Endometrial expression of Isg15 occurs during early pregnancy in several mammals; including the cow [3
], ewe [4
], sow [5
] mouse [6
]) and primates [1
]. Interferon-stimulated gene product 15 is hypothesized to play an intrinsic role during implantation and placentation by forming an isopeptide bond with intracellular proteins, potentially altering their activity. An Isg15-specific protease, Ubp43, is also up-regulated by type I IFNs in a manner that is similar to up-regulation of Isg15 [42
]. Therefore, it was postulated that regulation of Ubp43 within the placenta could play a significant role during pregnancy and fetal development.
Previous reports by our group [6
] and others [7
] described increased Isg15 mRNA within AM decidua when compared to deciduoma of psuedopregnant mice on 7.5 dpc. We also reported that AM decidual Isg15 mRNA increased from 4.5 to 7.5 and from 7.5 to 9.5 [6
]. Expression of Isg15 mRNA was restricted to maternal tissue and was not evident in conceptus-derived tissue on 7.5 dpc. [43
]. During later stages of pregnancy, MM decidua also expressed Isg15 and its conjugates on 12.5 and 17.5 dpc regardless of fetal Ubp43 genotype. The fact that free and conjugated Isg15 were present through 17.5 dpc in mice may suggest that not only is Isgylation necessary in the uterus during early pregnancy, it may also influence placental function, implantation and fetal development throughout pregnancy.
The level of Isg15 within the anti-mesometrial uterine tissue was greater on 12.5 dpc versus 17.5 dpc, which was similar to the temporal pattern of expression inMM decidua. However, Isg15 and its conjugates were increased in anti-mesometrial tissue surrounding the null fetuses in contrast to wild-type or heterozygous fetuses. Increased anti-mesometrial uterine Isg15 and conjugates surrounding null fetuses suggests that fetal deletion of Ubp43 can influence maternal endometrial levels of conjugated Isg15, even when tissues are not directly adjacent to one another.
Isg15 and its conjugates were also present in the vascular MM tissue surrounding the fetus and this level of expression was greater when compared to anti-mesometrial tissue on 12.5 and 17.5 dpc. As expected, there was an increase in conjugated Isg15 in null fetal-derived placental tissue in contrast to tissue from wild-type and heterozygous fetuses. This was probably caused by an accumulation of Isgylated proteins because of lack of action of the de-isgylating enzyme through Ubp43 gene deletion. Increased transcription of the Isg15 gene in response to Ubp43 -/- is more difficult to explain. However, this might be an indirect compensatory mechanism because of a lack of return of conjugated Isg15 to the free pool of Isg15. Because free Isg15 is essentially consumed, perhaps the cell responds to the Ubp43 null by increasing transcription of the Isg15 gene in order to continue to provide free Isg15 that can then enter the conjugating pathway.
Previous researchers have shown that deletion of Ubp43 in knockout mice that survived to term led to accumulation of Isg15 conjugates in the ependymal lining of the brain, whereas wild-type and heterozygous litter mates had undetectable levels of conjugated Isg15 proteins [30
]. Null mice had an increased incidence of hydrocephalus and premature death suggesting that Ubp43 has an important role in postnatal brain development. Under our conditions we were never able to produce viable Ubp43 null offspring indicating that Ubp43 deletion may also play a major role in placental function and/or fetal development. Ubp43 deficient mice are hypersensitive to type I IFN and have enhanced and extended IFN signaling responses that lead towards augmentation of apoptotic responses [13
]. The fetal Ubp43 -/- mice in our study may have been subjected to increased apoptotic activity as a result of the type I IFN hypersensitivity. Disregulation of Isgylation in Ubp43 deficient fetal mice may have caused fetal death due to enhanced activation of Stat 1 tyrosine phosphorylation, DNA binding and IFN-mediated gene activation [13
]. Fetal death may also have been due to non-Isg15 responses such as a lack of direct interaction of Ubp43 with the IFN receptor [35
Messenger RNA for Isg15 was increased in Ubp43 deleted placental tissue as well. Increased expression of Isg15 in the absence of Ubp43 suggests that Ubp43 may regulate Isg15 expression directly or have an indirect effect by influencing the level of free Isg15 within tissues. It is more likely that the latter is true since previous reports have indicated that lack of Ubp43 expression enhanced sensitivity to type I IFN signaling and led to increased expression of interferon-stimulated genes, including Isg15 [13
Implantation sites appeared disrupted in Ubp43 null when compared to wt mice. The junctional zone and the decidua were less densely compact in Ubp43 null when compared to wt fetuses. This disregulation of Isg15 through deletion of Ubp43 is hypothesized to contribute to 75% fetal mortality on day 12.5 of pregnancy.
