In the present study, we aimed to clarify the influence of corticotropin-releasing hormone (CRH) exposure on the syncytialisation rate of isolated primary villous trophoblastic cells. Moreover, we investigated whether CRH induces alterations in gene expression of specific endocrine placental regulators in vitro. Our results showed a significantly higher leptin expression in trophoblastic cells, which concomitantly resulted in a significant induction of leptin protein secretion into the supernatant after 48 h of CRH stimulation, compared to unstimulated control cells. Moreover, 11β-HSD2 expression was dose-dependently induced by 2.0 μg/ml CRH after 48 h. Formation of a functional syncytiotrophoblast occurred after 24 h in the CRH-stimulated and the control group to the same degree, as determined by increasing β-hCG secretion without a concomitant increase of cell lysis reflected by constant LDH levels in the supernatant throughout the experiment . Syncytin-1 expression, a key regulator gene of trophoblast syncytialisation [33, 35, 37], was dose-dependently induced after 48 h of CRH stimulation only.
The finding that leptin expression increases with the progression of trophoblast syncytialisation in both groups is in line with results from Ashworth et al. , who showed that placental leptin expression is an exclusive feature of the syncytiotrophoblast (SCT), with a reduced leptin expression in undifferentiated cytotrophoblasts. Likewise, CRH was described as a syncytical peptide . CRH-treated and vehicle stimulated trophoblasts showed an equal increase of ß-hCG concentration in the culture supernatant, while cell lysis was low and not different (as determined by LDH). This finding is indicative that the rate of trophoblast syncytialisation gradually increased over time in both groups . The induction of leptin expression after 24 h of culture of CRH- and vehicle treated trophoblastic cells can therefore be attributed to trophoblastic differentiation into functional syncytium. The significant difference in leptin gene expression in CRH-treated and vehicle treated control groups at 48 h and the induction of leptin protein secretion into the cell culture supernatant, however, do not seem to be solely an indirect effect of syncytialisation, as ß-hCG levels of CRH-treated trophoblastic cells were not significantly different from the ß-hCG levels of vehicle treated controls. Hence the significant increase in leptin expression and secretion of stimulated cells at 48 h rather seems to be a direct effect of CRH, possibly via activation of CRH-R1α, which was readily detectable in trophoblasts [10, 41] and towards which CRH shows a ten times higher affinity as compared to CRH-R2 , whose expression was significantly lower. The fact that we were able to detect CRH-R1α and CRH-R2 in vitro does not rule out the possibility of CRH signalling via its other known placental isoforms that were described in vivo [10, 41]. Interestingly Karteris et al. found significant levels of receptor hybridization foremost in syncytiotrophoblast and to a lesser extend in scattered cytotrophoblasts, which is supported by almost exclusive binding of 125I]CRH to purified syncytiotrophoblast . This could suggest CRH might rather excert its functions after spontaneous syncytialisation of cytotrophoblasts in vitro than on single cytotrophoblasts.
To address the functional relevance of our findings studies with CRH antagonists, such as CRH antagonists antalarmin (CRH-R1) and antisauvagine (CRH-R2) would be needed.
We found that CRH (2 μg/ml) dose-dependently induced CRH -R1α and –R2 gene expression at 48 h (Figure 1, p < 0.029). The stimulatory effect of CRH on leptin expression was abolished at 72 h. CRH-R internalization could be a possible mechanism to explain this phenomenon, as previously described by others [44, 45]. CRH-R internalization could also account for the absence of an expressional effect on the studied target genes following repeated CRH stimulation in our pilot study. Such an induction of CRH-R expression levels following exposure to higher doses of CRH might optimize CRH signal transduction. Interestingly, higher concentrations of CRH (2.0 μg/ml) also significantly induced 11ß-HSD2 expression at 48 h (Figure 1, p < 0.029), while 1.0 μg/ml showed no such effect. Hence higher levels of auto-/paracrine CRH might hypothetically prepare the syncytium to cope with a CRH-triggered increase of maternal cortisol more efficiently.
The above findings regarding leptin are in line with findings from our previous experiments showing a close relationship of trophoblast leptin expression and secretion rate in vitro following dexamethasone stimulation . Interestingly, dexamethasone stimulation (10 μM) produced a more pronounced leptin secretion (~120 pg/ml at 72 h) when compared to CRH (1.0 and 2.0 μg/ml) stimulation (~56-61 pg/ml at 72 h, respectively). However, dexamethasone clearly induced ß-hCG secretion. Hence in contrast to CRH the effect of dexamethasone on leptin secretion seems to be partly attributable to an increased rate of trophoblast differentiation and maturation, as also seen by Audette et al. . They were also able to demonstrate a trend to Syn1 induction in placental explants following dexamethasone treatment.
