Early Canine Pregnancy – A Battle for Successful Growth and Angiogenesis

Authors


Authors’ address (for correspondence): S Schäfer-Somi, Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria. E-mail: sabine.schaefer@vetmeduni.ac.at

Contents

Currently, no early pregnancy marker has been identified in the bitch. However, significantly decreased concentrations of heat-shock protein (HSP) 70 and increased activity of matrix metalloproteinases (MMP) 2,9 were detected in serum from bitches during the pre-implantation period between days 5 and 13 after mating, that is, 2–3 days after ovulation as determined by the measurement of progesterone and vaginal cytology. Especially during the implantation period and thereafter (days 15–55), high serum concentrations of antibodies against desmin are present, which is believed to indicate or regulate decidualization. Pre-implantation embryos express mRNA for enzymes and cytokines, known to promote and regulate trophoblast growth, and some intrauterine changings like the increased activity of MMP 2,9 in maternal endometrium are dependant on the presence of embryos. Some mechanisms that protect canine embryos from attack by the maternal immune system can also be identified. The embryos express CD4, a receptor known to interact with immune cells. They, furthermore, do not express MHC I and II, which might prevent them from being recognized as foreign antigen. Pre-implantation embryos express FasL, which probably renders them able to destroy Fas-bearing cytotoxic T cells. Furthermore, the uterus during pre-implantation and implantation expresses cytokines that modulate the intrauterine milieu towards a predominance of Th2 cells. During pre-implantation and implantation, an increased uterine expression of platelet activating factor (PAF) and PAFR, vascular endothelial growth factor (VEGF) and EGFR2 as well as epithelial growth factor (EGF) is characteristic. Towards placentation, the upregulation of leukaemia inhibiting factor (LIF) and at placentation the expression of insulin-like growth factor(IGF)2 and granulocyte–macrophage colony-stimulating factor (GM-CSF) are striking. Progesterone receptor (PR) appears to be downregulated inside the uterus except at placentation sites, presumably where it is essential for maintenance of pregnancy. In addition, receptor-bound P4 regulates the activity of MMP 2,9. Apoptosis seems to be a further regulatory mechanism. Expression of Fas and FasL mRNA in uterine tissue is maximum until implantation, both factors then decreased significantly. These changings might indicate increased endometrial apoptosis and defence against maternal cytotoxic T cells, probably promoting trophoblast invasion. In human decidual stromal cells, GnRH is involved in the regulation of apoptosis, which is proposed to be similar in pregnant bitches, as GnRH-R is expressed at canine implantation sites. Our work investigating immunological changes in pregnant bitches has elucidated aspects of the complex physiology of implantation but raises important questions about the mechanisms involved.

Immunological Changings in the Maternal Blood

In (1998, Morton) discovered a small molecule with a molecular weight of only 10 kDa in serum of pregnant women. This heat-shock protein (HSP 10) proved to be an early pregnancy marker and is known to affect immunosuppression and growth promotion, thereby contributing to maintenance of human pregnancy (Noonan et al. 1979). Further, HSPs with higher molecular weights like HSP 60 and HSP 70 are believed to exert an additional protective effect during pregnancy. The HSP 60 is presumed to participate in pro- and anti-apoptotic processes inside cells. HSP 10 and HSP 60 protect cells from damage caused by high temperatures and stress and support the repair of denatured proteins inside damaged cells (Neuer et al. 2000). HSP 70 was proven to exert a strong cytoprotective effect; it inhibits among others apoptosis by blockade of the apoptosis-signal regulating kinase 1 (ASK1; Gao et al. 2010).

In a recent study (Wondra 2012), we assessed blood samples from early pregnant (n = 23) and non-pregnant bitches (n = 8) for the presence of HSP 10, HSP 60 and HSP 70 as well as IGF II. The samples were taken between the pre-implantation (days 5–13 after mating) and placentation period (until day 45 after mating). Bitches were mated 2–3 days after ovulation as determined by the measurement of serum progesterone (P4) and vaginal cytology. In serum samples from bitches that proved to be pregnant by embryo flushing at days 5–13 after mating, HSP 70 was significantly decreased in comparison with bitches that were sampled at the same time but proved to be non-pregnant (p < 0.05). This is similar to the findings in normotensive pregnant women when compared with women with pre-eclampsia (Molvarec et al. 2007). The other proteins did not differ between pregnant and non-pregnant bitches at any time investigated.

