5 still increased the expression. On the other hand F10 and F20 had an opposite effect, significantly decreasing the level of PR, showing that the effect of FKN was concentration dependent (Figure 4B,I). SRC-1, a progesterone co-receptor, was elevated by F20 at 6 h, while F10 and F20 treatments reduced SRC-1 protein level at 24 h, and elevated it at 48 h (Figure 4B,N). These changes were parallel to the alterations found at PR. In the case of CX3CR1, the strongest effect was mediated by F10 which significantly reduced CX3CR1 expression at 24 h and 48 h (Figure 4B,J). The activin receptor was downregulated by F10 and F20 at 6 h and 24 h, but it was significantly elevated at 48 h by all three

cell types influences the effect of FKN and the translational rate of the examined proteins.

h, the protein level was significantly reduced (Figure 4A,E).

effective in upregulating the protein levels.

Based on the results, it is proven that F10 and F20 treatments triggered parallel alterations at protein level. The only exception is CX3CR1, which was downregulated by F10 but was upregulated by F20 treatments (Figure 4B,J). The latter result suggests that the regulation of CX3CR1 by FKN is concentration dependent. The fluctuations of PR, CX3CR1 and activin receptor expression may provide protection against the overactivation of signalling pathways regulating the same signal transduction proteins, MAPKs, SMAD transcription factors, the same genes (e.g., MMPs) or cellular process (e.g., invasion). These changes in protein levels can contribute to maintaining the proper implantation mechanism of trophoblast cells. 48 h. Based on the results, it is proven that F10 and F20 treatments triggered parallel alterations at protein level. The only exception is CX3CR1, which was downregulated by F10 but was upregulated by F20 treatments (Figure 4B,J). The latter result suggests that the regulation of CX3CR1 by FKN is concentration dependent. The fluctuations of PR, CX3CR1 and activin receptor expression may provide protection against the overactivation of signalling pathways regulating the same signal transduction proteins, MAPKs, SMAD transcription factors, the same genes (e.g., MMPs) or cellular process (e.g., invasion). These changes in protein levels can contribute to maintaining the proper implantation mechanism of trophoblast cells.

**Figure 4.** Western blot analyses of implantation-related proteins in fractalkine treated mono- (**A**) and co-cultured (**B**) JEG-3 cells. JEG-3 cells were collected and pelleted after fractalkine treatments. Following lysis, protein contents were measured. The same amount of protein (15 µg) from each sample was separated by SDS-PAGE using 10% or 12% polyacrylamide gel, transferred by electroblotting to nitrocellulose membranes. The membranes were probed with anti-progesterone receptor, anti-fractalkine receptor, anti-activin receptor, anti-MMP2, anti-MMP9 and anti-SRC-1 polyclonal rabbit antibodies according to the manufacturer's protocols. The experiments were repeated three times. (**C**–**H**) Optical density analyses of the examined proteins in JEG-3 monocultures. (**I**–**N**) Optical density analyses of the examined proteins in co-cultured JEG-3 cells. The analyses were carried out using ImageJ software, the optical densities of the examined proteins were expressed as percentage of target protein/β-actin abundance. The bars represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n* = 3). The \* mark *p* < 0.05 compared to the controls. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20.

*2.5. The Presence of HEC-1A Cells Contributes to the Action of Fractalkine on JEG-3 Cells by Changing the Expressions of Activin, Follistatin and BMP2*

Based on our results, it seems that the presence of HEC-1A cells in the co-cultures contributed to the action of fractalkine on JEG-3 cells. Therefore, we examined whether HEC-1A-expressed implantation-related genes showed alterations suggesting a cooperation in the implantation process at fractalkine treatment. We determined the mRNA expression levels of activin and follistatin regulating activin receptor, and BMP2 regulating cell growth and differentiation. Interestingly, follistatin and activin levels changed inversely in the co-culture: increased follistatin mRNA level and decreased activin level were found at 6 h and 48 h, and decreased follistatin level and increased activin mRNA level were detected at 24 h (Figure 5A,B). The expression of BMP2 that is also regulated by follistatin binding protein was significantly higher during the whole experiment compared to the control (Figure 5C). These results suggest that the interaction between JEG-3 and HEC-1A cells influences the expressional changes occurring in JEG-3 cells in fractalkine treatment. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 9 of 19

