Differential senescence in feto-maternal tissues during mouse pregnancy

Abstract

Background: Human studies show that fetal membranes have a limited lifespan and undergo telomere-dependent cellular senescence that is augmented by oxidative stress and mediated by p38 mitogen activated protein kinase (MAPK). Further, these studies suggest that fetal membranes are anatomically and physiologically positioned to transmit senescence signals that may initiate parturition at term.

Methods: Longitudinal evaluation of feto-maternal tissues from mouse pregnancies was undertaken to determine the molecular progression of senescence during normal pregnancy. On days 10-18 of gestation, C57BL/6 mice were euthanized. Fetal membranes, placenta, and decidua/uterus were collected. Tissues were examined for Telomere length (TL) and the presence of Phosphorylated (P) p38MAPK and p53, p21 and senescence associated β-Galactosidase (SA- β-Gal).

Findings: Linear regression modeling of observed telomere length as a function of gestational age revealed that beta (β), the slope of the linear regression was negative and significantly different from zero for each tissue (fetal membranes, β = -0.1901 ± 0.03125, p < 0.0001; placenta β = -0.09000 ± 0.03474, p = 0.0135; decidua/uterus β = -0.1317 ± 0.03264, p = 0.0003). Progressive activation p38MAPK was observed in all tissues from days 10 to day18, with the highest activation observed in fetal membranes. Activation of p53 was progressive in fetal membranes. In contrast, active p53 was constitutive in placenta and decidua/uterus throughout gestation. Detection of p21 indicated that pro-senescent change was higher in all compartments on day 18 as compared to other days. The number of SA-β-Gal positive cells increased in fetal membranes as gestation progressed. However, in placenta and uterus and decidua/uterus SA-β-Gal was seen only in days 15 and 18.

Conclusions: Telomere dependent p38 and p53 mediated senescence progressed in mouse fetal membranes as gestation advanced. Although senescence is evident, telomere dependent events were not dominant in placenta or decidua/uterus. Fetal membrane senescence may significantly contribute to mechanisms of parturition at term.

Keywords: Aging; Fetal membranes; Mouse; Parturition; Telomere; p38MAPK; p53.

Figure 1. Telomere length analysis of fetal membranes, placenta and decidua/uterus on different gestational ages

Shown are plots of telomere length (T/S Ratio) as a function of gestational age in days and the results of linear regression analysis, where Y is the value of T/S and X is the day of gestation. Negative values for the slope indicate gestational age dependent reduction in telomere length. Figure 1A: Mouse fetal membrane telomere length analysis. Number of samples used for analysis: day 8 = 1; 9 =2, day 10 = 10, day 12 = 3, day 15 = 10, day 17 = 4 and day 18 = 8. Linear regression: Y = −0.1901*X + 4.193, R²=0.5067. Slope of line is significantly different from 0, p < 0.0001. Figure 1B: Mouse placenta telomere length analysis. Number of samples used for analysis day 9 =3, day 10 = 10, day 12 = 4, day 13 = 6, day 15 = 2, day 16 = 5, day 17 = 3 and day 18 = 7. Linear regression: Y = −0.09000*X + 2.645, R²= 0.1501. Slope of line is significantly different from 0, p=0.0135. Figure 1C: Mouse deciduas/uterus telomere length analysis. Number of samples used for analysis day 8 =3, day 9 =2, day 10 = 8, day 12 = 6, day 13 = 5, day 15 = 2, day 17 = 3 and day 18 = 8. Linear regression: Y = −0.1317*X + 3.244, R²= 0.3305. Slope of line is significantly different from 0, p= 0.0003.