The fact, that we found an increase in Syn1 expression following CRH stimulation without a concomitant increase in ß-hCG might point to a differential regulation of the two genes. A common pathway for both Syn1 and ß-hCG stimulation is the forskolin triggered induction of cAMP : An activation of adenyl cyclase (AC) raises intracellular cAMP levels and leads to PKA activation via interactions with AKAPs and downstream phosphorylation of p38MAPK and ERK1/2. Accordingly, CRH was found to induce cAMP in human endometrium via CRHR1 triggered protein-kinase A (PKA)  and we recently found that Syn1 is induced via the cAMP pathway in endometrial carcinoma . However, as ß-hCG was not induced, CRH might either use alternative signalling pathways, such as signalling via PKC [45, 50], or the detected Syn1 expression might come from a source other than the cytotrophoblast. However our cytotrophoblast isolation is ~90% pure. Thus, we cannot exclude possible minor fractions of extravillous trophoblasts (EVT) in our cell culture. EVT express CRHR1  and show Syn1 expression [20, 52]. Therefore, we cannot completely rule out that the Syn1 induction measured is derived from EVT. But due to their extremely small fraction in the isolation, a six-fold increase in Syn1 expression by EVT following CRH-stimulation seems rather unlikely. Another explanation could be, that CRH fosters maintenance fusion events instead of functional fusion processes, that would be reflected in ß-hCG secretion. Importantly, Syn1 is related to trophoblast processes beyond its fusogenic nature. Possible functions of the syncytin proteins are suppression of the maternal immune response against the developing fetus  and induction of placental immunity against vertical transmission of retroviral infections . We observed a stimulatory effect of CRH (2.0 μg/ml after 48 h) on the expression of 11ß-HSD2 in primary cultured cytotrophoblasts. Like leptin, the induction of 11β-HSD2 by CRH subsided at 72 h, possibly due to CRH-receptor internalization, as discussed above. In a previous study using the same in vitro setup, we were able to show that dexamethasone (10 μM) similarly stimulates both leptin and 11β-HSD2 expression in primary trophoblastic cells . 11ß–HSD2 gene expression in human placental trophoblasts grown in primary culture has been shown to maintain the same pattern as in vivo  and dexamethasone stimulation regularly results in an increase in 11ß–HSD2 protein expression in trophoblasts . Upon the finding that CRH induces 11β-HSD2 expression one cannot draw conclusions about the activity of placental glucocorticoid metabolism. Interestingly, Friedberg et al.  found a CRH-induced reduction of 11ß-HSD1 activity in human adipocytes in vitro. In isolated cytotrophoblasts Sharma et al.  were unable to induce 11ß-HSD2 activity using CRH concentrations of 1-100 ng/ml, however, they were able to identify the CRH downstream signalling protein p38MAPK  as an essential regulator for 11ß-HSD2 activity. The fact, that we observed expressional changes of 11β-HSD2 following CRH treatment at much higher CRH dosages (2.0 μg/ml) could however imply a possibility of a CRH-driven glucocorticoid induced feed-forward mechanism on 11ß-HSD2 activity. Although such a mechanism has not been described for the placenta yet, the subsequent reduction of cortisol availability might be an intriguing regulatory function shielding the fetus of placental CRH-induced maternal glucocorticoids.
Our study focused on the auto- and paracrine effects of CRH on leptin production in isolated trophoblasts, as the placenta co-expresses both leptin (ObR-L)  and CRH receptors . We were able to show a significant increase in leptin expression in syncytialised trophoblastic cells following CRH treatment. While the exact auto- and paracrine mechanisms and the functional role of the interaction of CRH and leptin at the level of the syncytiotrophoblast remain to be determined, an increase of endocrine CRH and leptin expression might translate into endocrine signals affecting both fetus and mother, besides their local influence on the trophoblast.
In this respect it is noteworthy, that the major fraction of placental leptin and CRH is secreted into the maternal circulation [60, 61]. Nevertheless, the syncytiotrophoblast is also involved in the maintenance of fetal leptin and CRH serum levels [62, 63]. Besides its role in fetal organ maturation via cortisol induction, there is in fact evidence, that placental CRH drives parturition via induction of adrenal DHEA-S on the fetal side followed by an increase in placental estrogen secretion .
In IUGR, a condition characterised by increased fetal serum CRH levels , we found unchanged leptin levels in fetal umbilical cord blood , despite an elevated placental leptin mRNA and protein expression [28, 64]. Hence, it seems likely that CRH and CRH-induced leptin (as suggested by our results) might interact on the maternal side.
White et al.  showed that leptin has lipolytic effects in rat placental tissue in vitro. CRH antagonises lipolysis via down-regulation of 11ß-HSD1 in adipose tissue . Hypothetically leptin and CRH might act together in regulating the maintenance of fetal nutrient supply at the placental level.