↓152 |
Mammalian pregnancy is a parabiotic union of two genetically different individuals, the fetus and the mother. At the feto-maternal interface, inflammatory processes can occur due to the invasion of microorganisms, but also due to an immune reaction against alloantigens on the fetus or trophoblast. Many studies in animals and humans indicate that some degree of systemic or uterine inflammation is necessary for both normal implantation and pregnancy, but if this inflammation becomes too excessive it can cause pregnancy complications such as abortion (reviewed in Christiansen et al., 2006). Many changes in uterine physiology that occur following the implantation of the blastocyst resemble classical inflammation at the mucosal surfaces of the female reproductive tract (Mellor and Munn, 2000). One of the first works showing the similarity between implantation of the blastocyst and inflammation was the demonstration in rats by Psychoyos (Psychoyos, 1976) that areas of the uterus containing implanting blastocysts stained blue following the intravenous injection of Pontamine Sky Blue. This demonstrated increased blood flow and permeability of the vessels supplying implantation sites, a response known to be the basis of the main early stages of inflammation (Finn, 1986). As pointed out in previous reports, heme oxygenase-1 (HO-1) plays a key role in inflammatory processes (rewieved in Ryter et al., 2006). Viewing pregnancy mainly as an inflammatory process had led us to the idea that HO-1 may play an important role in pregnancy. Previous works from our group and from others have shown that HO1 as well as HO2 are expressed in different trophoblast cells (Lyall et al., 2000; Barber et al., 2001; Zenclussen et al., 2002b and 2003b), and diminished levels of both can be found in immunological pregnancy complications in humans as well as in animals models (Zenclussen et al., 2002b and 2003b). This suggests that the heme oxygenase system is necessary at the feto-maternal interface, and that its degree of expression may be related to the success or failure of pregnancy.
Pregnancy is viewed by many from a histoincompatibility point of view. Many propose the histoincompatibility between maternal and paternal antigens that provokes a semiallogeneic fetus as one of the main causes for fetal acceptance or rejection. In this regard, it is thought that an exacerbated immunological reaction towards the fetus may provoke an immunological misbalance that would lead to fetal rejection. According to this, fetal rejection mechanisms would resemble the mechanisms involved in the rejection of an allograft. In the transplantation field, HO-1 has also been shown by others to play an important role and to have beneficial effects when up-regulated (Soares et al., 1998; Coito et al., 2002; Tullius et al., 2002).
↓153 |
Considering the important role of HO-1 in inflammatory processes as well as in transplantation, and regarding the resemblance of both entities to different stages of pregnancy, the main aim of this work was to analyze the role of HO-1 in the different processes related to pregnancy by means of functional studies employing in vivo as well as in vitro models. In the first part of the work we aimed to clarify whether a specific upregulation of HO-1 may be able to rescue mice from spontaneous abortion using a well established abortion model. The HO-1 up-regulation was performed by applying an adenoviral vector containing HO-1. We further aimed to obtain T cells over-expressing HO-1 as a therapeutic tool. In the second part of this thesis, considering that trophoblasts are important sources of HO-1, we aimed to clarify whether HO-1 is indispensable in the process of trophoblast survival and differentiation into giant cells. For this, we used a trophoblast stem cell line which differentiates into trophoblast giant cells in order to in vitro resemble the process of placentation. In the last part of this work, mice deficient in Hmox1 were used for clarifying the role of HO-1 in earlier stages of pregnancy, namely fertilization and ovarian follicle development. Besides, different mating combinations of mice partially or totally deficient in Hmox1 were used to analyze the pregnancy outcome, to try to clarify if HO-1 is necessary in the female, in the male or in both for successful pregnancy to occur. All these points are discussed below.
Role of HO-1 in immunological spontaneous abortion
Previous studies from our group and from others support the concept that, during pregnancy, different types of trophoblast cells are important sources of HOs (Ihara et al., 1998; Barber et al., 2001; Zenclussen et al., 2003; Zenclussen et al., 2005) and would participate in the catabolism of the heme protein, avoiding accumulation or recirculation of free heme, which could be extremely toxic for the mother and for the fetus. HO-1 and HO-2 down-regulation has a high potential to be harmful at the feto-maternal interface, since high amounts of free heme readily incorporate into endothelial cells, leading to oxidative injury and enhanced adhesion molecule expression (Balla et al., 1991; Wagener et al., 1997; Vachharajani et al., 2000), allowing the migration of inflammatory lymphocytes into the feto-maternal interface. Interestingly, previous works have reported down-regulated HO-1 and HO-2 levels at the feto-maternal interface from mice undergoing abortion (Zenclussen et al., 2005). Accordingly, low HO-1 and HO-2 levels were found in placental tissues from patients undergoing abortion when compared to normal pregnant patients (Zenclussen et al., 2003).
