J. Endocrinol. Invest. 13: 353-371, 1990

REVIEW ARTICLE

New aspe.cts of placental endocrinology F. Petraglia*, L. Calza**, G.C. Garuti*, L. Giardino**, B.M. Oe Ramundo*, S. Angioni* *Department of Obstetrics and Gynecology, University of Modena, School of Medicine, 41100 Modena, and ** Department of Physiology, University of Cagliari, School of Medicine, 09100 Cagliari, Italy

INTRODUCTION

and growth factors show a preferential localization within the cytotrophobasts (Fig. 1). However, at present it is simplistic to correlate the transcription of a hormonal gene to a single stage of trophoblast differentiation. The evidences of a placental production of new hormones arised aseries of researches to define their biological roles. A local action of placental neuro hormones, neuropeptides, growth factors or cytokines has been suggested. In particular, because several hypophyseotropic releasing or inhibiting factors have been found in human placenta and they modulate other placental hormones release, the intraplacental mechanism of control has been compared to the organization of the hypothalamus-pituitary-target organ axes. Moreover, growth factors and cytokines also influence the endocrine activity of placental cells. Therefore, a complex local mechanism of control regulating placental hormone release has been hypotesized. This last hypothesis is based upon aseries of results obtained in vitra. Using isolated cotyledon perfusion, placental explants, transformed placental trophoblast maintained in long-term culture, malignant choriocarcinoma cells, or primary placental monolayer culture studies on putative secretagogues or suppressants have been conducted. Other than a local effect, placental neuro hormones, neuropeptides, growth factors or cytokines mayaiso enter the maternal and fetal circulation and exert a role on other target functions in these organisms. The major efflux seems to be toward the maternal bloodstream. The generally elevated plasma levels decrease abruptly following parturition. Changes of plasma neurohormone levels have been described during labor or in the maternal or fetal pathological conditions. Once in the circulatory system, the placental hormones may act on their usual target, i.e pituitary gland for hypophyseotropic neurohormones, or on the other specific receptors.

The classical knowledge indicated that: 1) human placenta produces aseries of protein and steroid hormones whose structure is similar to that of some pituitary, gonadal and I or adrenal hormones; 2) an autonomous mechanism of control regulates placental endocrine function (1-4). These assumptions are going to be modified and largely amplified: the numer of hormones produced by human placenta increases year by year, and new functional aspects have been proposed. Recent findings showed that human placenta produces peptide hormones typically synthesized by neurons. Indeed, in addition to pituitary- or gonadallike hormones, human placenta synthesizes hypothalamic-like neurohormones and neuropeptides. Moreover, growth factors and cytokines are also produced in human placenta and influence the endocrine function. The placental tissue with endocrine competence is the trophoblast, while cytokines are produced by the immune cells present in the placental villi. MorphQlogical differences permits to distinct various cell populations in the trophoblast: cytotrophobl'ast, intermediate trophoblast and syncytiotrophoblast (5, 6). A continuous differentiation process involves these cells during pregnancy. Indeed, mononucleated cytotrophoblast differentiate to intermediate cells and successively to multinucleated syncytial cells. In the structure of placental villi, syncytial teils form the most external layer while cytotrophoblasts are in the inner part. Steroid (progesterone, estrogens) and pituitary-like hormones [human chorionic gonadotropin (hCG), human placental lactogen (hPL), ACTH] are mostly found in the syncytiotrophoblast. Neurohormones

Key-words: placenla, GnRH. CRH. aclivin. inhibin growth laclors. cytokines. Correspondence: F. Pelraglia. MD., Depl. Obslel. Gynecol., Univ. 01 Modena School 01 Medicine, Via dei Pozzo 71. 41100 Modena. Ilaly.

353

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GnRH in cultured placental cells indicates a local effect of placental GnRH. The stimulatory effect of GnRH on the release of hCG from placental cultures has been confirmed by several studies (17 -21 ). On the other hand, the lack of any effect reported by some Authors may be related to the different culture preparations (22, 23). The highest GnRH-induced hCG increase is shown in cultures prepared from placentas collected from the first trimester of pregnancy. More controversial are the results on the action of GnRH on placental steroid release. An increase of estradiol and progesterone (24) or the lack of any effect (25), or a decrease of progesterone (26) release following the addition of GnRH. to cultured placental cells have been described. Since the evidence that human placental cells synthesize and release GnRH, studies have been conducted on the possible mechanisms regulating its secret-ion. A significant release of placental GnRH is induced by the activation of Ca++ influx, a mechanism analogous to that involved in hypothalamic GnRH release (27). GnRH secretion depends on the activation of the adenylate cyclase system, as occurs for other placental hormone secretions (28). Prostaglandins (PGE 2 and PGF2a ) and epinephrine increase GnRH release from cultured placental cells throughout the cAMP pathway. The effect of epinephrine is associated to the presenCe of specific beta adrenergic receptors in the placental tissue. Even in absence of nerve endings in trophoblastic cells, the findings of metabolizing enzymes and of binding sites for neurotransmitters in placental tissue suggest that circulating neurotransmitters may act on placental target functions (29, 30). Other peptides and hormones act on the GnRH release from cultured placental cells insulin and VIP stimulate, while substance P, oxytocin and neuropeptide Y (NPY) do not significantly modify placental GnRH release (31 ). Some of the factors regulating GnRH release from placental cells (Fig. 2) are similar to those actively involved in the control mechanisms regulating hypothalamic GnRH release. An action of endogenous opioid peptides is supported by the evidence that cultured human placental cells incubated in the presence of various doses of morphine (mu receptor agonist) and U-50, 488H (kappa receptor agonist), but not M-Enk (delta receptor agonist), showed a significant decrease of GnRH release. These results suggest that endogenous ß-endorphin at:1d dinorphin may play an inhibitory control on placental GnRH release. Other important

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Fig. 1 - Representative scheme showing the putative cellular localization of steroid and protein hormones, neurohormones, neuropeptides, growth factors and cytokines in human placental villi.

In the present review, the identification and the putative biological roles of neurohormones, neuropeptides, growth factors and cytokines present in human placenta will be described. PLACENTAL AS ORGAN PRODUCING NEUROHORMONES 1- Gonadotrapin-releasing hormone (GnRH) Human placenta produces gonadotropin-releasing hormone (GnRH) with chemical characteristics and biological activity identical to the hypothalamic counterpart. Placental extracted GnRH stimulates the in vivo and in vitra release of pituitary gonadotropins (7 -10). Interestingly, the isolation of cloned genomic and cDNA sequences encoding the structure of GnRH precursor molecule has been conducted in human placenta (11). The presence of GnRH in human placenta has been confirmed by immunohistochemical findings: cytotrophoblast cells mainly localize GnRH material (12-14). The presence of specific GnRH binding sites (15, 16), and the increase of hCG release the following addition of

354

Placental hormones

Indeed, the blockade of GnRH receptors via specific antagonists causes a decrease of hCG release from placental explants (34) and a reduced neonatal viability when administered to pregnant monkeys (35). Further supporting the existence of a GnRHhCG axis, immunoreactive GnRH in the maternal circulation shows the highest values levels in the first trimester of gestation as weil as hCG levels (36).