Furthermore, during the pre-implantation period (days 5–12 after mating, that is, 2–3 days after ovulation), the intrauterine activity of matrix metalloproteinases (MMP) 2,9 was so high (Beceriklisoy et al. 2007) that in serum of these animals, significantly higher MMP 2,9 activity was measured than in non-pregnant bitches (p < 0.05; Schäfer-Somi et al. 2005).

The role of autoantibodies (AABs) throughout canine pregnancy is unclear. In pregnant women, some AABs are reported to exert a physiological regulatory function, whereas others like antithyroid AAB may jeopardize pregnancy (Kaider et al. 1999). We therefore assessed the presence of AAB against contractile structure proteins of the cytoplasm in pregnant (n = 11, days 5–55 after mating, that is, 2–3 days after ovulation) and non-pregnant bitches (n = 11, mated but proved to be non-pregnant). We found that the majority of pregnant bitches had higher end point titres than the non-pregnant controls, and these differences were mainly AAB against desmin and during placentation. It is presumed that this indicates or regulates decidualization. Furthermore, we also assessed for thyroid-specific AAB; however, only two of the pregnant bitches had low titres of anti-T3 AAB (Schäfer-Somi et al. 2006).

So, do pregnancy markers exist? Considering excellent work of other groups (Concannon et al. 2001), we conclude that there are significant changes in the blood of pregnant bitches during the pre-implantation and post-implantation period.

The Canine Pre-Implantation Embryos – Probable Tasks and Defence Mechanisms

Pre-implantation canine embryos were found to express enzymes and cytokines, known to promote and regulate trophoblast growth, and some intrauterine changes in maternal endometrium are dependent on the presence of embryos (Schäfer-Somi et al. 2008). In the following paragraphs, the pre-implantation period is defined as days 5–13 after mating, that is, 1–3 days after ovulation as determined by the measurement of progesterone and vaginal cytology.

We assessed mRNA for matrix metalloproteinases (MMP) 2,9, well known to promote trophoblast invasion in other species. The embryos expressed mRNA for cytokines, including tumour necrosis factor(TNF)α and interleukin(IL)6 known to increase the gelatinolysis of maternal endometrial tissue, and increased activity of MMP 2,9, which has been detected in canine maternal uterine tissue during pre-implantation and placentation (Beceriklisoy et al. 2007; Schäfer-Somi et al. 2008). The pre-implantation embryos in addition expressed transforming growth factor(TGF)ß and leukaemia inhibiting factor(LIF); both factors are known to inhibit and probably regulate gelatinolysis, which might indicate a self-regulatory mechanism. Secretory products were not isolated and their function not proven; however, the described intrauterine changes are clearly dependent on the presence of pre-implantation embryos – during the pre-implantation stage, they are so strong that in serum of pregnant animals, significantly higher MMP2,9 activity was found than in non-pregnant bitches (Schäfer-Somi et al. 2005). Interesting was the strong expression of COX 2 in the embryos, probably indicating PGF2α synthesis. This prostaglandin probably initiates cytokine synthesis inside the embryos and the maternal endometrium.

Some of the detected genes probably contribute to the defence against attack from the maternal immune system. The embryos, for example, expressed CD4, a receptor that interacts with immune cells; in human embryos, several CD molecules including CD4 are proposed to exert a role in cell-to-cell interaction during fertilization and implantation. Investigations concerning its functions in canine embryos are ongoing. A further defence mechanism could be the lack of MHC I and II as no mRNA was detected in canine pre-implantation embryos; potentially, this could provide shelter from maternal cytotoxic cells. Only recently, we investigated whether the apoptotic Fas/FasL system might play a regulatory role during implantation of canine embryos. We detected that pre-implantation embryos express FasL but not Fas (Schäfer-Somi 2011). This is similar to the findings in human embryos; by binding via the FasL to invading Fas-bearing T cells, embryos are able to destroy these potentially cytotoxic cells. Furthermore, the pre-implantation and implantation uterus expresses cytokines that modulate the intrauterine milieu towards a predominance of Th2 cells, which is a prerequisite for survival and further development of the embryos.

The role of maternal hormones on embryo growth and secretion of cytokines, growth factors and enzymes still has to be investigated. In previous work, we did not find mRNA for progesterone (P4) receptor (PR) in pre-implantation embryos (Schäfer-Somi et al. 2008). However, other hormone receptors like estradiol and GnRH-R as well as receptors for growth factors would be of interest.

Growth and Angiogenesis inside the Uterus

In all following paragraphs, the pre-implantation period is defined as days 5–13 after mating, that is, 2–3 days after ovulation, and the implantation and post-implantation period as days 15–55, as determined by the measurement of progesterone and vaginal cytology.