↓154 |
Although the presence and the putative protective role of HO-1 during pregnancy have been previously reported (Ihara et al., 1998; Barber et al., 2001; Zenclussen et al., 2003; Zenclussen et al., 2005c), it was still unknown if an up-regulation of HO-1 around implantation window would have beneficial effects on pregnancy outcome. During the preparation of this thesis, it was shown by another work from our group that an up-regulation of HO-1 through chemical induction by means of CoPPIX can diminish the abortion rate (Sollwedel et al., 2005). Most interestingly, the down-regulation of HO-1 through ZnPPIX during implantation boosted abortion up to 60%, suggesting that HO-1 plays an important role in avoiding fetal rejection (Sollwedel et al., 2005). However, the mechanisms by which HO-1 exerted its action were not clear in the employed onset, since many of the effects observed can be accounted to secundary effects of the chemical compound CoPPIX and in many cases were similar to the effects observed by ZnPPIX application.
In the first part of the present work, the CBA/J x DBA/2J combination, an established mouse model to investigate immunological pathways leading to spontaneous abortion was employed (Chaouat et al., 1988), to analyze the effect of HO-1 up-regulation on pregnancy outcome. Pregnant mice were injected with an adenovirus coding for HO-1 and the reporter gene GFP. As indicated by similar abortion rates to the PBS-treated abortion-prone animals, the injection of a vector containing EGFP alone did not provoke any deleterious effects in the pregnancy outcome. We could show that the application of viral vectors containing putative protective genes provides a useful and valid system to glance at their effects on pregnancy outcome in this experimental model.
Here, it could be shown for the first time that a specific increase in the systemic HO-1 expression following gene transfer of 1.105 PFU AdHO1/GFP improves pregnancy outcome in a murine model of abortion by diminishing fetal rejection rate. The avoidance of accumulated free heme might be one main reason for the success of the therapy, since an excess of free heme can cause cell damage and conduce to tissue injuries by the formation of reactive oxygen species (Vercellotti et al., 1994). Nevertheless, mice receiving 1.108 PFU AdHO-1/GFP showed no significant diminution in the abortion rate. One has to keep in mind that all the heme-degradation products are potentially toxic, depending on the generated amounts and the microenvironment (Wagener et al., 2003). Too high HO-1 expression may cause tissue injury by generating high levels of iron or resulting in bilirubinemia (Suttner and Dennery, 1999; Tyrrell, 1999). Interestingly, mice receiving high doses of the AdHO-1/GFP vector showed icteric sera and amniotic liquid, suggesting a too high HO-1 expression and subsequently hyperbilirubinemia and toxicity against the fetus.
↓155 |
The fact that no significant differences regarding HO-1 levels were found at mRNA or protein levels after AdHO-1/GFP treatment may be related to the time point chosen for analysis (9 days after adenovirus injection). It is known that adenoviruses are cleared between 5 and 20 days post-infection (Dai et al., 1995), due to the strong immune that they generate in the host. Having sacrificed the animals 9 days after the injection implies the possibility that most of the transduced cells were already cleared and therefore no major differences regarding HO-1 expression could be found. It would have been interesting to sacrifice the animals 2 or 3 days after injection of the adenovirus to analyze the pattern of HO-1 expression in different organs. However, if that would have been the case, it would have been impossible to analyze the pregnancy outcome, since at day 8 of pregnancy it is still impossible to differentiate between a healthy fetus and a resorption. However, some transduced cells were found in some of the treated animals. As it was shown by fluorescence microscopy, trophoblast cells were efficiently transduced by the adenovirus, whereas no GFP expression was found in fetal tissue. This is in accordance with previous reports pointing out that fetal membranes may act as a barrier, which may naturally prevent adenoviral particles from passing between embryonic cavities (Laurema et al. 2004). On the other side, when analyzing GFP expression by PCR, most of the GFP expression was found in the maternal liver. This is due to the fact that the liver sequesters the majority of systemically administered Ad particles via hepatic macrophages (Kupffer cells) (Glasgow et al., 2006) and is in accordance with the already described hepatic tropism of adenoviruses. However, it was surprising to find comparable levels of GFP DNA expression in the fetuses and placentas. The fact that no GFP expression was observed by fluorescence microscopy in the fetuses may suggest that adenoviral vectors may have been able to infect some cells of the fetus, but the protein was not expressed. Interestingly, a report by Everett et al. analyzing the persistence of adenoviral vectors in different strains of animals have shown that despite the persistence of the vector genome in the liver, a precipitous loss of LacZ protein expression was found. One of their explanations was that the differential elimination of transgene expression may have been due to a strain-specific differential down-regulation of the CMV enhancer/promoter element utilized to drive the LacZ gene in their Ad vector (Everett et al., 2004). One alternative speculation for the promoter shutdown was a differential rate of down-regulation of transcriptional factors induced by adenoviral infection. It is tempting to speculate that in the work presented in this thesis, a differential down-regulation of the CMV-promoter took place in the fetuses, making the GFP protein not expressed in this tissue. The GFP expression found by PCR can also arise from fetal skin, since Senoo and co-workers have also been able to find low adenoviral expression in fetal skin (Senoo et al., 2000).