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2- Corticotropin-releasing hormone (CRH) Corticotropin-releasing hormone (CRH) is localized in the cytotrophoblast layer of term placental villi (37,38). CRH extracted fram term placenta is identical to the hypothalamic counterpart and stimulates the release of ACTH and ß-END fram cultured rat pituitary cells (39-41). The presence of a specific CRH mRNA strongly supports the local synthesis of the neurohormones. The CRH mRNA is expressed in human placenta fram early gestation (7 weeks) and increases more than 20-fold during the last 5 weeks of pregnancy (42, 43). Placental release of CRH into maternal circulation during pregnancy is also supported by the parallel increase of placental CRH content and of maternal plasma CRH levels (42). The addition of CRH in a system of cultured placental cells stimulates ACTH release with a dosedependent mechanism. The effect of CRH is completely reversed by the addition of a synthetic CRH antagonist (37). Similarly to the pituitary effect, CRH

opiates

Fig. 2 - Summary of the substances which increase or decrease the release of GnRH from cultured human placental cel/s.

factors regulating GnRH release are activin and inhibin af1d GnRH release. The addition of activin, a gonadal glycoprotein which increases pituitary FSH release, stimulates the release of GnRH from human cultured placental cells (32). This effect is reversed by inhibin, a glycopratein hormone with decreases pituitary FSH release. Inhibin and opioid peptides also reduce the 8br-cAMP-induced GnRH release (Figure 3). The putative importance of opioid peptides and of inhibin glycoproteins in modulating placental GnRH release in vitro is emphasized by the evidences that they are produced by human placenta. Steroid hormones are the most largely produced by placental tissue and their possible role in regulating GnRH release form cultured placenta cells has beel'l recently investigated. Indeed, a local interaction between steroids and peptides modulate the GnRH release fram cultured human placental cells (33). The addition of estradiol or estriol significantly augments the activin-induced increase of GnRH release fram cultured placental cells. The effect of estriol is antagonized by tamoxifen or pragesterone. Progesterone reduces the activin- or 8br-cAMP- induced release of GnRH from cultured placental cells (33). The action of steroid hormones and activin in modulating GnRH release emphasizes a possible paracrine-autocrine regulation of placental hormone praduction (Fig. 3). The positive action of GnRH on hCG release (1721) suggests a GnRH-hCG axis in human placenta. In view of the important role of hCG in the physiology of pregnancy, it may be assumed a relevant rale of GnRH in the function of the endocrine function.

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355

F Petraglia, L. Calza, G.G. Garuti, et al.

increases also ß-endorphin (ß-END) and MSH release from perifused placental cells (44). Indeed human placenta produces the precursor molecule, proopiomelanocortin (POMC) (45, 46), and releases the related hormonal end praducts (47-50). In view of the presence of specific CRH binding sites in placental tissue (51,52), placental CRH may be the local secretagogue of the POMC-related hormones. In contrast with the function of the hypothalamicpituitary-adrenal axis, the addition of glucocorticoids to the culture medium does not decrease ACTH release and does not modify the effect of CRH (37, 44). CRH also increases the placental release of prostaglandins allowing to the hypothesis of a further possible paracrine or autocrine interaction of CRH in placental tissue (53). Local mechanisms may regulate the release of CRH from placental cells. Prostaglandins, neurotransmitters and peptides stimulate the release of CRH fram cultured placental cells. Norepinephrine (NE) and acetylcholine (ACh) are the most active neurotransmitters in increasing CRH release (54). ACh seems to act via a muscarinic receptor. This action .is of particular interest in view of the high levels of ACh concentrations and cholinergic receptors in human placenta (57). A similarity between mechanisms regulating hypothalamic and placental CRH release has been suggested, since NE and ACh increase hypothalamic CRH release. Moreover, also the neuropeptides which modulate placental CRH release are known to act on hypothalamic CRH. Angiotensin 11, arginine vasopressin or oxitocin increase the release of placental CRH from cultured trophoblast. Prastaglandins (37), interleukin-1 (IL1) (54) and glucocorticoids (55, 56) stimulate CRH

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Fig. 4 - Putative site(s) of action of placental CRF in the maternalplacental-fetal unit.

356

release or CRH mRNA levels in cultured placental cells at term. Because these molecules are known to increase during pregnancy and labor they may be suggested as endogenous factors regulating placental CRH release in vivo. In healthy pregnant women the gestational-related increase of maternal plasma and amniotic fluid CRH levels correlates with circulating ACTH and cortisol levels (58-62). During spontaneous labor, plasma maternal CRH levels progressively and significantly increase until parturition, in parallel with increasing cervical dilatation (52, 63-65). In view of the sudden decrease of CRH levels after placental delivery, placenta· is most likely the source of circulating CRH during labor (52, 58-62). Stress of labor seems to be very important when we consider the lack of changes at elective cesarean section (52). Various hypotheses may be done on the possible site(s) of action of placental CRHduring pregnancy and at parturition (Fig. 4). Placental CRH may stimulate ACTH release fram placenta, fetal and/or maternal pituitary. The local placental CRH-ACTH axis has been shown in vitro. A possible local effect is also supported by the evidence that the number of placental CRH binding sites is higher after vaginal delivery than after cesarean section (52). Fetal pituitary may be a target of CRH circulating in umbilical cord plasma. This hypothesis is supported by the evidence that CRH levels in umbilical vein are higher than in umbilical artery (60). The parallel increase of maternal plasma CRH and ACTH/cortisol plasma levels during pregnancy suggests that maternal pituitary is one target of placental CRH. This hypothesis is supported by the impaired dexamethasone-induced suppression of plasma cortisol (66, 67) and by the impairedh response of plasma ACTH and cortisol to acute CRH administration in pregnant women (64) This insensitivity of the axis to the glucocorticoid feedback inhibition persists for three weeks after parturition (68) and the reduced response to CRH for 3 days (31). The presence of a high affinity binding prateins for CRH (molecular weight 38,000 Daltons) has been found in human plasma (68-71) blocks the possible development of a Cushing's syndrome. Most of plasma CRH in pregnant women is bound to this protein and the capacity of CRH-binding pratein to bind exogenous CRH decreases during the third trimester of pregnancy (70), even if it has the capacity to bind additional CRH(71). However, at the time of labor and parturition the rapid increase of circulating

Placental hormones

action of somatostatin on hPL release has not been cOnfirmed in cultured placental cells at term (73).

CRH may overcome the buffering capacity of the binding protein. The evidences that CRH stimulates placental and decidual prastaglandins release (53) and that may increase uterine contractility (Petraglia et al. in preparation), further support maternal site(s) of action. In agreement with the hypothesis of a possible role of placental CRH in the mechanism leading to the onset of labor patients with preterm labor show plasma CRH levels higher than healthy pregnant women (58).

4- Growth hormone-releasing factor (GHRH) Rat but not human placenta contains the GH-releasing factor (GHRH) (76, 77). The molecule has not the same immunological characteristics and biological activities as the hypothalamic GHRH. It is measurable only from midgestation at term, and is not present in pregnant rat plasma (76, 77). 5- Thyreotropin-releasing factor (TRH) A peptide active to stimulate TRH release from cultured pituitary cells is present in human placenta (78, 79). The chemical identity with hypothalamic TRH has been discussed and two different molecules have been isolated (80, 81).

3- Somatostatin Somatostatin, the GH-inhibiting factor, has been isolated fram human placental tissue (72-74). Radioimmunological and immunohistochemical studies revealed a somatostatin-like material in human placenta during the first trimester, with the most intense staining in the cytotrophoblast and in the stroma at six/twelve weeks of gestation (72-74) . Accordingly, amniotic fJuid somatostatin levels are higher in the first trimester of pregnancy (75). The gestational changes of somatostatin are inversely correlated to the circulating hPL, the placental counterpart of pituitary GH (1-4). The hypothesis of an inhibitory

6- Oxytocin A molecule showing the immunoreactive and bioactive characteristics of oxytocin has been described in human placenta (82, 83). An action of oxytocin in mOdulating placental hormone production is suggested by several studies. The increase of ACTH (39) ß-END (46) or prostagiandin (84) release from

Fig. 5 - Fluoreseenee photomierograph ot human plaeental tissue

at term. In the inner part ot plaeental villi there is a positive staining tor the antiserum raised against neuropeptide Y (panel B). Indeed, eytotrophoblast and intermediate trophoblast show a positive staining tor monoelonal vimentin antiserum (Panel A), a eellular marker ot these eell populations (5, 6). On the other hand, syneytiotrophoblast cells show a positive staining for the monoelonal keratin antiserum (Panel G) (5, 6).