Vascular endothelial growth factor is upregulated in response to hypoxia during physiological conditions, including such events as wound healing [45
]. Vascular endothelial growth factor is expressed and localized within trophoblast cells of various species [47
] and is considered to be a very powerful mitogenic and angiogenic factor [50
]. In vitro studies on VEGF165
incubation with human trophoblast cells inhibited cell migration through an extracellular matrix chamber [52
]. The increased levels of VEGF165
in the placenta may inhibit appropriate implantation resulting in fetal mortality. Albeit, since the mortality rate was 75 and 100% on 12.5 and 17.5 dpc, respectively, the increased VEGF may be a result of fetal resorption activity or it may have increased in response to hypoxia. Der and co-workers [42
] reported that VEGF-C mRNA is up-regulated in response to IFN-α and -γ. Therefore another possible cause for increased VEGF in null fetal-derived tissue may be an indirect result of increased sensitivity to IFNs due to Ubp43 deletion.
Furthermore Ubp43 -/- mice are hypersensitive to Type I IFN, implicating a role for Ubp43 to downregulate IFN responses (Ritchie et al., 2002). These investigators later identified that Ubp43 attenuated IFN signaling by direct interaction with the region of the IFNAR2 receptor subunit that interacts with JAK1 [35
]. Subsequently interaction of Ubp43 to the IFNAR2 receptor suppressed JAK1 interactions and concomitantly decreased downstream phosphorylation cascades and other IFN-responsive signaling events. These actions of Ubp43 are independent of its effects on Isg15.
An expected Mendelian ratio of 1:2:1 wt:het:null live progeny from het interbreedings of Ubp43 knockout mice was achieved previously using C57 BL/6 × 129 background with the pGK-Neo fragment [30
]. However, during colony expansion we were never able to produce any viable null offspring at the University of Wyoming (UW) facility by breeding het (C57 BL/6 × 129 crossed to Swiss Webster to remove the pGK Neo fragment) pairs. In addition to loss of null fetuses by 12.5 dpc, we also observed loss of +/- fetuses by 17.5 dpc. We estimate that the het animals at the UW facility had less than 50% Swiss Webster genetic contribution.
Ubp43 +/- interbreedings (C57 BL/6 × 129 × Swiss Webster; n = 10 litters) were followed to term, and from these offspring 16 were +/+ and 31 were +/-, implicating that the Mendelian ratio for offspring was 1:2 for wt:het, as would be expected when considering the lack of null offspring. Therefore, we recorded fetal genotype ratios on 12.5 and 17.5 dpc in utero to investigate the loss of null offspring. On 12.5 dpc we had a ratio of 2:5:1 from a total of 30 live and dead fetuses (4 litters). And on 17.5 dpc the ratio was 2:2:1 calculated from 35 live and dead fetuses (6 litters). On 12.5 dpc only 25% of the null fetuses were viable and by 17.5 dpc all null fetuses were non-viable. There also was a noticeable loss of +/- fetuses by 17.5 dpc. The loss of heterozygous fetuses by 17.5 dpc was unexpected and might be caused by a Ubp43 gene dosage due to loss of one allele. When considering live fetuses only, genotypes did not follow normal expected Mendelian ratios on 12.5 and 17.5 dpc.
The same mice at the Scripps Research Institute produced Ubp43 -/- mice from heterozygous breedings. At the age of genotyping (3–4 wks old), a ratio of 1:4:1 from 58 pups was observed. However, work done at Scripps found that backcrossing Ubp43 het mice (C57 BL/6 × 129) with C57 BL/6 mice to F10 generation did not produce any viable Ubp43 -/- pups. Similar to reports by the UW facility, homozygous Ubp43 null mice died in utero by 12.5 dpc. It is believed that genetic drift, influenced by the contribution from C57/BL 6 lineage, altered the outcome at the UW facility. The founder animals at the UW facility could have influenced the expressionicity of the +/- interbreedings by either increased genetic contribution from C57 BL/6 or potential deletion of the 129 pGK-Neo fragment by crossing into the Swiss Webster strain.
Differences in viable offspring due to location may be a reflection of facility environmental cues. However, bacterial and viral analyses of Ubp43 mice from our facility were negative suggesting that other causes may be responsible for fetal loss. The slight changes in angiogenic and hypoxia markers provided preliminary evidence that hypoxia played a major role in fetal loss, however future experiments are planned to further study this possibility. Mouse genetic background and associated genetic modifiers plays a significant role in sensitivity to interferon [53
] and may also explain why fetuses died in the present experiment, but apparently were born and then died post-natally in other Ubp43 -/- experiments [30
]. The genetic drift due to a mixed background may actually have caused the unexpected fetal loss.