Regarding the mechanisms by which the adenoviral vector containing HO-1 is pregnancy-protective in this model, one of them seems to be a Th2 polarization. In this work, augmented levels of IL-4 as well as diminished levels of IFN-γ were found systemically and locally after application of AdHO1/GFP. The “Th1/Th2/Th3 paradigm” proposes that a balance between Th1 and Th2/Th3 cytokines is critical for pregnancy development. Th2 and Th3 type cytokines such as IL-4, IL10 (Th2) and TGF-β (Th3) were proposed to favour the maintenance of mammalian pregnancy (Lin et al., 1993; Chaouat et al., 1995), whereas the excessive production of Th1 cytokines (IL-2, IFN-γ, TNF-α) would mediate the rejection of the fetus at the feto-maternal interface (Lin et al., 1993; Raghupathy, 1997). Down-regulated HO levels were related with increased Th1 levels in transplantation immunology (Woo et al., 2000) and the up-regulation of HO-1 by CoPPIX diminished Th1-cytokines levels while being successful in allowing graft acceptance (Woo et al., 2000). Here, we found diminished IL-4/IFN-γ (Th2/Th1) ratio at the fetal-maternal interface from abortion-prone mice, confirming previous results of the group (Zenclussen et al., 2005c). Treatment of mice undergoing abortion and presenting elevated Th1 levels with 1.105 PFU AdHO-1/GFP provoked an augmentation in Th2 secretion accompanied by a diminution in the Th1 production, as shown by an augmented IL4/IFN-γ#SYMBOL#ratio when compared to animals treated with PBS or the control vector. Here, enhanced Th2/Th1 ratio was found in spleen as well as in decidual lymphocytes from mice in which the therapy was successful in avoiding fetal rejection, suggesting a local and systemic effect of the therapy on the Th2/Th1 secretion. It is tempting to speculate that the up-regulation of HO1 protects the tissues by stimulating cells to shift their cytokine production to a Th2 profile. Regarding the expression of IL-10 and TNF-α, a tendency towards an augmentation of the IL-10/TNF-α ratio was found in spleen lymphocytes from mice treated with AdHO-1/GFP. In decidual lymphocytes, however, normal pregnant mice presented a diminished IL-10/TNF-α ratio when compared to the abortion prone-group. In accordance with the levels found in normal pregnant mice, decidual lymphocytes from mice receiving AdHO-1/GFP presented a diminished IL-10/TNF-α ratio when compared to the abortion-prone mice group. Nevertheless the differences were not significant for any of the cases (spleen and decidual lymphocytes). Even though the results were not as expected, the IL-10/TNF-α levels in decidual cells of the group receiving the AdHO-1/GFP behaved in the same way as the decidual cells from normal pregnant animals. Our data on the influence of HO-1 on the Th1/Th2 ratio are in agreement with data by Woo and co-workers showing that an up-regulation of HO-1 by CoPPIX diminished the IFN-γ production in a graft-versus-host disease model (Woo et al., 2000).
To analyze whether the Th2 augmentation responds to a major number of Th2 lymphocytes migrating to the fetal-maternal interface or to a polarization of lymphocytes to Th2, lymphocyte infiltration was measured in placental and decidual tissue. In placental tissue, no differences were found between the groups, suggesting that HO-1 application did not lead to an augmented lymphocyte infiltration into the fetal component of the feto-maternal interface. In decidual tissue, however, a significant augmentation of the CD3 mRNA expression was found in the group of mice receiving the AdHO-1/GFP vector. This suggests an augmented infiltration into the maternal part of the feto-maternal interface. Considering the already mentioned Th2 shift, it is tempting to speculate that these lymphocytes were Th2.
↓156 |
We next analyzed whether HO-1 application has any effect on a specific lymphocyte sub-population, namely regulatory T cells (Treg) as Treg were proposed to be important for pregnancy maintenance (Zenclussen, 2005; Saito et al., 2005). Interestingly, it has been proposed that it may be a relationship between Treg and HO-1 (Pae et al., 2003; Choi et al., 2005), whereas others do not find any relation (Zelenay et al., 2007). We have shown that mice that were rescued from abortion by the adoptive transfer of Treg presented augmented levels of HO-1 at the feto-maternal interface (Zenclussen et al., 2006). This suggests that Treg are able to induce HO-1 expression at the feto-maternal interface. On the other hand, we could also show that HO-1 up-regulation by means of CoPPIX augmented the levels of the Treg marker Neuropilin-1 (Sollwedel et al., 2005), suggesting a bi-directional relationship between HO-1 and Tregs in pregnancy. In this work, no relation could be found between HO-1 and Treg as the application of an adenoviral vector containing HO-1 did not modify the levels of the markers analyzed (Foxp3 and TGFβ).