357

F. Petraglia, L. Calza, G.c. Garuti, et al.

cultured placental cells has been shown in presence of oxytocin. PLACENTA AS ORGAN PRODUCING NEUROPEPTIDES

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1- Neuropeptide Y (NPY) Neuropeptide Y (NPY) is a 36-amino acid peptide extensively distributed in the brain (85). NPY has been found in neurons throughout the central and peripheral nervous system. Among the central effects, NPY plays a role in the regulation of the activity of hypothalamus-pituitary-gonadal axis. In the autonomic nervous system NPY appears to be in close association, but not exclusively within catecolamine containing nerves (86, 87). NPY is found in sympathetic neurons innervating the cardiovascular and respiratory systems as weil as the gastrointestinal and genitourinary tract. The human placenta contains NPY and specific NPY-binding sites at term (88). The peptide is localized in cytotrophoblast and intermediate trophoblast cells (Fig. 5,. Extracted NPY elutes with the same chromatographic profile as native NPY. The binding sites for NPY are widely distributed wfthin the trophoblastic tissue. The activation of these receptors by the addition of NPY stimulates the release of CRF from human placental preparations. This effect is doseand time-dependent. Norepinephrine potentiates the action of NPY on CRH release (88). Other than a local action, placental NPY is released in maternal circulation and/or in amniotic fluid. High levels of NPY are found in pregnant women throughout physiological pregnancy, without significant changes in gestation (89). High NPY levels are also measurable in the amniotic fluid (89). Maternal plasma NPY levels increase during labor and the most relevant rise coincides with the most advanced stages of cervical dilatation, at vaginal delivery (89) (Fig. 6). Since plasma NPY levels do not increase in patients undergoing elective caesarean section, a stress-related mechanism occurring during labor has been hypothesized (89). This hypothesis fits with the observation that in healthy subjects metabolic and physical stresses are followed by significant increase of plasma NPY levels (90, 91). According to some experimental observations, pla~ental N.PY durin~ pregnancy and labor may be Inv?lved In. r~gulatlng local blood vessel contractility or In modlfYlng the muscle tonus of myometrium (92,93).

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2- Beta-endorphin (ß-END) Beta-endorphin (ß-!:ND) was the first opioid peptide detected in human placenta. It may be included either among neuropeptides or pituitary hormones. As previously stated, the mRNA of the precursor molecule, POMC, has been found in rat, mouse and human placenta (49). The human placental cell cultures release ß-END, ACTH and MSH (44, 45), indicating that placental processing of the POMC is similar to that occurring in brain or pituitary cells. This is further suggested by the evidence that CRF or oxytocin stimulate the POMC-related hormone release from human cultured placental cells (37, 44f ß-END is a neuropeptide involved in several biological functions. Therefore, multiple sites of action may be hypothesized for placental ß-END. Plasma ß-END levels increase during pregnancy and labor and several authors have related these changes to a possible action in the regulation of endogenous analgesia (94). 3- Methionine-enkephalin (M-EnK) ~et~ionine-enk~phalin (M-EnK), the most wideiy dlstnbuted EOP In the brain, has also been shown in human placenta (95). M-EnK plasma levels in pregnant women do not show significant difference from non-pregnant women (95). 4- Dynorphin (DYN) Dynorphin (DYN) has been characterized in human

358

Placental hormones

placenta (96, 97). Opiate subtype Kappa-receptors are found in abundance in the human placenta (98). Maternal plasma Dyn levels in the third trimester and at delivery are higher than in nonpregnant subjects (97).

at term (108). The addition of purified inhibin to cultured human placental cells reduces the GnRHinduced hCG release (110). On the other hand, activin increases GnRH and progesterone production and potentiates the GnRH-induced release of hCG (110). As described for the mechanisms regulating placental GnRH release, the activin-induced GnRH release is modulated by steroid hormones. Estrogens increase and porgesterone reduces the effects of activin on GnRH release (33). The regulation of inhibin secretion from placental cells is identical to that shown in gonads, and various substances may influence placental inhibin activity. cAM P has been proposed as the second messenger mediating placental inhibin release (108). The increase of cAMP concentration in the placental cells may be the cause of the release of inhibin induced by the hCG addition to the placental cell cultures (108). Various substances (NPY and VIP) increase the release of placental inhibin from cultured placental cells (20). Placenta is the source of increased maternal plasma levels during pregnancy. Two major evidences support such hypothesis: 1) women with premature ovarian failure and undetectable inhibin showed a constant increase of plasma inhibin levels when they become pregnant for oocyte donation (112); 2) pregnant women with placental tumors showed very high plasma inhibin levels and decreased after chemotherapy (113).

5- Inhibin and aetivin Human placenta produces inhibin and activin. Inhibin and activin are glycoprotein hormones with hypophyseotropic action modulating FSH release from pituitary gland (99-102). Even if they have also been found in the central nervous system, they are different from the other hypophyseotropic neurohormones being gonads the major source. Moreover, because inhibin and activin also act as growth factors, they are presented both as hormones and growth factors. Inhibin is a heterodimeric glycoprotein composed of 2 subunits: one cx and one ß subunits. The a subunit is constant, while 2 forms of ß subunits are recognized: A and B. Since now, two different forms of inhibins (inhibins AaßA and inhibin B: aß B) are known. Activin is a homodimeric protein composed of 2ß subunits; 3 forms (AA, AB, BB) have been since now recognized (103,104). Inhibin subunits are expressed in gonads and in extragonadal tissues (105) and have opposing activities in several biological systems. While inhibin inhibits, activin increases FSH release from cultured pituitary cells. In addition to its dual endocrine role on the pituitary gland, inhibin and activin exert paracrine and / or autocrine effect in gonads and bone marrow (103). It has been demonstrated that inhibin a, ßA ßB subunits are present in human placenta (108, 109). Inhibin a subunit is localized mostly in the cytotrophoblast cells while inhibin ßB subunit is distributed in the syncytial layer of placental villi. Both cell populations contain inhibin ßA subunit immunoreactivity. Following the evidence that human placenta expresses inhibin a subunit (106), the presence of bioactive and immunoreactive inhibin in placental extracts (110) and the release of inhibin from cultured placental cells (108) confirmed the production of inhibin by human placenta. Inhibin subunit mRNAs are expressed from early pregnancy and are maximal at term. Atthe present time, placental inhibin and activin 3eem to be involved in the regulation of local GnRH and hCG release. Indirect evidences obtained by passive immunoneutralization indicated that placental inhibin locally inhibits the release of hCG and GnRH from human placenta

PLACENTA AS AN ORGAN WH/CH PRODUCES GROWTH FACTORS Human placenta produces and is a target for many growth factors. Since now the presence and/ or the putative role(s) of insulin -like growth factors (IGFs), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), transforming growth factors (TGF) and platelet derived growth factor (PDGF) have been shown.