**Figure 5.** Expression analysis of activin, follistatin and BMP2 in co-cultured HEC-1A cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. β-actin was used as housekeeping gene for the normalization. The relative expression of untreated controls was regarded as 1. The mRNA expressions of the treated cells were compared to their appropriate controls (6 h, 24 h or 48 h). HEC-1A cells were collected by trypsinization directly from the coverslips. The cells were lysed and their protein contents were measured. The same amount of protein (15 µg) from each sample was separated by SDS-PAGE using 10% or 12% polyacrylamide gel, transferred by electroblotting to nitrocellulose membranes. The membranes were incubated with anti-follistatin and anti-BMP2 polyclonal rabbit antibodies according to the manufacturer's protocols. The experiments were repeated three times. **(A**–**C)** mRNA expression levels of activin, follistatin and BMP2. (**D**) Western blot analyses of follistatin and BMP2. **(E**–**F)** Optical density analyses of the examined proteins in HEC-1A cells. The analyses were carried out using ImageJ software; the optical densities of the examined proteins were expressed as percentage of target protein/β-actin abundance. The columns represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n*  = 3). The \* mark *p* < 0.05 compared to the controls. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20. **Figure 5.** Expression analysis of activin, follistatin and BMP2 in co-cultured HEC-1A cells. Real-time PCR was performed with SYBR green protocol using gene specific primers. β-actin was used as house-keeping gene for the normalization. The relative expression of untreated controls was regarded as 1. The mRNA expressions of the treated cells were compared to their appropriate controls (6 h, 24 h or 48 h). HEC-1A cells were collected by trypsinization directly from the coverslips. The cells were lysed and their protein contents were measured. The same amount of protein (15 µg) from each sample was separated by SDS-PAGE using 10% or 12% polyacrylamide gel, transferred by electroblotting to nitrocellulose membranes. The membranes were incubated with anti-follistatin and anti-BMP2 polyclonal rabbit antibodies according to the manufacturer's protocols. The experiments were repeated three times. (**A**–**C**) mRNA expression levels of activin, follistatin and BMP2. (**D**) Western blot analyses of follistatin and BMP2. (**E**,**F**) Optical density analyses of the examined proteins in HEC-1A cells. The analyses were carried out using ImageJ software; the optical densities of the examined proteins were expressed as percentage of target protein/β-actin abundance. The columns represent mean values and error bars represent standard deviation (SD) for three independent experiments (*n* = 3). The \* mark *p* < 0.05 compared to the controls. Abbreviations of fractalkine treatments: 5 ng/mL-F5; 10 ng/mL-F10; 20 ng/mL-F20.

**3. Discussion**  The early stages of pregnancy comprise the attachment of embryo to the uterine epithelium, invasion of embryo into the uterine stroma and the decidualization of the stroma. Multiple molecules forming a complex network regulate implantation. The interplay between endometrial tissue and At protein level, we found that follistatin level decreased at 24 h that may influence the regulation of activin receptor activity in JEG-3 cells. BMP2 protein level was elevated at 6 h and 48 h. The latter shows that there is a delay between mRNA expression and protein synthesis (Figure 5C,D,F). The decreasing level of BMP2 protein at 24 h is supposed to occur due to the secretion of protein into the culture

conceptus is critical to induce uterine receptivity to implantation [52]. The pre- and early implantation periods require alterations in the gene expression both in the epithelium and in the trophoblast cells. medium (Figure 5D,F). Our results suggest that HEC-1A cells contribute to the implantation by altering the expression of implantation-related genes and proteins, which could contribute to the action of fractalkine in trophoblast cells.
