DeMayo 2012). Since we have previously observed a decrease in progesterone receptor protein expression in the Mtrr+/+, Mtrr+/gt, or Mtrrgt/gt uteri at estrus, we wanted to determine
whether a similar finding occurred at GD6.5 with the potential to affect decidualisation during pregnancy. We assessed Pgr mRNA expression via RT-PCR in whole implantation sites at GD6.5 derived from mating C57Bl/6 males with C57Bl/6, Mtrr+/+, Mtrr+/gt, Mtrrgt/gt,
F1 Mtrr+/+, or F1 Mtrr+/gt females. There was a decrease in Pgr mRNA expression in
implantation sites of Mtrr+/gt and Mtrrgt/gt females compared to implantation sites of C57Bl/6
females (p=0.0002; Figure 4.4A). No change in Pgr mRNA expression was observed in Mtrr+/+ females compared to the C57Bl/6 (Figure 4.4A). Furthermore, a decrease in Pgr
mRNA expression was observed in implantation sites of F1 Mtrr+/gt females compared to
C57Bl/6 females (p=0.003; Figure 4.4B). No change in Pgr mRNA expression was observed in F1 Mtrr+/+ females (Figure 4.4B). Altogether, these data suggest that intrinsic but not
paternal abnormal folate metabolism affects Pgr mRNA expression at GD6.5. PGR protein expression in implantation sites at GD6.5 should be assessed in the future.
To further assess molecular differences in the decidua of implantation sites at GD6.5 caused by the maternal Mtrr genotype, we assessed the expression of genes important for
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decidualisation (e.g., Hoxa10, Hand2, Nrf2f, Bmp2, Sfrp5 and Cdh1) via qPCR analysis. HOXA10 is a regulator of progesterone responsiveness of uterine stromal cells, and therefore important during implantation and decidualisation (Lim, Ma, et al. 1999). Hand2 is also a gene that is regulated by progesterone during decidualisation (Okada et al. 2014). However, Hoxa10 and Hand2 mRNA expression did not change in implantation sites at GD6.5 derived from Mtrr+/+, Mtrr+/gt, or Mtrrgt/gt females (Figure 4.4C, D). These data are inconsistent with
Pgr mRNA expression levels in Mtrr+/gt and Mtrrgt/gt implantation sites (Figure 4.4A),
although PGR protein levels should be assessed to address this inconsistency. Interestingly, Hoxa10 mRNA expression was decreased in the implantation sites of F1 Mtrr+/gt mothers
compared to those from either C57Bl/6 or F1 Mtrr+/+ females (p=0.0005; Figure 4.4I), a
similar trend to Pgr mRNA expression in similar implantation sites (Figure 4.4B). Hand2 expression was only slightly decreased in F1 Mtrr+/gt implantation sites but the result was not
statistically significant (Figure 4.4J). The inconsistency of Hoxa10 and Hand2 expression in implantation sites at GD6.5 from Mtrr+/gt females derived from either two Mtrr+/gt parents or
only an Mtrr+/gt father needs to be resolved.
NR2F2 is a transcription factor that is highly expressed in uterine stroma cells and is important for decidualisation (Kurihara et al. 2007). It is known to activate Bmp2
transcription (Wetendorf & DeMayo 2012). We observed a decrease in Nr2f2 and Bmp2 mRNA expression specifically in implantation sites from Mtrr+/gt and Mtrrgt/gt females
compared to C57Bl/6 controls (p=0.0012; Figure 4.4E-F). Bmp2 mRNA was lower in implantation sites from Mtrr+/+ females than controls (Figure 4.4F) even though Nr2f2
expression did not change (Figure 4.4E). These data suggest that only an intrinsic Mtrrgt
allele is sufficient to affect Nr2f2 expression and that another transcription factor beyond NR2F2 might regulate Bmp2 during decidualisation. In support of this finding, normal levels of Nr2f2 mRNA were found in implantation sites from F1 Mtrr+/+ and F1 Mtrr+/gt females
(Figure 4.4K) despite decreased Bmp2 mRNA levels (Figure 4.4L). SFRP5 is an inhibitor of WNT signalling (Bovolenta et al. 2008; Stuckenholz et al. 2013) and is involved in stromal cell differentiation and decidualisation (Li et al. 2007). Expression of Sfrp5 mRNA was within the normal range in all of the Mtrr implantation sites assessed compared to the controls (Figure 4.4G, M). Lastly, CDH1, also known as E-cadherin, is a protein important for decidualisation (Reardon et al. 2012). While Cdh1 mRNA expression was unaltered in all Mtrr uteri during estrus (Chapter 3), we observed a decrease in Cdh1 mRNA expression at GD6.5 only in Mtrr+/gt and Mtrrgt/gt uteri compared to the C57Bl/6 controls (p=0.03; Figure
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Figure 4.4 Relative mRNA expression of decidualisation markers in GD6.5 implantation sites.
Graphs depicting relative mRNA expression of genes involved in decidualisation in
implantation sites from mothers derived from either (A, C-H) Mtrr+/gt intercrosses or (B, I-N)
from an Mtrr+/gt father. Polr2a mRNA was used as a housekeeping gene. n=5-7 implantation
sites from each adult female genotype. Data are presented as fold change compared to C57Bl/6 controls (normalised to 1). Statistical test: One-way ANOVA, a p<0.05 when compared to b and c; b p<0.05 when compared to a and c; c p<0.05 when compared to a and b.
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4.4H). A downward trend in Cdh1 mRNA expression was also observed in Mtrr+/+ uteri at
GD6.5, though this did not reach statistical significance compared to C57Bl/6 uteri (Figure 4.4N). Cdh1 mRNA expression was similar in C57Bl/6, F1 Mtrr+/+ and F1 Mtrr+/gt females at
GD6.5, suggesting a combined effect of a maternal and an intrinsic Mtrrgt allele to disrupt its
expression.
In summary, even though histological sections did not show gross alterations in
decidualisation at GD6.5, gene expression of decidualisation markers were disrupted by one or more intrinsic Mtrrgt allele. While Pgr mRNA expression is altered, this effect on
decidualisation may in part be independent of progesterone signalling, since we observe normal Pgr mRNA expression, but abnormal expression of Bmp2 mRNA in Mtrr+/+ and F1
Mtrr+/+ females. For instance, BMP2 is a downstream mediator of Egfr signalling and
ablation of Egfr in mice results in defects in decidualisation (Large et al. 2014), and this is independent of Pgr signalling. The misexpression of some, but not all, decidual markers suggests that specific pathways important for proper decidualisation might be affected (e.g., BMP2 signalling).