1- Insulin-like growth faetar (IGFs) Insulin growth factors land 11, or somatomedins, belong to a family having primary structural homology with proinsulin and tertiary structural similarity with insulin.IGFs have insulin-like biological effects and stimulate the proliferation of various cell types (114, 115). The expression of IGF mRNAs demonstrated that both IGF land IGF II are synthesized by human

359

F. Petraglia, L. Calza, G.c. Garuti, et al.

While there are no evidences of EGF synthesis in human placenta, high levels of EGF receptors are shown in placental tissue (129). In fact, EGF receptors are localized on human placental syncytiotrophoblast plasma membranes and on EGF-stimulated tyrosine kinase activity has been reported (129). Moreover, EGF receptors have also been revealed in a number of intracellular organelles, such as lysosomes, rough and smooth endoplasmic reticulum and Golgi apparatus. The function of these binding sitesin the intracellular organelles may be explained by three theories: 1) they may be catabolic sites for internalized EGF molecules: 2) they may represent newly synthesized receptors or 3) they may represent the triggers for biological signals of growth factor actions (130). Placental EGF receptors levels are significantly higher in early pregnancy than in term pregnancy placentas (131) and in male fetuses than in female fetuses placentas (132). The presence of binding sites and the different levels between the placentas from male and female, lead to the hypothesis that EGF and its receptor might be involved both in the sexual dimorphic traits and in the weight of neonates (132). The gestational-related difference of EGF receptors has been correlated with the morphological differentiation from cyto to syncytiotrophoblast induced by EGF in cultured early pregnancy placental cells (131, 133). Moreover, EGF enhances hCG and hPL release (131, 133). The increase of hCG and hPL release induced by EGF in term piacenta cultures was evident with a lag period of 3 days, thus suggesting a mechanism other than simple releasing of intracellular polypeptide (131 ). Undetectable levels of hCG ß mRNA, and a 6-fold decrease of hCG a mRNA in term may account for the lack of EGF effect on hCG release by term placentas. The different response of term placental cells to EGF in producing hCG a, may be due to differences in levels of EGF receptors. It is, therefore, possible hypothesize that the changes of hCG during pregnancy may be related to the decrease EGF receptors in term placental syncytiotrophoblast (129, 131, 134). Finally, using placental explants no effects of EGF on the release of hCG and hPL has been observed (135, 136). The different methodological procedures may explain such discrepancies. Indeed, EGF, without affecting placental cell proliferation, seems to playa role in placental development and function of differentiated trophoblast (131, 133).

placenta (116). The IGFmRNA expression undergoes to developmental changes since its levels are higher in second trimester than at term (117). Regulatory mechanisms for IGFs synthesis and release by placental cell are not clearly understood. The fetal prodiction of GH and human placenta lactogen (hPL) may be factors involved in the control of IGFs synthesis. Moreover a placental variety of GH has been recently identified (118). IGFs act on placental cells via specific cell membrane receptors, which are different for IGF land II (119). The presence oflGF binding sites on placental cells suggest an autocrine/paracrine action of IGFs in human placenta (120). Placental IGF I receptors bind, with decreasing affinity, IGF '1, IGF 11 and insulin (121 ). Two distinct IGF I receptors are present in human placental tissues, thus suggesting the possibility that tissue-specific isotypes of the IGF I receptor could mediate the different biological actions IGF 1(122). IGF 11 receptor binds IGF II with higher affinity than IGF I but does not bind insulin. Placental IGF 11 receptors have the same characteristics as the other receptor structures in the periphery (121 ). IGFs actions have been studied in cultured choriocarcinoma cells (Jeg-3 cells) and in normal cultured human trophoblast cells (121-123). Like insulin, IGFs increase hPL release, from cultured placental cells. This effect is dose- and glucose- dependent as demonstrated by the decrease of the stimulatory effect of insulin in presence of glucose transport inhibitors (124). Moreover, IGF I modulates also steroid hormone release blocking the aromatase activity (inhibition of androstenedione to estrogen conversion and stimulating P450 side-chain cleavage enzyme (increase of progesterone secretion) (125). Finally, IGFs are thought to playa key role in early proliferative phase of embryonic and fetal development. In fact, because IGFs receptor levels are higher in early placenta, it is reasonable to speculate that IGFs may regulate the early proliferative phase of fetal growth, whereas insulin influences later growth (117, 118). 2- Epidermal growth factor (EGF) Epidermal growth factor (EGF) is a polypeptide possessing a variety of biological activities (mitogenic activity, membrane proteins phosphorilation, stimulation of RNA and DNA synthesis and cell multi plication in various cell types) (126, 127) mediated by a specific cell membrane receptor (128).

360

Placental hormones

The developihg human placenta may therefore represent a ca se of autocrine growth regulation in normal tissue, in which cells bearing receptors for a growth factor can also synthesize and respond to it. The endocrine effects of GFs most probably refleet the local response of trophoblastic cells, and represent a local factor participating to the mechanism regulating placental hormone release (Fig. 7).

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Fig. 7 - Hormonal effects of grawrn tactors on human placental cells in vitra.

3- Fibroblast growth faetar (FGF) Two different forms of FGF are present in various tissues: acid (the major FGF component in the brain) and basic. Acid and basic FGF are structurally related (shearing 55% of sequence homology), interact with the same cell receptors and have biological activities in common, i.e. stimulating of angiogenesis, cell differentiation and proliferation (137, 138). Basic FGF is present in human placenta and acting via autocrine/paracrine mechanism, stimulates hCG ß synthesis (139). This effects: 1) on "de nava" synthesis since intracellular content of hCG ß is relatively constant during incubation with FGF; 2) it is sustained, since FGF levels continue to be elevated in spite of the withdrawal of the growth factor; 3) it is enhanced by insulin, althoughthe relative mechanism of this enhancement is still unknown (139). Because of the angiogenic effect, FGF mayaiso cause a high degree of neovascularization during the initial stages of placental development. This action mayaiso playa role in the release of hCG induced by FGF.

Pl.ACENTA AS AN ORGAN WH/CH PRODUCES CYTOK/NES

Human placenta contains several cytokines, the hormonal messenger of the immune system. It is not clear whether trophoblast or immune cells deriving from the circulation are the source of cytokines found in placental extracts. Some reperts indicate that 65% of placental cells resemble mononuclear phagocytes (144). Some recent re ports are showing the action of cytokines on placental hormone pro~ duction. 1- Interleukin 1 (IL -1 ) Interleukin 1 (IL-1), a cytokine maily produced by macröpahges, is one of the key mediators of the body's response to immunological reactions and has a wide range of biological functions. Recombinant IL-1 produces endogenous fever, stimulates PGE 2 and induces the proliferation of fibroblastand T Iymphocytes (145). IL-1 also induces the release of several hypothalamic and pituitary hormones, i.e. CRF, somatostatin, ßEND and ACTH (146, 147). Recently, it has been shown that monocytes derived from human placenta produce IL-1 and cultured placental cells release IL-1 (148, 149). The addition of estradiol or progesterone increases the IL-1 production from cultured human placenta monocytes, suggesting a local regulatory influence of steroid hormones on placental IL-1 production (150). The placentallL-1 has the same molecular weight, isoelectric point, and activity as the monocyte content. Several hypotheses have been done on the possible role of IL-1 in pregnancy: 1) increases body temperature; 2) increases plasma insulin and glucagon; 3) decreases plasma albumin; 4) influences circulating iron, copper and zinc level changes. Because these changes normalize with the completion of pregnancy, they may correl~te with placental monocytes (148). It has been hypo~hesized that placental IL-1 may regulate the synthesis of some placental hormone.

