CORTICOTROPIN RELEASING HORMONE (CRH) IN NORMAL AND PREGNANT UTERUS: PHYSIOLOGICAL IMPLICATIONS

7. CRH IN THE NON-PREGNANT UTERUS

7.1 Expression and localization of endometrial CRH
The CRH mRNA and its peptide product are both present in normal human glandular endometrial cells as well as in neoplastically transformed human endometrial cells (Ishikawa cell line) (49). The size of the CRH transcript is about 1.3 kilobases (kb), similar or identical to that of its human placenta and rat hypothalamus counterparts. The epithelial cells of rat uterus also express the CRH gene, in common with epithelial cells of human endometrium (50). A CRH transcript is identified in RNA extracts of rat uteri having a size of about 1.3 kb, i.e. similar or identical to that present in human placenta. immunofluorescence for CRH in normal human glandular endometrium and in Ishikawa cells reveals a cytoplasm rich in granules positive staining for ir-CRH. The bulk of ir-CRH present in extracts from normal human endometrium and in Ishikawa cell extracts and culture media, has the same chromatographic profile as synthetic 41-amino acid CRH, suggesting that endometrial ir-CRH is authentic CRH. However, in gel filtration chromatography from normal epithelial cells as well as from Ishikawa cells, a substantial amount of ir-CRH is detected, with an apparent molecular size of approximately 10 kd. This may correspond to the precursor preproCRH. The finding that most ir-CRH in the normal human endometrial cells corresponds to the 41-amino acid sized hypothalamic CRH suggests that the cycling human endometrium possesses all the necessary enzymes for the posttranslational processing of preproCRH, thus giving rise to bioactive end product. Ishikawa cells also retain this characteristic. Additionally the bulk of ir-CRH present in extracts from rat uteri has the same chromatographic profile as synthetic 41-amino acid CRH suggesting that the rat uterine ir-CRH is also the authentic CRH. Ir-CRH is localised only in the glandular cells while the stroma is negative in ir-CRH staining. In addition, immunohistochemical data show that both epithelial and decidualized stromal cells of the early pregnant rat uterus contain ir-CRH, suggesting that epithelial endometrial cells are the main source of intrauterine CRH in the non-pregnant uterus, whereas decidualization of normal stromal cells, which do not express CRH, results in the induction of the expression of this gene in both humans and rodents (48,50).

7.2 Potential physiological implications of endometrial CRH
The role of endometrial CRH within the uterine cavity is still largely unknown. Clues regarding its physiological function derive from the study of the regulation of endometrial CRH gene promoter. In homologous transfection experiments with the 0.9 kb flanking region of hCRH gene in Ishikawa human endometrial cells, we have demonstrated that estradiol, dexamethasone and indomethacin inhibit while prostaglandin PGE₂ induces in a dose dependent fashion the activity of the hCRH promoter (table 2). These findings provide evidence for direct transcriptional regulation of endometrial CRH gene by these agents and suggest that endometrial CRH is under the negative control of estrogens and glucocorticoids and under the positive control of PGE₂ (51). In addition, Br-cAMP and forskolin (FSK), two inducers of protein kinase A (PKA), increase the activity of the CRH promoter. Furthermore, epidermal growth factor (EGF), a known activator of protein kinase C (PKC), stimulates it.

The expression of the CRH gene is induced in the implantation sites of the early pregnant rat uterus, since the CRH mRNA and its peptide product present in the implantation sites are in significantly higher concentrations compared to the inter-implantation regions (50). Human endometrial cells express the proopiomelanocortin (POMC) gene and synthesize its peptide end products (52,53) the secretion of which is inhibited by estrogens and glucocorticoids. The co-expression in endometrial epithelial cells of both CRH and POMC suggests that uterine CRH may have an autocrine/paracrine effect on locally produced POMC-derived peptides, as in the case for other peripheral tissues, such as placenta and testes (29,40). Thus, the inhibitory effects of estrogens or glucocorticoids on endometrial POMC could be the result of a preceding inhibition of endometrial CRH.

It is postulated that CRH participates in the inflammatory phenomena, possessing procytokine-like properties. Actually, CRH is detectable in rat and human inflammatory sites. Immunoneutralization of this CRH attenuates the inflammatory response (11). The reaction of uterus to the invading blastocyst has many characteristics of inflammation (54). Indeed, several immune mediators of the inflammatory response, such as Interleukin-1 (IL-1) and Interleukin-6 (IL-6), are produced in the endometrium and IL-1 and Tumor Necrosis Factor-alpha (TNF-alpha) receptors are expressed in endometrial cells (55,56). The implanting blastocyst secretes inflammatory mediators, including IL-1 and PGE₂ and has been suggested that blastocyst-deriving IL-1 plays an essential role in implantation, since in mice blockage of its action by the antagonist IL-1ra inhibits it (57). Furthermore, measurement of IL-1 levels in the periimplantation embryo culture fluid has predictive value for the successful outcome of implantation (58). It is now known that IL-1 and PGE₂ are major inducers of CRH expression in human placenta and rat hypothalamus (59,60). Moreover, the implantation sites of the early pregnant rat uterus, contain significantly higher concentrations of ir-CRH and CRH mRNA compared to the uterine tissue in-between the implantation sites, suggesting that uterine CRH may play a role in endometrial decidualization and egg implantation. Thus, the following sequence of events may take place during implantation: the blastocyst secretes PGE₂ at the site of nidation which, among other effects, induce through the uterine epithelium the local production of pharmacodynamicaly potent concentrations of CRH, facilitating the subsequent endometrial inflammatory reaction associated with egg implantation and the formation of egg nidus. At the same time, endometrial CRH may also regulate the induced inflammatory reaction by augmenting the production of local (uterine) ß-endorphin which may facilitate immunosupression at the site of nidation inhibiting the rejection of the semi-xenograft. At the same time, blastocyst-deriving estrogens could exert a local inhibitory effect on endometrial CRH establishing a local regulatory system of endometrial reaction to the implanting blastocyst.

Additionally, the repressive effects of dexamethasone and RU486 on the activity of the CRH promoter suggest that these molecules could regulate the CRH related intrauterine immune phenomena. Recently the presence of a rich network of uterine lymphokines, has been described (55). These lymphokines are mainly synthesized from endometrial stromal or epithelial cells, they affect blastocyst attachment and implantation, trophoblast outgrowth or menstruation and they are inhibited by glucocorticoids and antiglucocorticoids e.g RU486 (61,62).

Another action of endometrial CRH could be its participation in endometrial vascularization. Indeed, peripheral CRH has been found to exert vasodilatory effects (63). On the other hand, during implantation, there is a major increase in vascular permeability surrounding the implanting embryo. Endometrial capillaries adjacent to the primary decidual zone become dilated. It has been suggested that local inflammatory mediators regulate the vascular changes in the endometrium during the implantation process. Thus, endometrial CRH may be part of these sequences of events that may be under the control of factor(s) originating from the blastocyst (fig.1).

Uterine CRH, apart from its effects on the endometrium, may play a local role in the regulation of myometrial tone. Specifically, multiple isoforms of the CRH receptor have been detected in human myometrium (17). Additionally, CRH induces the release of prostaglandins PGE₂ and PGF_2a, strong inducers of myometrial tone. Furthermore, CRH potentiates the stimulatory effect of oxytocin on myometrial tone (64). CRH may also play a role in the regulation of myometrial tone through its effect on endometrial ß-endorphin since ß-endorphin has a relaxant effect on smooth muscles (fig.1) (65).