Figure 2. Western blot analysis to determine the presence of Phospho (active) p38 mitogen activated protein kinase (MAPK) in fetal membranes, placenta and decidua/uterus on different gestational ages

Figure 2A and D: Western blot analysis of P-p38MAPK in fetal membranes: Progressive increase in P-p38MAPK was seen in fetal membranes starting at day 10 with maximum activation seen on day 18. 2D: P-p38 /total p38 MAPK. Plot of the densitometric units observed after scanning of the relevant band as a function of gestational day. Inset, log₁₀-transformed data with R² and p value for testing hypothesis that the slope of the line generated is statistically significant from 0. Linear regression (not shown) of the non transformed data shows the slope of the line to be 0.1351, R2=0.8576, p=0.0239. Figure 2B and E: Western blot analysis of P-p38MAPK in placenta: Progressive increase in P-p38MAPK was seen in placenta starting at day 12 and with maximum activation seen on day 18. 2E: Plot of the densitometric units observed after scanning of the relevant band as a function of gestational day. Inset, log₁₀-transformed data with R² and p value for testing hypothesis that the slope of the line generated is statistically significant from 0.D Figure 2C and F: Western blot analysis of P-p38MAPK in deciduas/uterus: Minimal activation of P-p38MAPK was seen on days 10 – 17 and maximum activation was seen on day 18. 2F: Plot of the densitometric units observed after scanning of the relevant band as a function of gestational day. Inset, log₁₀-transformed data with R² and p value for testing hypothesis that the slope of the line generated is statistically significant from 0.

Figure 3. Western blot analysis to determine the presence of Phospho (active) p53 fetal membranes, placenta and decidua/uterus on different gestational ages

Figure 3A and C: Western blot analysis of P-p53in fetal membranes (n=3 sets): Progressive increase in P-p53 was seen in fetal membranes starting at day 10 with maximum activation seen on day 18. 3D: Plot of the densitometric units observed after scanning of the relevant band as a function of gestational day. Inset, log₁₀-transformed data with R² and p value for testing hypothesis that the slope of the line generated is statistically significant from 0. Figure 3B and E: Western blot analysis of P-p53 in placenta (n=3 sets): P-p53 was constitutive in placenta on all 5 gestational days (10, 12 15, 17 and 18) tested 3E: Plot of the densitometric units observed after scanning of the relevant band as a function of gestational day. Inset, log10-transformed data with R² and p value for testing hypothesis that the slope of the line generated is statistically significant from 0. Figure 3C and F: Western blot analysis of P-p53 in decidua/uterus (n=3 sets): P-p53 was constitutive in tissues on all 5 gestational days (10, 12 15, 17 and 18) tested 3F: Plot of the densitometric units observed after scanning of the relevant band as a function of gestational day. Inset, log₁₀-transformed data with R² and p value for testing hypothesis that the slope of the line generated is statistically significant from 0. Neither the transformed (not shown) nor the transformed data showed a significant linear change with time.

Figure 4. Immunolocalization of p21 in fetal membranes, placenta, and deciduas/uterus from tissues isolated on days 10 and 18 of gestation

A: Fetal membranes on day 10 and 18 (n=3 sets): p21 (total and active) was localized in fetal membranes on day 10 and 18. Day 18 membranes had higher staining intensity than day 10. B: Placenta on day 10 and 18 (n=3 sets): p21 (total and active) was localized in fetal membranes on day 10 and 18. Day 18 placenta had slightly higher staining intensity than day 10 placenta. C: Decidua/uterus on day 10 and 18 (n=3 sets): p21 (total and active) was localized in tissues on day 10 and 18. Based on staining intensities, no obvious differences in staining intensities were noted between day 10 or 18.

Figure 5. Determination of senescence in fetal membranes, placenta and decidua/uterus by staining for senescence associated β-Galactosidase (SA-β-Gal). Blue staining cells visualized under light microscope (20X images) on different gestational days are shown in 5A – 5C

Figure 5A: SA-β-Gal staining of fetal membranes on day 10, 12, 15 and 18 (n=3 sets): SA- β-Gal positive cells were absent in fetal membranes on day 10 and appeared on day 12. Majority of cells were SA-β-Gal positive by day 15 and 18. Figure 5B: SA-β-Gal staining of placenta on day 10, 12, 15 and 18 (n=3 sets): SA-β-Gal positive cells were absent in placenta on day 10 and 12 appeared on day 15. Several of cells were SA-β-Gal positive by day 15 and 18. Figure 5C: SA-β-Gal staining of decidua/uterus on day 10, 12, 15 and 18 (n=3 sets): SA-βGal positive cells were absent in placenta on day 10 and 12. Several of cells were SA-β-Gal positive by day 15 and 18.