4- Other growth faetars

Nerve growth factor (NGF) mRNA and immunoreactive NGF have been found in human placenta (140). The actions of NGF on placental tissues are unknown. A placental angiogenic factor (PAF) has been isolated from term human placenta. It seems to be a low molecular weight peptide, expressing both angiogenic and mitogenic activity (141 ). Both transforming growth factor /TGF) a and ß have been isolated from extracts of human placenta (142). C-sis, a proto-oncogene encoding for platelet derived growth factor (PDGF) ß chain, is expressed by first trimester human placenta. In fact, the levels of PDGF-like activity in the medium of placental explants are significantly high, PDGF receptors are abundant in cultured trophoblast that respond to exogenous PDGF by activating the c-myc oncogene (143).

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IL-1

CRM~"---

such as interferons, and colony stimulating factors (156). Human placental syncytiotrophoblast expresses IL-2 mRNA thr.ough the entire period of gestation (157). IL-2 may playa role in protecting the feto-placental unit when infections occur or to modulate the immune interactions between uterus and the feto-placental graft (157). No endocrine effects have been described for IL-2 in placental cell cultures (54).

PGs

3- Interferons (IFNs) The interferons are a group of powerful molecules than can be produced by virtually all cells. Their most relevant roles are to activate the natural process of defence from viral infections and to modulate cell differentiation (158). Among the three major antigenic types, i.e. a (Ieucocyte), ß (fibroblast) and (immune). Since now, IFN-a has been found in feto-placental unit (159). While it was not detected in maternal blood radioimmunometric assays, it was present in all fetal tissues, including blood, placenta, fetal membrane and maternal decidua. It is not clear at this moment what is the stimulus that force fetoplacentalleucocytes to produce such a relevant IFN-a concentration and what is its action.

ACTH Fig. 8 - Possible mechanisms ot action ot interleukin-1 (IL -1) in increasing placental CRF and ACTH release trom cultured placental cel/s.

The addition of IL-1 stimulates hCG release from human first trimester cultured trophoblast cells (151) and increases the release of CRF from cultured placental cells (152). This last effect is reversed by a CRF antagonist and is partially reversed by a prostagiandin synthesis inhibitor (152). The interaction between IL-1 and prostaglandins allows to stimulate the CRF / ACTH release from placental cells (Fig. 8). This mechanism may work at the time of labor. IL-1 is also produced by human decidua (153) and stimulates prostagiandin production by human amnion and decidua (154). IL-1 is measurable in amniotic fluid and high IL-1 levels in women at term and in spontaneous labor have been found (155). In particular, in women in preterm labor associated with intraamniotic infection a strong correlation between increased amniotic fluid IL-1 and prostagiandin levels has been described (155). Therefore it has been supported that IL-1 might serve as a signal for the onset of labor:

Colony stimulating factor-1 (CSF-1) is a glycoprotein growth factor required for the proliferation and differentiation of mononuclear phagocytes cells. A placental CSF-1 has been purified fram conditioned media (160). CSF-1 mRNA has been detected by in situ hybridization in the uterus of pregnant mice, and CSF-1 concentration is regulated by synergistic action of E2 and P (161). The major the site of CSF1 synthesis are the uterine luminal and glandular secretory cells the presence of specific receptors in trophoblastic cells and the increase of placental cells to enter DNA synthesis may suggest a role of CSF-1 in the apposition of the uterine secretory epithelium to placental trophoblast. Therefore, uterine CSF-1 synthesis in response to E2 and P may regulate placental trophoblast proliferation and differentiation (161).

2- Interleukin 2 (IL-2) Interleukin 2 (IL-2) is a 15.000 Dalton glycoprotein that is produced by T Iymphocytes after antigen or mitogen stimulation and required for proliferation of activated T cells. IL-2 also affects other cells such as ß-Iymphocytes and macrophages and it stimulates or increases the production of other cytokines,

5- Tumor necrosis factor-cr (TNF-cr) Tumor necrosis factor-a (TNF-a) is another importa nt cytokine. It participatesin the immune response by modulating repair processes. Like IL-1, TNF-a is produced by activate macrophages. Recently, TNF-a has been correlated proposed as factor involved in the onset of parturition. In fact,

4- Colony stimulating factor 1 (CSF-1)

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Placental hormones

REFERENCES

TNF-a is known to stimulate the synthesis of prostaglandins (162). The presence of TNF-a mRNA has been detected in human placental tissue. Treatment with loacterial endotoxin (Iipopolysaccharide) increases TNF-a mRNA levels in cultured decidual cells (162). Infection-associated preterm labor ha high TNF-a amniotic fluid levels, following decidual and / or placental macrophages release in response to the action of factors derived from microorganisms (162). Cytokines, as IL-1 and TNF-a, may represent the biochemical link between infection and preterm labor.

1. Osathanondh R., Tulchinsky D. Placental polypeptide hormones. In: Tulchinshy 0., Ryan K.J. (Eds.), Maternal-fetal endocrinology. Saunders, Philadelphia, 1980, p. 26.

2. Simpson E.R., McDonald P.C. Endocrine physiology on the placenta. Annu. Rev. Physiol. 43: 163, 1981. 3. Jaffe R.B. Endocrine physiology of the fetus and fetoplacental uni!. In: Yen S.S.C., Jaffe R.B. (Eds.), Reproductive endocrinology. Saunders, Philadelphia, 1986, p. 737.

CONCLUSION The endocrine functioning of human placenta results patticularly complex in view of these recent knowledge. The hormonal messen gers of the endocrine, neuroendocrine and immune systems interact for determining a correct placental growth and differentiation. From the time of implantation of trophoblast in uterus until parturition, these large number of molecules represent the critical factors mediating the feto-maternal weil being. Placenta may play the pivotal role in the endocrinology of pregnancy. The definition of placenta as neuroendocrine organ, source of brain hormones (163-166) has been shown to be correct. However, actually this concept is limited and is going to be enlarged taking into account the other groups of molecules produced by human placenta. These new aspects of placental endocrinology other than showing placenta as source of messages directed to the mother and to the fetus, also as target of hormonal, neuronal and immune messages originating from maternal and fetal circulation. Therefore, a correct placental functioning appears to be fundamental for initiation and maintenance of pregnancy. The potential clinical applications in the diagnosis and treatment of human pregnancy remain to be investigated.

4. Yen S.S.C. Endocrinology of pregnancy. In: Creasy R.K., Resnik R. (Eds.), Maternal-fetal medicine: principles and practice. Saunders, Philadelphia, 1989, p. 375. 5. Butterworth B.H., Khong T.v., Loke y.w., Robertson WB. Human cytotrophoblast populations studied by monoclonal using single and double biotin-avidin-peroxidase immunocytochemsttry. J. Histochem. Cytochem. 33: 977,1985. 6. Kurman R.J., Main C.S., Chen H.C. Intermediate trophqblast: a distinctive form of trophoblast with specific morphological, biochemical and functional features. Placenta 5: 349, 1984. 7. Gibbons J.M., Mitnick M., Chieffo V. In vitro biosynthesis of TSH- and LH-releasing factors by the human placenta. Am. J. Obstet. Gynecol. 121: 127, 1975. 8. Khodr G.S., Siler-Khodr TM. Placental LRF and its synthesis. Science 207: 315, 1989. 9. Tan L., Rousseau P. The chemical identity of the immunoreactive LHRHlike peptide biosynthesized in the human placenta. Biochem. Biophys. Res. Commun. 109: 1061, 1982. 10. Lee J.N., Seppala M., Chard 1. Characterization of placental luteinizing hormonereleasing factor-like material. Acta Endocrinol. 96: 394, 1981.

