Ubp43 gene expression is required for normal Isg15 expression and fetal development

Abstract

Background: Isg15 covalently modifies murine endometrial proteins in response to early pregnancy. Isg15 can also be severed from targeted proteins by a specific protease called Ubp43 (Usp18). Mice lacking Ubp43 (null) form increased conjugated Isg15 in response to interferon. The Isg15 system has not been examined in chorioallantoic placenta (CP) or mesometrial (MM) components of implantation sites beyond 9.5 days post coitum (dpc). It was hypothesized that deletion of Ubp43 would cause disregulation of Isg15 in implantation sites, and that this would affect pregnancy rates.

Methods: Heterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc).

Results: Free and conjugated Isg15 were greater on 12.5 versus 17.5 dpc in MM. Free and conjugated Isg15 were also present in CP, but did not differ due to genotype on 12.5 dpc. However, null CP had greater free and conjugated Isg15 when compared to het/wt on 17.5 dpc. Null progeny died in utero with fetal genotype ratios (wt:het:null) of 2:5:1 on 12.5 and 2:2:1 on 17.5 dpc. Implantation sites were disrupted within the junctional zone and spongiotrophoblast, contained less vasculature based on lectin B4 staining and contained greater Isg15 mRNA and VEGF protein in Ubp43 null when compared to wt placenta.

Conclusion: It is concluded that Isg15 and its conjugates are present in implantation sites during mid to late gestation and that deletion of Ubp43 causes an increase in free and conjugated Isg15 at the feto-maternal interface. Also, under mixed genetic background, deletion of Ubp43 results in fetal death.

Figure 1

Illustration of fetal development and Ubp43 genotype on day 17.5 of pregnancy (A) and description of how implantation sites were collected (B). A litter from a day 17.5 pregnant mouse shows that all Ubp43 null offspring were dead. Genotype ratio was 2:2:1 (wt:het:null) on day 17.5. On day 12.5, 75% of Ubp43 null mice were dead with a genotype ratio of 2:5:1. There also was a significant loss of +/- fetuses between 12.5 and 17.5 suggesting that Ubp43 gene dosage may also be involved. Panel B shows a cross-sectional representation of a mouse uterus illustrating the various tissues collected for protein and RNA analysis. Anti-mesometrial (AM) represents uterine tissue surrounding the fetal compartment. Fetal chorioallantoic placenta (CP) represents fetal-derived placental tissue. Mesometrial decidua (MM) represents uterine tissue in direct contact with fetal-derived placenta.

Figure 2

Northern blot analysis for Ubp43 and Isg15 (panel A). Fetal chorioallantoic placental RNA was analyzed for Ubp43 and Isg15 using cDNA probes. Isg15 mRNA was present on 12.5 and 17.5 dpc in all genotypes. Ubp43 mRNA was present in wt and het tissues at the correct size. 18S rRNA was not different. Panel B represents a graphical interpretation of the expression of Isg15 in fetal chorioallantoic placenta. Isg15 mRNA was greater in null derived placenta in comparison to wt or het placenta. Means are LSM ± SE and represent 3 +/+, 3 +/-, and 2 -/- on 12.5 dpc, and 3 +/+, 2 +/- and 2 -/- fetal chorioallantoic placenta. * denotes P < 0.07.

Figure 3

Fetal chorioallantoic placental concentrations of Isg15. Panel A represents a western blot of placental free Isg15 (~17-kDa) and conjugated Isg15 (> 30-kDa) on 17.5 dpc. Panel B represents quantitation of free placental Isg15. Panel C represents quantitation of conjugated placental Isg15. Null fetal-derived placental tissue had greater concentrations of Isg15 when compared to pooled wt/het placenta. An interaction (P < 0.05) of genotype by dpc suggested that null fetal-derived placental tissue had greater concentrations of Isg15 conjugates on 17.5 dpc in contrast to other genotypes or null tissue on 12.5 dpc. Means are LSM ± SE. * denotes P < 0.05.

Figure 4

Mesometrial (MM) concentrations of free Isg15 (A) and conjugated Isg15 (B). Based on western blot detection of Isg15, MM had greater concentrations of Isg15 on 12.5 versus 17.5 dpc (A). MM also had greater concentrations of conjugated Isg15 on 12.5 dpc in contrast to 17.5 (B). Means are LSM ± SE. * denotes P < 0.05. Arbitrary densitometric units (ADU).

Figure 5

Anti-mesometrial (AM) tissue concentrations of free Isg15 (A) and conjugated Isg15 (B). Concentrations of Isg15 determined by western blot did not differ across genotype, but were greater on 12.5 dpc in AM tissue than on 17.5. AM concentrations of conjugated Isg15 did not differ across dpc. However, AM tissue had increased concentrations of conjugated Isg15 in tissue surrounding null fetuses in contrast to tissue overlying wt/het fetuses. Means are LSM ± SE. * denotes P < 0.05.

Figure 6

Concentrations of chorioallantoic placental VEGF¹⁶⁵ as determined by western blot (panel A). Concentrations of VEGF¹⁶⁵ were greater in null fetal placenta in comparison to pooled wt/het-derived placenta (panel B). Means are LSM ± SE. * denotes P < 0.05.

Figure 7

General morphology (hematoxolin-eosin; top panels) and labyrinthe endothelial cell staining (isolectin B4; bottom panels) in 12.5 dpc implantation cross sections. ____ = 200 μm.