Since HO-1 is known to have anti-apoptotic properties (Soares et al., 1998), the possible influence of HO-1 up regulation on apoptosis-related events at the fetal-maternal interface was investigated. AdHO-1 treatment could slightly down-regulate the placental caspase-3 activity when compared to abortion-prone mice treated with PBS or with a control vector, suggesting diminished apoptotic activity at the feto-maternal interface. Moreover, the TUNEL staining allowed us to confirm a significant diminution in the number of apoptotic cells in placenta from mice receiving low doses of AdHO-1/GFP when compared to PBS- or EGFP-treated abortion-prone mice, confirming therefore the anti-apoptotic effect of the HO-1-therapy at the feto-maternal interface. Since the caspase-3 activity was not statistically diminished after AdHO-1 therapy, while the number of apoptotic cells was, it is tempting to speculate that apoptosis in this system is not caspase-3 dependent. Other works have already pointed out that the anti-apoptotic effects of HO-1 may act through a caspase-3 independent pathway (Lang et al., 2005). The anti-apoptotic effect observed in our model is also in accordance with anti-apoptotic effects of HO-1 observed in transplantation (Coito et al., 2002; Braudeau et al., 2004). Interestingly, a work of Redaelli and co-workers in transplantation showed like we did that HO-1 had anti-apoptotic effects in a model of liver graft, and this was associated with significantly lower number of apoptotic cells (detected by TUNEL) and slightly diminished levels of caspase-3 (Redaelli et al., 2002). The work of Coito and coworkers also suggest that HO-1 may also act by up-regulating anti-apoptotic molecules like Bcl-2 and Bag-1 (Coito et al., 2002). Accordingly, we found augmented mRNA levels of the anti-apoptotic molecule Bag-1 which reinforces an anti-apoptotic/cytoprotection hypothesis. This data strongly suggest that Bag-1 over expression following HO-1 treatment is directly implied in the avoidance of abortion. As Bag-1 mRNA is not significantly downregulated in the abortion-prone combination compared to the normal one, the finding that AdHO-1 induces Bag-1 expression may be specific to gene over-expression. In fact, Bag1 was already described to be augmented after upregulation of HO-1 by gene therapy in tolerant grafts (Coito et al., 2002). Since the first demonstration of Bag-1 as an anti-apoptotic molecule (Takayama et al., 1995), Bag-1 over-expression has been shown to inhibit apoptosis induced by a wide range of inducers in various cell types (reviewed in Townsed et al., 2003). The mechanisms accounting for the effects of Bag-1 on apoptosis induced by several stimuli are not fully characterized yet. One potential target is the Bcl-2 protein, a Bag-1 interaction partner (Townsed et al., 2003). Thus, it is possible that the broad effects of Bag-1 derive from its targeting of this key Bcl-2-regulated mitochondrial apoptosis checkpoint. Further, Bag-1 interacts with the HSC70 and HSP70 heat shock proteins and promotes cell survival by coordinating the function of these chaperones with the proteasome to facilitate protein degradation (Townsed et al., 2004). Interestingly, Townsend et al. have demonstrated that Bag-1 has a novel cytoprotective function, e.g. protecting cardiac myocytes from apoptosis induced by simulated ischemia/reperfusion, mediated via association with HSC70/HSP70, which is critical upon cytoplasmic localization but independent of the BAG-1 ubiquitin-like domain (Townsed et al., 2004). Accordingly to our data, we speculate that Bag1 is mediating fetal tissue protection. This data is in accordance with other data from our group, where the protective effect of HO-1 through CoPPIX application was also related to augmented levels of Bag-1 at the feto-maternal interface (Sollwedel et al., 2005).
Heme oxygenase-1 has also been described to be involved in angiogenesis. In this regard, Dulak and co-workers have shown that these angiogenic effects are related to the modulation of vascular endothelial growth factor (VEGF) synthesis by HO-1 activity (Dulak et al., 2002). In pregnancy, the endometrium, decidua and placenta are sources rich if angiogenic growth factors (Zygmunt et al., 2003), and VEGF production by the trophoblasts during pregnancy has paracrine effects on maternal endothelial cells resulting in vascular remodelling and/or permeability within the microcirculation. Besides, trophoblast derived VEGF acts by increasing the efficiency of vascular-mediated exchange between the mother and the fetus. In this work, an up-regulation of HO-1 led to a slight augmentation in the number of VEGF positive cells in placental tissue, suggesting that HO-1 may have had angiogenic effects in this model. Knowing the relationship between VEGF and HO-1 expression, it is tempting to speculate that HO-1 up-regulation during implantation may have augmented VEGF expression within the feto-maternal interface, favouring a proper vasculogenesis and angiogenesis that would lead to a proper implantation of the fetuses and consequently to an avoidance of fetal rejection.
↓157 |
The protective effect of up-regulated HO-1 levels in our treatment could be also related to increased carbon monoxide (CO) production (Sato et al., 2001). Although CO was considered for years a very dangerous gas (Johnson et al., 1999), it is now clear that CO can reduce inflammatory responses by inhibiting pro-inflammatory genes and by augmenting antiinflammatory cytokine production (Otterbein and Choi, 2000; Sarady et al., 2002; Song et al., 2003). Interestingly, inhibition of HO-1 by SnPPIX did not conduce to graft rejection if the mice were simultaneously exposed to CO. Besides, both, gene transfer of HO-1 or CO application inhibited chronic graft rejection (Chauveau et al., 2002). These data indicate a crucial role for CO in transplant acceptance and suggest that the HO-1-related beneficial effects are related to CO production. Data obtained in our group by Ivonne Wollenberg (Diploma thesis), showed that exposure to 250 p.p.m. of CO during implantation (days 4 and 5 of pregnancy) could completely avoid abortion in the CBA/J x DBA/2J model, indicating a beneficial effect of CO during pregnancy. However, CO inhalation during the whole experiment (days 0-13 of pregnancy) led to detrimental effects on the fetuses and the mother, and even to unsuccesful pregnancies (no viable litters). This indicates that CO in specific amounts and in specific time points of pregnancy can be protective, and that may also suggest that the effects seen in this approach may be due to the effects of CO. If biliverdin/bilirubin or free iron were involved in the protective effect of our work is unclear. However, it has been shown in other models that biliverdin can imitate the effects of HO-1 by inducing tolerance to cardiac allografts (Yamashita et al., 2004), suggesting that some of the effects seen by the upregulation of HO-1 can be explained by augmented biliverdin levels. The third by-product of the HO-1 enzymatic reaction, free iron, is potentially toxic but can give antioxidant protection when coupled with proteins that promote their sequestration or export of the liberated iron. In this regard, ferritin has been also proposed as a cytoprotective molecule and as a contributory mechanism underlying HO-dependent protection (reviewed in Ryter, 2006). It was not found in the literature if an up-regulation of biliverdin or ferritin can be protective in models of pregnancy complications, and it is unknown if in our model the upregulation of HO-1 acted through its compounds.