ACKNOWLEDGMENTS This study has been in part supported by the Consiglio Nazionale delle Ricerche (P.F. FATMA, SP. 7.2.3) and by the NIH grants No. HO 13527 and OK 26741. The collaboration of A.R. Genazzani, A Volpe, A Segre, M.C. Galassi, S. Bettelli,B. Preti, AC. Mancini, W. Vale, J. Rivier, A.T.w. Lim, PM Sawchenko, S. Sulton, V. Roberts, J. Vaughan, A Corrighan, R. Kaizer, S. Guerra is particularlyacknowledged.

11. Seeburg P.H., Adelman J.P. Characterization of cD NA for precursor of human luteinizing hormone releasing hormone. Nature 311: 666, 1984. 12. Khodr G.S., Siler-Khodr TM. Localization of luteinizing hormone releasing factor

363

F. Petraglia, L. Calza, G.c. Garuti, et al.

luteinizing hormone-releasing hormone, and aromatase inhibitor on simultaneous outputs of progesterone, estradiol, and human chorionic gonadotropin by term placental explants. J. Clin. Endocrinol. Metab. 55: 213, 1982.

(LRF) in the human placenta. Fertil. Steril. 29: 523, 1978. 13. Miyake A., Sakumoto T., Aono T., Kawamura Y., Maeda T., Kurachi K. Changes in luteinizing hormone-releasing hormone in human placenta throughout pregnancy. Obstet. Gynecol. 60: 444, 1982.

23. Haning RV. Jr., Breault P.H., DeSilva M.v., Hackett R.J., Pouncey C.L. Effects of fetal sex, stage of gestation, dibutyryl cyclic adenosine monophosphate, and gonadotropin releasing hormone on secretion of human chorionic gonadotropin by placental explants in vitro. Am. J. Obstet. Gynecol. 159: 1332, 1988.

14. Seppala M., Wahlstrom T., Lehtovirta P., Lee J.N., Leppalouto J. Immunohistochemical demonstration of luteinizing hormone-releasing factor-like material in human syncytiotrophoblast and trophoblastic tumours. Clin. Endocrinol. 12: 441, 1980.

24. Siler-Khodr T.M., Khodr G.S., Valenzuela G., Rhode J. GnRH effects on placental hormones during gestation. 11 Progesterone, estrone, estradiol and estriol. Biol. Reprod. 34: 225, 1986 b.

15. Belisle S., Guevin J.F., Bellabarba D., Lehoux J.G. Luteinizing hormone-releasing hormone binds to enriched human placental membranes and stimulates in vitro the synthesis of bioactive human chorionic gonadotropin. J. Clin. Endocrinol. Metab. 59: 119, 1984.

25. Branchaud C.L., Goodyer C.G., Lipowski L.S. Progesterone and estrogen production by placental monolayer cultures: effect of dehydroepiandrosterone and luteinizing hormone-releasing hormone. J. Clin. Endocrinol. Metab. 56: 761, 1983.

16. Currie A.J., Fraser H.M., Sharpe R.M. Human placental receptors for luteinizing hormone releasing hormone. Biochem. Biophys. Res. Commun. 99: 332, 1981.

26. Wilson E.A., Jawad M.J. Luteinizing hormone-releasing hormone suppression of human placental progesterone production. Fertil. Steril. 33: 91, 1980.

17. Butzow R. Luteinizing hormone releasing factor increases release of human chorionic gonadotropin in isolated cell columns of normal and malignant trophoblasts. Int. J. Cancer. 29: 9, 1985.

27. Petraglia F., Lim A.TW, Vale W. Adenosine 3',5'-monophosphate, prostaghindins, and epinephrine stimulate the secretion of immunoreactive gonadotropin-releasing hormone from cultured human placental cells. Clin. Endocrinol. Metab. 65: 1020, 1987.

18. Khodr G.S., Siler-Khodr T.M. The effect of luteinizing hormone-releasing factor on human chorionic gonadotropin secretion. Fertil. Steril. 30: 301, 1978.

28. Nulsen J.C., Woolkalis M.J., Kopf G.S., Strauss J.F. Adenylate cyclase in human cytotrophoblasts: characterization and its role in modulating human chorionic gonadotropin secretion. J. Clin. Endocrinol. Metab. 66: 258, 1988.

19. Kim S.J., Namkoong S.E., Lee J.w., Jung J.K., Kang B.C., Park J.S. ResponsErof human chorionic gonadotropin to luteinizing hormone-re leasing hormone stimulation in the culture media of normal human placenta, choriocarcinoma cell lines and in the serum of patients with gestational trophoblastic disease. Placenta 8: 257, 1987.

29. Nandakumaran M., Gardey C., Challier C., Olive G. Placental monoamine oxidase conte nt and inhibition: effect of enzyme inhibition on maternofetal transfer of noradrenaline (norepinephrine) in the human placenta in vitro. Placenta 4: 57, 1983.

20. Petraglia F., Vale W. Role of inhibin-related peptides in human placenta. In: Hodgen G., Rosenwaks Z., (Eds.), Nonsteroidal gonadal factors: physiological roles and possibilities in contraceptive development. Institute Press, NOrfolk, 1988, p. 181.

30. Whitsett JA, Johnson C.L., Noguchi A., DarovecBeckerman C., Costello M. ß-adrenergic receptors and catecholamine-sensitive adenylate cyclase of the human placenta. J. Clin. Endocrinol. Metab. 50: 27, 1980.

21. Siler-Khodr T.M., Khodr G.S. Dose response analysis of Gn-RH stimulation of hCG release trom human term placenta. Biol. Reprod. 25: 353, 1981.

31. Petraglia F., Volpe A., Genazzani A.R., Rivier J., Sawchenko P.E., Vale W. Neuroendocrinology of the human placenta. Front. Neuroendocrinol. (in press), 1990.

22. Haning R.V., Choi L., Kiggens A.J., Kuzma D.L., Summerville J.w. Effect of dibutyryl adenosine 3'-5' -monophosphate,

32. Petraglia F., Vaughan J., Vale W.

364

Placental hormones

43. Grino M., Chrousos G.P., Margioris A.N. The corticotropin in releasing hormone gene is expressed in human placenta. Biochem. Biophys. Res. Commun. 148: 1208, 1987.

Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells. Proc. Natl. Acad. Sci. USA 86: 5114, 1989. 33. Petraglia F., Vaughan J., Vale W. Steroid hormones modulate the release of immunoreactive gonadotropin-releasing hormone from cultured human placental cells. J. Clin. Endocrinol. Metab. (in press) 1990.

44. Margioris A.N., Grino M., Protos P., Gold PW., Chrousos J.P. Corticotropin-releasing hormone and oxytocin stimulate the release of placental proopiomelanocortin peptides. J. Clin. Endocrinol. Metab. 66: 922, 1988.

34. Siler-Khodr T.M., Khodr G.S., Vockery B.H., Nester J.J. Inhibition of hCG, a-hCG and progesterone release from human placenta tissue in vitro by a GnRH antagonist. Life Sci. 32: 2741, 1983.

45. Liotta A.S., Krieger DT In vitro biosynthesis and comparative pGst translational processing of immunoreactive precursor cor~ ticotmpin/ ß endorphin by human placenta and pituitary cells. Endocrinology 106: 1504, 1980.

35. Siler-Khodr TM., Kuehl TJ., Vickery B.H. Effects of gonadotropin releasing hormone antagonist on hormonal levels in the pregnant baboon and on fetal outcome. Fertil. Steril. 41: 448, 1984.