The pre-implantation uterus expressed similar factors as the dioestrous uterus; however, factors exclusively expressed in the pregnant uterus were interleukin(IL)-4, CD8 and interferon(INF)ϒ (Beceriklisoy et al. 2009). These factors are known to modulate the intrauterine milieu towards a predominance of Th2 cells, which is essential for maintenance of pregnancy in other species.

We investigated uterine tissue of pregnant bitches for the expression of mRNA for certain growth and angiogenesis factors (platelet activating factor PAF, epithelial growth factors EGF, vascular endothelial growth factor VEGF) and their receptors. The pre-implantation group was detected by embryo flushing between days 10–12 after mating, which was 1–2 days after ovulation as determined by the progesterone measurement and vaginal cytology. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 occurs during the pre-implantation stage than in all other stages, and a strong upregulation of EGF during implantation was characteristic. In the embryos, mRNA from all factors except VEGF was detected. This may be explained by an increased need for these factors during pre-implantation and implantation (Schäfer-Somi et al. 2012). During the implantation and placentation stage, a significant upregulation of intrauterine LIF was striking, which was paralleled by a significant decrease in PR (Schäfer-Somi et al. 2009). Furthermore, the expression of insulin-like growth factor(IGF)2 and granulocyte–macrophage colony-stimulating factor (GM-CSF) occurs at placentation sites (Beceriklisoy et al. 2009).

Regulatory Mechanisms

Interesting are the probable mechanisms responsible for regulating growth and angiogenesis. Progesterone receptor (PR) was downregulated inside the pregnant uterus except at placentation sites, presumably as this is essential for maintenance of pregnancy. We recently assessed that the decrease in uterine expression of PR was paralleled by a significant increase in LIF towards placentation (Schäfer-Somi et al. 2009); however, a direct regulatory mechanism has to be proven. When an antiprogesterone was given during the implantation and post-implantation stage (days 25–45 after an unwanted mating; gestational age as given by the owner and surveyed sonographically), we measured a significantly increased activity of endometrial collagenases (MMP 2,9). Receptor-bound P4 was therefore proven to participate in the regulation of these enzymes. However, the activity was significantly higher during natural abortions probably indicating a more inflammatory mechanism than during antiprogesterone-induced abortions (Kanca et al. 2011). Therefore, we have recently commenced investigations into the role of apoptosis as a further regulatory mechanism during implantation, as has been described in humans. During a pilot study, expression of Fas and FasL mRNA was assessed in uterine tissue from pregnant bitches until the placentation stage. In uterine pre-implantation tissues, expression of FasL exceeded that of non-pregnant bitches and then decreased significantly. Expression of Fas did not change significantly. Fas was always more strongly expressed than FasL (Schäfer-Somi 2011). These changes might indicate increased endometrial apoptosis during the pre-implantation and implantation period and a defence mechanism against maternal cytotoxic T cells, all together probably promoting trophoblast invasion. However, this mechanism clearly loses importance with advancing decidualization during placentation, indicated by a significant decrease in both factors at that time. In human decidual stromal cells, GnRH was recognized to be involved in the regulation of apoptosis via binding to local GnRH-R. During an in vitro study, blockade of the receptor with a GnRH antagonist induced apoptosis of human decidual cells (Wu et al. 2012). The role of GnRH as a regulatory hormone during early canine pregnancy has not been investigated yet. However, only recently, we assessed that expression of mRNA for GnRH-R at canine implantation sites is upregulated (S. Schäfer-Somi, S. Sabitzer, D. Klein, unpublished observations).

In conclusion, a multitude of immunological changes can be found in the serum and uterus of early pregnant bitches, some probably initiated by the pre-implantation embryo. Significant changes in serum during the pre-implantation period include decreased concentrations of HSP70 and increased activity of MMP2,9. Some mechanisms that protect the embryos from attack by the maternal immune system could be elucidated. Expression of PR is conserved at placentation sites only, and gelatinolysis is regulated by, among others, receptor-bound P4. Until implantation, an increased expression of factors important for growth and angiogenesis was found inside the uterus. The Fas and FasL apoptosis system seems to participate in the regulation of implantation. At least, as GnRH-R was detected at implantation sites, a probable regulatory function of GnRH should be further investigated.

Conflicts of interest

None of the authors have any conflicts of interest to declare.

Author contributions

The author is responsible for the manuscript.

Ancillary