Summarizing, the use of an adenoviral vector containing HO-1 allowed us to glance at the mechanisms by which HO-1 would have a protective role during pregnancy. Taken together, our findings point out a protective effect of systemic HO-1 up regulation on pregnancy outcome. A complex picture of regulatory interactions between the HO system and apoptotic and cytokine networks is suggested. These data could further open new opportunities finding novel therapeutic directions in immunological pregnancy failure.
The use of an adenoviral vector over-expressing HO-1 was used in order to elucidate the important role of HO-1 in avoiding fetal rejection, and not intended to be proposed as a therapy. A more therapeutical approach was intended to do using T cells over-expressing HO1. The use of retroviral vectors, reported by others as being a successful method for the transduction of T cells (Hammer et al., 2002; Hori et al., 2003), was in our hands not successful enough. One of the reasons why this approach could not be established may be that cells from CBA/J females were poorly stimulated with paternal antigens, since retroviral vectors are known to transduce only dividing cells (Somia and Verma, 2000). The attempt of stimulating cells with anti-CD3 and anti-CD28 did improve the proliferation of these cells, but the transduction efficiency was not significantly improved. The other possibility for the lack of success of this approach may be the vector (pLXSN), since works from our group (Paul Wafula, ongoing PhD thesis) using other retroviral vector (MIGR-1) are currently being successful in transducing CBA/J cells stimulated with anti-CD3 and anti-CD28 (but not stimulated with paternal antigens). Since the maximal transduction efficiency obtained in this approach was less than 8%, it was not feasible to obtain enough amounts of cells to be transferred in sufficient number of animals. For this reason, the approach using protein transfection was performed.
↓158 |
The protein transfection has been used to transfect mainly adherent cells (Morris et al., 2001) and has even been used in vivo (Aoshiba et al., 2003). However, no report has been found to date about the transfection of murine primary T cells. In the context of this PhD work, a successful transfection has been achieved in CD4+ T cells. The practical convenience of this method relies on the time that is saved when compared to retroviral transduction, and the percentage of efficiency obtained. CD4+ cells have been efficiently transfected using the positive control (80% of transfection), and have been also efficiently transfected with HO-1. When used in vitro, however, these cells were not showing any particular effect on the proliferation of cells from a CBA/J female. The effects observed were always comparable to those observed with cells treated with the transfection agent alone (TMOCK). In vivo (data not shown) these cells were also not effective, reason why the experiment was not fully completed. One possible explanation for this may be that the protein transfected into the cells is not active. The HO-1 protein used for this approach was commercially available, and the company was not able to inform us whether the protein is active or not. The other possibility may be that CD4+ T cells are not the right target for the transfection of HO-1. Alternative targets for the HO-1 transfection could be dendritic cells, since HO-1 is known to act on dendritic cells by keeping them in an immature state (Chauveau et al., 2005).
Role of HO-1 in trophoblast survival and differentiation
After implantation, the trophectoderm surrounding the blastocyst goes on to differentiate into a variety of trophoblast cell subtypes with different functions. Trophoblast stem cells emerge from the polar trophectoderm that overlies the inner cell mass of the blastocyst, and they proliferate in response to close contact to the inner cell mass (Cross, 2005). From the different types of trophoblasts in the mouse, the giant cells are the first to arise, already at the blastocyst stage. These cells exit the mitotic cell cycle and stop dividing, enlarge, and they undergo endoreduplication to become polyploid (MacCauley et al., 1998). The function of trophoblast giant cells is to first mediate the process of implantation and invasion of the conceptus into the uterus. Later they produce several hormones and cytokines to promote local and systemic physiological adaptations in the mother including the regulation of maternal blood flow to the implantation site, and production of progesterone from the ovary (reviewed in Cross, 2005). Due to the vital importance of the giant cells in the formation of the placenta and in the establishment of a successful pregnancy, the main aim of this part of the study was to analyze in vitro whether HO-1 is necessary for trophoblast stem cell differentiation into giant cells.
↓159 |
The Rcho-1 cell line is usually used as an in vitro model for studying trophoblast cell differentiation (Faria and Soares, 1991). These cells can be manipulated to proliferate or differentiate into trophoblast giant cells by altering culture conditions. Rcho-1 trophoblast stem cell differentiation recapitulates in vivo trophoblast giant cell development, and is a valuable in vitro tool for studying the process of trophoblast cell differentiation (Sahgal et al., 2005). So far, HO-1 was reported to be expressed in different trophoblast cells of human and murine placentas (Ihara et al., 1998; Barber et al., 2001; Zenclussen et al., 2003b; and 2005b), and its expression in the murine placenta is normally the highest in giant cells (personal observation). However, no report was found in the literature showing that HO-1 is necessary during the differentiation process of trophoblasts. In the experiment showed in this thesis, the main aim was to analyze whether a down-regulation of HO-1 renders these cells unable to proliferate into giant cells. This would help us understand if HO-1 is really necessary for the differentiation of trophoblast stem cells, and therefore to the proper formation of a placenta when translated into an in vivo situation.
Interestingly, the down-regulation of HO-1 expression by ZnPPIX led already to a significantly diminished viability of precursor stem cells, suggesting that HO-1 is necessary even for the survival of trophoblast stem cells. The application of CoPPIX did not lead to significant changes in the viability of Rcho-1 cells, suggesting that the effect seen with ZnPPIX may not be due to the toxic effect of the porphyrin per se. When analyzing the ability of these cells to differentiate into giant cells, the application of ZnPPIX led them unable to transform into giant cells, whereas cells treated with CoPPIX did not show any problem in the differentiation process. These results suggest that HO-1 may play an important role in the differentiation process of trophoblast cells. No results have been found in the literature on this regard, making this a novel discovery in the role of HO-1 in formation of placenta.
The fact that mice lacking Hmox1 do not get pregnant also suggests that without HO-1 no placentation is able to occur.
↓160 |
Role of partial or total loss of HO-1 on the outcome of pregnancy
As already described by others (Poss and Tonegawa, 1997, Yet et al., 1999) and as mentioned before, no progeny can be achieved when mating Hmox1 -/- mice. Interestingly, the mating of heterozygous females and males leads to only 3-10% of knockout progeny, instead of the 25% expected Mendelian rate. Both facts suggest that Hmox1 plays a very important role in pregnancy, but so far no explanation for this problem in the maintenance of the colony was found in the literature. For this reason, we aimed to analyze the origin of this problem by analyzing if the low progeny of the heterozygous combination is due to rejection of the fetuses or to other reasons. Besides, different combinations of wild type, heterozygous and knockout animals were performed in order to assess whether the problem resides in the females, in the males, or in both.
When females partially deficient in Hmox-1 (Hmox1 +/- ) were mated with Hmox1 -/- males, only 4 out of 11 females that showed insemination plug were indeed pregnant, and showed an abortion rate of 38.7 %. The fact that only 4 out of 11 females were pregnant suggests that Hmox1 -/- males may present fertilization problems. Similarly, when Hmox1 +/+ females were mated with Hmox1 -/- males, 3 out of 5 females mated were pregnant, suggesting here that Hmox1 -/- males may have fertilization problems. However, Hmox1 +/+ males also showed fertilization problems when fertilizing Hmox1 +/- or Hmox1 +/+ females, since not all females showing insemination plug were pregnant. This suggests that these fertilization problems may not be exclusively associated with the lack of Hmox1 in the male.
↓161 |
Analyzing the situation from the female site, an interesting result when using the heterozygous females was observed, namely that the abortion rate is higher when less HO-1 is present in the male, thus leading to less HO-1 in the F1 tissue (placenta). This was as follows: when heterozygous females were mated with Hmox1 +/+ males, they presented an abortion rate of 18.4 %, with Hmox1 +/- males the abortion rate was of 22.4 %, whereas with Hmox1 -/- males the abortion rate increased to 38.7%. This clearly shows a trend towards an augmentation on the abortion rate with a diminution of HO-1 in the system. When Hmox1 +/+ females were mated with all three types of males, they showed very similar abortion rates in all three types of combinations. All these data together shows that, when the female present normal HO-1 levels, the male HO-1 contribution is not relevant for the success of pregnancy. However, when the female is already partially lacking HO-1, the less HO-1 the males are contributing the more abortion can be observed. This data strongly suggests that HO-1 is very important in the maternal site for a successful pregnancy, whereas its absence in the paternal site may not be as important as like in the maternal site.
No references in the literature were found regarding the necessity of HO-1 in the oocytes or sperm for reproductive processes. For HO-1-related molecules, however, interesting information has been published. It has been pointed out that a low concentration of nitric oxide may have some physiological role in fertilization through the enhancement of sperm´s capacity to bind to the zona pellucida of the oocyte (Kazuo et al., 1998). On the other hand, mice deficient in Cox-2, a molecule related to HO-1, are known to have multiple reproduction failures including problems in ovulation, fertilization, implantation and decidualization (Lim et al., 1997). Interestingly, mice deficient in Cox-2 showed a blunted and delayed induction of HO-1 in a model of endotoxemia (Ejima and Perrella, 2004), suggesting altered HO-1 expression in these animals. It is not mentioned in the literature whether HO-1 is involved in the process of fertilization, but considering the relationship of HO-1 with the nitric oxide system and with Cox-2, it is tempting to speculate that the lack of HO-1 leads to impaired or altered expression of NO and/or Cox-2, which in turn leads to infertility. We did not address this point in the present work and this may lead to interesting results and merits further studies. One can also speculate that animals deficient in Cox-2 present fertilization problems an impaired fertility due to altered expression of HO-1, as we clearly show that HO-1 is important in several steps of reproduction. This work clearly opens the possibility of analyzing this process more accurately in future projects.
When performing an allogeneic combination using Hmox1 +/+ C57/BL6 females mated either with Hmox1 -/- or Hmox1 +/+ BALB/c males, most of the females presenting insemination plug were pregnant. When the male was deficient in Hmox1, an 8.6% of abortion was observed, against 0% of abortion obtained with the Hmox1 +/+ males. These percentages of abortion are very similar, and suggest again that the lack of HO-1 in the male does not significantly affect pregnancy outcome when HO-1 is present in the female.
↓162 |
We next genotyped the offsprings (healthy fetuses as well as unviable implantations) in order to analyze whether animals deficient in HO-1 were implanted and rejected, which may explain the low percentage of knockouts in the Hmox1 +/- x Hmox1 +/- matings. The analysis of the genotype of the fetuses and resorptions showed that, surprisingly, the mating of Hmox1 +/- females with Hmox1 -/- males lead only to heterozygous offspring. This result strongly suggests that Hmox1 -/- blastocysts were unable to implant or were rejected in a very early time point and not visible at the time of preparation, since fetuses as well as resorptions were heterozygous.
The mating of Hmox1 +/- females with Hmox1 +/- males led to the typical percentage of knockouts obtained from these matings (personal communication from Prof. Soares and personal observation from the maintenance of the colony), and this percentage is lower than the expected Mendelian rate of 25%. Interestingly, the resorptions were again exclusively heterozygous suggesting that the lower percentage of Hmox1 -/- fetuses obtained from the Hmox1 +/ x Hmox1 +/- mating is not due to immunological rejection of these fetuses but to the lack of implantation of the Hmox1 -/- blastocysts. This can be due to the fact that, as reported by Chen and co-workers in 2005, blastocysts normally up-regulate HO-1 immediately after hatching (Chen et al., 2005), and in Hmox1 deficient blastocysts this can not occur, but may be compensated by the presence of HO-1 in the maternal uterus. In the case of heterozygous uteruses, the lack of Hmox1 in the blastocysts may not be always sufficiently compensated by the HO-1 expression from the uterus, and that may explain why so few Hmox1 -/- blastocysts are efficiently implanted.
Role of HO-1 in early stages of pregnancy (ovulation, fertilization, implantation)
↓163 |
So far, HO-1 in pregnancy was thought to be important for implantation. The data obtained with the IVF now brings new light into the role of HO-1 in pregnancy as we could confirm that Hmox-1 -/- females are not able to produce as many oocytes as wild type females, indicating that HO-1 is necessary even at very early stages of reproduction. The difference in the oocyte production may be due to differences in the reaction of the Hmox1 -/- females to hormonal treatment, since ovaries from Hmox1 +/+ and Hmox1 -/- females showed the same total number of follicles per ovary. The changes that take place on the ovary at the site of follicular rupture are pathophysiological in nature. This local damage induces hemorrhage in the vicinity of the lesion on the surface of the ovary (reviewed in Espey et al. 2004). Considering the importance of the up-regulation of HO-1 in inflammatory processes, especially in events related to hemorrhage, it is tempting to speculate that the lack of HO-1 may impair the ovulation process in a way that these animals do not manage to induce the follicular rupture. Without HO-1, the ability to counteract the inflammatory process related to it is missing. In other words, due to the inflammatory nature of ovulation, mature follicles lacking HO-1 may found it more difficult to induce the rupture of the tissue due the inflammatory nature of the process, and may be then less secreted than follicles expressing HO-1. Interestingly, NO, an inflammatory mediator normally associated with HO1, has been described to be involved in ovulation. Studies using eNOS -/- mice have shown that they have reduced fertility due to impaired ovulatory efficiency, abnormalities in meiotic maturation, increased oocyte apoptosis and altered estrous cyclicity compared to their wild type littermates (Jablonka-Shariff and Olson, 1998; Drazen et al., 1999; Hefler and Gregg, 2002). Studies using iNOS -/- mice show that iNOS deficiency does not alter ovulatory capacity, but it may play a role in fertilization (Yang et al., 2005). Furthermore, the cyclooxygenase (COX) pathway, which is responsible for prostaglandin (PG) synthesis, is also important in inflammatory responses, as the HO and NO pathways are. Additionally, they have constitutive and inducible isoforms (McGarry et al., 2005), and they are closely related (Ejima and Perrella, 2004). The two enzymes responsible for the synthesis of PGs are COX1 and COX2, being the first constitutive and the second inducible form (Venturini et al., 1998). Although Cox1 -/- mice have normal fertility, Cox2 -/- females are infertile and exhibit abnormalities in ovulation due to PG deficiency (Matsumoto et al., 2001). Regarding the HO system, interestingly, Hmox2 -/- mice do not present reproductive problems (Poss et al., 1995), whereas Hmox1 -/- do present problems, as shown in the results presented in this thesis. These results open the possibility of further analyze the problem in a more accurate way, and these studies are being currently done in our group.
We then analyzed the ovaries from the females used as oocyte donors in order to analyze whether the lack of HO-1 is associated with morphological changes. Ovaries from both types of females were morphologically similar, but a significant diminution in the number of corpus luteum of Hmox1 -/- ovaries when compared to Hmox1 +/+ ovaries was obtained. This is in accordance to the lower number of oocytes obtained from Hmox1 -/- females. As the number of total follicles per ovary was similar when comparing both groups, the difference in the number of corpus luteum was compensated by a slight augmentation in the number of primordial and secondary follicles in Hmox1 -/- ovaries when compared to Hmox1 +/+ ovaries. It would be very interesting to know if Hmox1 -/- females can even ovulate without hormonal stimulation. This will be performed in our institute as soon as sufficient amount of knockout females are available. It will be interesting also to know if these females present normal hormonal levels, and if they present normal estrous cyclity.
The oocytes obtained from Hmox1 -/- females showed additionally a very poor fertilization rate (19.78%), suggesting that Hmox 1 may also be necessary (but not indispensable) for the fertilization. This may also suggest that Hmox-1 -/- oocytes are not as viable as wild type oocytes, and may be therefore the reason why the mating of mice heterozygous for HO-1 may yield so few Hmox-1 -/- progeny (around 310% instead of the 25% expected Mendelian rate), and why Hmox-1 -/- animals do not give viable progeny when paired with Hmox-1 -/- males. In order to finally confirm that the reason as to why Hmox1 -/- animals are not fertile and Hmox +/- do not yield the expected Mendelian rate, current experiments with Hmox1 deficient animals are being carried out. However, taking into account the results from the different combinations, I would speculate that Hmox1 is more important in the oocyte for a successful fertilization.
↓164 |
Another possible explanation for the lack of progeny of Hmox1 -/- could be an impaired implantation. In order to test this hypothesis, the embryos in two-cell stage obtained by in v i tro fertilization were transferred into recipient females. To test whether Hmox1 is necessary in the mother, in the fetus or in both for implantation to occur, Hmox1 -/- oocytes were transferred into a Hmox1 +/+ or into a Hmox1 -/- recipient female. Additionally, Hmox1 +/+ oocytes were transferred into a Hmox1 /- or into a Hmox1 +/+ female. The fact that Hmox1 -/- oocytes were able to implant in a Hmox1 +/+ uterus confirms that Hmox1 -/- oocytes do not present impaired hatching and can therefore implant in a uterus in which HO-1 is expressed. A work from Chen et al. shows that, after hatching, oocytes up-regulate HO-1 (Chen et al., 2005). In the context of the experiments done for this thesis, the inability to upregulate HO-1 in Hmox1 deficient blastocysts may be compensated by the HO1 expression in the maternal uterus. On the other hand, the fact that Hmox1 +/+ or Hmox1 -/- oocytes were unable to implant in Hmox1 -/- uteruses suggests that HO-1 is indispensable in the uterus for implantation to occur. Further, the sole expression of HO-1 by the blastocyst may not be sufficient to compensate the inflammatory milieu that the implantation of the embryo induces in the maternal uterus and cannot therefore contribute to the tolerance state needed after implantation for pregnancy to be maintained.
Having observed that HO-1 expression in the uterus is important or even indispensable for implantation to occur and that a lack of HO-1 leads to both, impaired implantation and impaired pregnancy, we next wondered whether HO-1 acts via T-cell dependent pathways. The results obtained when transferring Hmox1 +/+ or Hmox1 -/- oocytes into SCID mice deficient in Hmox1 are still very preliminary to make strong conclusions. By using SCID mice lacking T and B cells we next aimed to have a closer look on the participation of T cells in HO-1-dependent pregnancy success. If HO-1 mediated rejection operates via T cells, the lack of T cells would then rescue from abortion. However, it was very interesting to observe that a higher percentage (around 13-15%) of Hmox1 -/- progeny is obtained if the mice are in a SCID background (personal communication from Prof. Soares). In the experiments done for this work, Hmox1 +/+ oocytes were able to implant in a SCID female lacking HO-1. However, the SCID female used as a control did not implant, making the results difficult for interpretation. Nevertheless, the fact that more Hmox1 -/- progeny is achieved in SCID background suggests that, in immune deficient animals, HO-1 may be not as necessary as in immune competent animals. This can be thought taking into account the immune cells present at the feto-maternal interface.
Summary and conclusion
↓165 |
This data opens vast opportunities of further analyzing the role of this molecule in a more closely way in the different stages of pregnancy, and also helps to elucidate unknown mechanisms related to a process so important as the beginning of a new life.
© Die inhaltliche Zusammenstellung und Aufmachung dieser Publikation sowie die elektronische Verarbeitung sind urheberrechtlich geschützt. Jede Verwertung, die nicht ausdrücklich vom Urheberrechtsgesetz zugelassen ist, bedarf der vorherigen Zustimmung. Das gilt insbesondere für die Vervielfältigung, die Bearbeitung und Einspeicherung und Verarbeitung in elektronische Systeme. | ||
DiML DTD Version 4.0 | Zertifizierter Dokumentenserver der Humboldt-Universität zu Berlin | HTML generated: 29.09.2014 |