46. Odagiri E., Sherrel B.J., Mount C.D., Nickolson W.E., Orth D.N. Human placental immunoreactive corticotropin, lipotropin, and ß-endorphin: evidence for a common precursor. Proc. Natl. Acad. Sci. 76: 2027, 1979.

36. Siler-Khodr TM., Khodr G.S., Valenzuela G. Immunoreactive GnRH levels in maternal ciruculation throughout pregnancy. Am. J. Obstet. Gynecol. 150: 376, 1984.

47. Rees L.H., Burke CW., Chard T., Evans SW., Letchworth AT Possible placenta origin of ACTH in normal human pregnancy. Nature 254: 620, 1975.

37. Petraglia F., Sawchenko P., Rivier J., Vale W. Evidence for local stimulation of ACTH secretion by corticotropin-releasing factor in human placenta. Nature 328: 717, 1987.

48. Genazzani A.R., Fraioli F., Hurlimann J., Fioretti P., Felber J.P. Immnuoreactive ACTH and cortisol plasma levels during pregnancy: detection and partial purification of corticotropin-like placental hormone: the human chorionic corticotropin (HCC). Clin. Endocrinol. 4: 1, 1975.

38. Saijonmaa 0., Laatikainen 1., Wahlstrom 1. Corticotrophin-releasing factor in human placenta: localization, concentration and release in vitro. Placenta 9: 373, 1988. 39. Shibasaki 1., Odagiri E., Shizume K., Ling N. Corticotropin-releasing factor-like activity in human placental extract. J. Clin. Endocrinol. Metab. 55: 384, 1982.

49. Fraioli F. Genazzani A.R. Human placenta ß-endorphin. Gynecol. Obstet. Invest. 11: 37, 1980. 50. Nakai Y., Nakao K., Oki S., Imura H. Presence of immunoreactive ß-lipoprotein and endorphin in human placenta. Life Sci. 23: 2013, 1978.

40. Sasaki A., Tempst P., Liotta A.S., Margioris A.N., Hood L.E., Kent S.B.H., Krieger DT Isolation and characterization of a corticotropin-releasing hormone-like peptide from human placenta. J. Clin. Endocrinol. Metab. 67: 768, 1988.

ß-

51. Suda 1., Iwashita M., Tozawa F., Ushiyama 1., Tomori N., Sumitomo 1., Nakagami Y., Demura H., Shizume

41. Chan E.C., Thomson M., Madsen G., Falconer J., Smith R. Differential processing of corticotrophin-releasing hormone by human placenta and hypothalamus. Biochem. Biophys. Res. Commun. 153: 1229, 1988.

K.

Secretion of corticotropin-releasing factor in pregnancy. In: Imura H., Shizume K., Yoshida S. (Eds.), Preocessin endocrinology. Excepta Medica, Amsterdam, 1988, vol. 1, p. 401.

42. Frim D.M., Emanuel R.L., Robinson B.G., Smas C.M., Adler G.K., Majzoud JA Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta. J. Clin. Invest. 82: 287, 1988.

52. Petraglia F., Giardino L., Coukos G., Calza L., Vale W., Genazzani A.R. Corticotropin-releasing factor and parturition: plasma and amniotic fluid levels and placental binding sites. Obstet. Gynecol. In press 75: 1990.

365

F Petraglia, L. Calza, G. C. Garuti, et al.

53. Jones S.A., Challis J.R.G. Local stimulation of prostagiandin production by corlicolropin-releasing hormone in human felal membranes and placenla. Biochem. Biophys. Res. Commun. 159: 192, 1989.

Sato S., Murakami 0., Go M., Shimizu Y., Hanew K., Yoshinaga K. Immunoreactive corticotropin-releasing hormone in human plasma during pregnancy, labor, and delivery. J. Clin. Endocrinol. Metab. 64: 224, 1987.

54. Petraglia F., Sutton S., Vale W. Neurotransmitters and peplides modulate the release of immunoreactive corticolropin-releasing faclor from human cullured placenlal cells. Am. J. Obstet. Gynecol. 160: 247,1989.

64. Sasaki A., Shinkawa 0., Yoshinaga K. Placental corticotropin-releasing hormone may be a stimulator of maternal pituitary adrenocorlicolropin hormone secretion in humans. J. Clin. Invest. 84: 1997, 1989.

55. Robinson B.G., Emanuel R.L., Frim D.M., Majzoub JA Glucocorticoid stimulales expression of corticotropin-releasing hormone gene in human placenta. Proc. Nall. Acad. Sci. USA 85: 5244, 1988.

65. Okamoto E., Takagi T, Makino T., Sata H., Iwata 1., Nishino E., Mitsuda N., Sugita N., Otsuki Y., Tanizawa

O. Immunoreactive corticotropin-releasing hormone, adrenocorticotropin and cortisol in human plasma during pregnancy and delivery and postpartum. Horm. Metab. Res. 21: 566, 1989.

56. Jones SA, Brooks A.N., Challis J.R.G. Steroids modulale corlicotropin-releasing hormone produclion in human felal membranes and placenta. J. Clin. Endocrinol. Metab. 68: 825, 1989.

66. Nolten W.E., Rueckert PA Elevated free cortisol index in pregnancy: possible regulatory mechanism. Am. J. Obstet. Gynecol. 139: 492-,1981.

57. Sastry B.V.R., Olubadewo J.O., Harbison RD., Schmid~ D.E. Human placenta cholinergic system: occurrence, distribution and variation with gestational age of acetylcholine in human placenta. Biochem. Pharmacol. 25: 425, 1976.

67. Smith R., Owens P.C., Brinsmead M.W., Singh B., Hall C. The nonsuppressibility of plasma cortisol persists after pregnancy. Horm. Melab. Res. 19: 41, 1987.

58. Campbell E.A., Linton E.A., Wolfe CDA, Scraggs P.R., Jones M.T., Lowry P.J. Plasma corticotropin releasing hormone concentrations during pregnancy and parturition. J. Clin. Ehdocrinol. Metab. 64: 1054, 1987.

68. Linton EA, Wolfe CDA, Behan D.P., Lowry P.J. A specific carrier substance for human corticotrophin releasing factor in late gestational malernal plasma which could mask the ACTH-releasing activity. Clin. Endocrinol. 28: 315, 1988.

59. Laatikainen T, Saijonmaa 0., Salminen K., Wahlstrom T Localization and concentrations of ß-endorphin and ß-lipotropin in human placenta. Placenta 8: 381, 1987.· 60. Goland R.S./Wardlaw S.L., Stark R.I., Brown L.S., Frantz A.G. High levels of corticotropin-releasing hormone immunoreactivity in maternal and fetal plasma during pregnancy. J. Clin. Endocrinol. Metab. 63: 1199, 1986.

69. Orth D.N., Mount CD. Specific high-affinity binding protein for human corticotropin-releasing hormone in normal human plasma. Biochem. Biophys. Res. Commun. 143: 411,1987. 70. Suda T, Iwashita M., Tozawa F., Ushiyama T, Tomori N., Sumitomo T, Nagakami Y., Demura H., Shizume

K.

Characterization of corticotropin-releasing hormone binding prolein in human plasma by chemical crosslinking and its binding during pregnancy. J. Clin. Endocrinol. Metab. 67: 1278, 1988.

61. Sasaki A., Liotta A.S., Lukey M.M., Margioris A.N., Suda T., Krieger D.T. Immunoreactive corticotropin-releasing factor is present in human malernal plasma during the third trimester of pregnancy. J. Clin. Endocrinol. Metab. 59: 812, 1984.

71. Suda T, Iwashita M., Ushiyama T, Tozawa F., Sumitomo T, Nakagami Y. Responses to corticotropin-releasing hormone and its bound and free forms in pregnant and nonpregna nt women. J. Clin. Endocrinol. Metab. 69: 38, 1989.

62. Laatikainen T, Raisanen I.J., Salminen K. Corticotropin-releasing hormone in amniotic fluidi during geslation and labor and in relation to fetal lung maturalion. Am. J. Obslel. Gynecol. 159: 891, 1988.

72. Kumasaka T, Nishi N., Yaoi Y. Demonstration of immunoreactive somatostatin-like subslance·in villi and decidua in early pregnancy. Am. J. Obstel. Gynecol. 134: 39, 1979.

63. Sasaki A., Shinkawa 0., Margioris A.N., Liotta A.S.,

366

Placental hormones

73. Macaron C., Kynel M., Rutsky L. Failure of somatostatin to affect human chorionic somatomammotropin and human chorionic gonadotropin secretion in vitro. J. Clin. Endocrinol. Metab. 47: 1141, 1978.

84. Ekblad U. The effect of oxitocin and betamimetic stimulation on prostagiandin release in perfused human fetal placenta. Eur. J. Obstet. Gynecol. Reprod. Biol. 23: 153, 1986.

74. Watkins W.B., Yen S.S.C. Somatostatin in cytotrophoblast of the immature human placenta: localitatiön by immunoperoxidase cytochemistry. J. Clin. Endocrinol. Metab. 50: 969, 1980.

85. Allen J.M., Bloom S.R. Neuropeptide Y: a putative neurotransmitter. Neurochem. Int. 8: 1, 1985.

75. Fitz-Patrick D., Patel Y.C. Measurement, characterization, and source of somatostatin-like immunoreactivity in human amniotic fluid. J. Clin. Invest. 64: 737, 1979.

86. O'Donohue T.L., Chronwall B.M., Pruss R.M., Mezey E., Kiss J.Z., Eiden L.E., Massari V.J., Tessel RE., Pickel V.M., Dimaggio DA, Hotchkiss A., Crowell W.R., Zukowska-grojec Z. Neuropeptide Y and peptide YY neuronal and endocrine systems. Peptides 6: 755, 1985.

76. Baird A., Wehremberg B., Bohlen P., Ling N. Immunoreactive and biologically active growth hormone-releasing factor in the rat placenta. Endocrinology 117: 1598, 1985.

87. Lundenberg J.M., Martinsson A., Hemsen A. Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem. Biophys. Res. Commun. 133: 30, 1985.

77. Meigan G., Sasaki A., Yoshinaga K. Immunoreactive growth hormone-releasing hormone in rat placental. Endocrinology 123: 1088, 1988.

88. Petraglia F., Calza L., Giardino L., Sutton S., Marrama P., Rivier J., Genazzani A.R, Vale W. Identification of immunoreactive neuropeptide-t in human placenta: localization, secretion, and binding sites. Endocrinology 124: 2016, 1989.

78. Shambaugh G., Kubek M., Wilson J.F. Thyrotropin-releasing hormone activity in the human placenta. J. Clin. Endocrinol. Metab. 48: 483, 1979.

89. Petraglia F., Coukos G., Battaglia C., Bartolotti A., Volpe A., Serge A., Genazzani A.R Plasma and amniotic fluid immunoreactive neuropeptide Y levels changes during pregnancy, labor and at parturition. J. Clin. Endocrinol. Metab. 69: 324, 1989.

79. Shambaugh G., Kubek M., Wilber J.F. Characterization of rat placental TRH-like material and the ontogeny of placental and fetal brain TRH. Placenta 4: 329, 1983.

90. Lundenberg J.M., Stjarne L. Neuropeptides Y (NPY) depresses the secretion of H-noradrenaline and the contractile response evoked by field stimulation in rat vas deferens. Acta Physiol. Scand. 120: 477,1988.

80. Youngblood WW., Humm J., Kizer J.S. TRH-like immunoreactivity in rat pancreas and eye, bovine and sheep pineals, and human placenta: non-identity with synthetic pyro-glu-his-pro-NH2 (TRH). Brain Res. 163: 101, 1979.

91. Takahashi K., Mouri T., Murakami O. Increases of neuropeptide Y -like immunoreactivity in plasma during insulin-induced hypoglycemia in man. Peptides 9: 433, 1988.

81. Youngblood WW., Humm J., Lipton M.A., Kizer J.S. Thyrotropin-releasing hormone-like bioactivity in placental: evidence for the existence of substances other than pyro-glu-his-pro-NH2 (TRH) capable of stimulating pituitary thyrotropin release. Endocrinology 106: 541, 1980.

92. Stjernquist M., Owan C. Interaction of noradrenaline NPY and VIP with the neurogenic cholinergic response of the rat uterine cervix in vitro. Acta Physiol. Scand. 131: 553, 1987.

82. Fields PA, Larkin L.H. Purification and immunohistochemical localization of relaxin in the human term placenta. J. Clin. Endocrinol. Metab. 52: 79, 1981.

93. Tenmoku S., Ottesen B., O'Hare M.M.T. Interaction of NPY and VIP in regulation of myometrial blood flow and mechanical activity. Peptides 9: 269, 1988.

83. Nakazawa K., Makino T., lizuka R., Kohsaka S., Tsukada Y. Immunohistochemical study on oxytocin-like substance in the human placenta. Endocrinol. Jpn. 31: 763, 1984.

94. Genazzani A.R, Petraglia F., Facchinetti F., Volpe A., Giarre G., Santi M., Baraldi M.

367

F Petraglia, L. Calza, G.c. Garuti, et al.

Central, peripheral, and placental opioids in pregnancy. In: Delitala G., Motta M., Se rio M., (Eds.), Opioid modulation of endocrine function. Raven Press, New York, 1984, p. 237.

Chemical and biokJgical characterization of the inhibin family of protein hormones. Recent Progr. Horm. Res. 44: 1, 1988.

95. Rama Sastry BV, Barnwell L.S., Tayeb O.S., Janson V.E., Owens K.L. Occurrence of methionine enkephalin in human JIllacental villus. Biochem. Pharmaco!. 29: 475, 1980. 96. Lemaire S., Valette A, Chouinard L., Dupuis N., Day R, Porthe G., Cros J. Purification and identification of multiple forms of dynorphin in human placenta. Neuropeptides 3: 181, 1983. 97. Valette A., Desprat R., Cros J., Pontonnier G., Belisle S., Lemaire S. Immunoreactive dynorphine in maternal blood, umbilical vein and amniotic fluid. Neuropeptides 7: 145, 1986. 98. Belisle S., Petit A, Gallo-Payet N., Bellabarba D., Lehoux J.G., Lemaire S. Functional opioid receptorsites in human placentas. J. Clin. Endocrino!. Metab. 66: 283, 1988.

104. Ying S.Y. Inhibins, activins, and follistatuns: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev. 9: 267, 1988. 105. Meunier H., Rivier C., Evans RM., Vale W. Gonadal and extragonadal expression of inhibin er, ßA and ßB subunits in various tissues predicts diverse function. Proc. Nat!. Acad. Sei. USA 85: 247, 1988. 106. Mayo K.E., Cerelli G.M., Spiess J., Rivier J., Rosenfeld M.G., Evans R.M., Vale W Inhibin A-subunit cDNAs from porcine ovary and human placenta. Proc. Nat!. Acad. Sci. USA 83: 5849, 1986 .. 107. McLachlan RJ, Healy D.L., Robertson D.M., Burger H.G., De Kretser DN The human placenta: a novel source of inhibin. Biochem. Biophys. