p38 MAPK regulates cavitation and tight junction function in the mouse blastocyst

Abstract

Blastocyst formation is essential for implantation and maintenance of pregnancy and is dependent on the expression and coordinated function of a series of proteins involved in establishing and maintaining the trans-trophectoderm ion gradient that enables blastocyst expansion. These consist of Na/K-ATPase, adherens junctions, tight junctions (TJ) and aquaporins (AQP). While their role in supporting blastocyst formation is established, the intracellular signaling pathways that coordinate their function is unclear. The p38 MAPK pathway plays a role in regulating these proteins in other cell types and is required for embryo development at the 8-16 cell stage, but its role has not been investigated in the blastocyst.

Hypothesis: p38 MAPK regulates blastocyst formation by regulating blastocyst formation gene expression and function.

Methods: Embryos were cultured from the early blastocyst stage for 12 h or 24 h in the presence of a potent and specific p38 MAPK inhibitor, SB 220025. Blastocyst expansion, hatching, gene family expression and localization, TJ function and apoptosis levels were analyzed.

Results: Inhibition of the p38 MAPK pathway reduced blastocyst expansion and hatching, increased tight junction permeability, affected TJP1 localization, reduced Aqp3 expression, and induced a significant increase in apoptosis.

Conclusion: The p38 MAPK pathway coordinates the overall events that regulate blastocyst formation.

Figure 1. p38 MAPK Regulates Blastocyst Expansion and Hatching.

p38 MAPK inhibition decreased embryo expansion and hatching. (A) After 12 h and 24 h embryos cultured in KSOMaa + SB 220025 were significantly less expanded than controls. Three replicates were performed and 30–35 embryos were measured in each group. SEM p≤0.05; letters (a–c) represent groups that are significantly different from one another. (B–C) Embryo recovery after 12 h (B) and 24 h (C) treatment of SB 220025. Embryos were washed in fresh KSOMaa after 12 h or 24 h and allowed to recover until 24 h, 28 h, and 36 h post collection. Embryos treated with SB 220025 for 12 h remained significantly less expanded compared to controls, however, they resumed cavitation and increased their expansion following treatment. Embryos treated with SB 220025 for 24 h remained significantly less expanded compared to controls, and were not able to re-expand. Three replicates were performed and 30–35 embryos were measured in each group. SEM p≤0.05; letters (a–c) represent groups that are significantly different from one another. (C) Inhibition of the p38 MAPK pathway blocked embryo hatching. Embryos cultured in SB 220025 for 12 h or 24 h displayed significantly reduced zona hatching compared to controls. Embryos were cultured in KSOMaa, SB 202474 or SB 220025 for 12 h and then washed in fresh KSOMaa and cultured until 24 h post collection and assessed for hatching. Embryos that had begun to hatch were counted as positive (SEM; p≤0.05; n = 4).

Figure 2. TE formation and apico-basal cell polarity in TE appear normal in p38 MAPK inhibited embryos.

CDX2 protein localization (A) following 24 h of treatment with KSOMaa, SB 202474, or SB 220025. White arrows indicate ICM; Yellow arrows indicate TE. Blue  =  nucleus; Red  =  F-actin; Green  =  CDX2; n = 10–15 embryos in each group; scale bar  = 10 microns. CDH1 protein localization (B) following 24 h of treatment with KSOMaa, SB 202474, or SB 220025. CDH1 protein localization pattern did not vary after 24 h. Blue  =  nucleus; Red  =  F-actin; Green  =  CDH1; n = 10–15 embryos in each group; scale bar  = 10 microns.

Figure 3. p38 MAPK inhibition compromises TJ integrity.

To test TJ integrity, embryos were treated in KSOMaa, SB 202474, or SB 220025 then exposed to 40 kDa FITC-dextran. After 12 h of treatment (A), a significant difference was observed in the number of embryos displaying 40 kDa FITC-dextran fluorescence within the blastocyst cavity between SB 220025 and control groups (p≤0.05) n = 3. After 24 h of treatment (B), a significant difference was observed in the number of embryos displaying 40 kDa FITC-dextran fluorescence within the blastocyst cavity between SB 220025 and control groups (p≤0.05) n = 3. Representative images of embryos following 12 h or 24 h treatment with (C) 2 mM EGTA (positive control), (D) KSOMaa, (E) SB 202474 or (F) SB 220025 using 40 kDa FITC-dextran. n = 3; ± SEM; scale bars  = 10 microns.

Figure 4. Inhibition of p38 MAPK affects TJP1 localization after 12 h and 24h.

TJP1 protein localization following treatment with KSOMaa, SB 202474, or SB 220025. Representative images of TJP1 after 12 h (A) and 24 h (B) exposure. SB 220025 treatment affected TJP1 localization after 12 h (A) and 24 h (B). In KSOMaa and SB 202474 images, white arrows indicate normal localization of TJP1 along adjacent cell borders. In p38 MAPK inhibited images, white arrows indicate punctate distribution of TJP1 compared to control embryos. In control embryos, yellow staining indicates co-localization of TJP1 and F-actin (yellow arrowhead). In treated embryos, there is reduced yellow staining, and increased red staining indicating a loss of co-localization of TJP1 and F-actin (yellow arrowhead). Blue  =  nucleus; Red  =  F-actin; Green  =  TJP1; n = 10–15 embryos in each group; scale bar  = 10 microns.

Figure 5. p38 MAPK inhibition does not affect mRNA expression of Tjp1, Atp1b1, or Occludin.

q-PCR was utilized to determine mRNA expression levels of Tjp1, Atp1b1, and Occludin. There was no significant difference in mRNA expression levels between treatment groups. (n = 4, mean ± SEM).

Figure 6. Inhibition of p38 MAPK affects Aqp3 mRNA expression and protein localization.

(A) Relative Aqp 3 and 9 mRNA levels following treatment. qRT–PCR was used to determine the relative mRNA levels of Aqp 3 after 12 h or 24 h in SB 220025. Aqp 3 and Aqp 9 mRNA was not significantly affected after 12 h; n = 3, mean ± SEM, P≤0.05. Aqp 9 mRNA was not significantly affected after 24 h; however, Aqp 3 mRNA was significantly decreased after 24 h. Each group is expressed relative to KSOMaa control. n = 3, mean ± SEM, P≤0.05. (B) AQP3 immunofluorescence after 24 h of p38 MAPK inhibition. AQP3 protein localization following treatment with KSOMaa, SB 202474, or SB 220025. Embryos treated with SB 220025 for 24 h displayed a clear and consistent reduction in AQP3 immunofluorescence compared to controls. Green  = AQP3; n = 10–15 embryos in each group; scale bar  = 10 microns.

Figure 7. Prolonged inhibition of p38 MAPK increased apoptosis.

(A) Apoptosis following KSOMaa, SB 202474 or SB 220025 treatment for 12 h and 24 h. TUNEL was used to assess the level of apoptosis in blastocysts after having been cultured in KSOMaa, SB 202474 or SB 220025 media for 12 h or 24 h. The number of individual cells that were TUNEL positive was counted in each blastocyst and is represented as the average number of cells that are TUNEL positive per blastocyst. Embryos cultured in KSOMaa, SB 202474 or SB 220025 for 12 h displayed the same average number of TUNEL positive cells. Embryos cultured in SB 220025 for 24 h displayed a significant increase in the average number of TUNEL positive cells compared with controls; mean ± SEM, P≤0.05. (B) Representative images of embryos from TUNEL assay. Negative: No fluorescence control; Positive: Embryos pretreated with DNAse to ensure TUNEL staining was successful. n = 4 replicates; 15–20 embryos in each group.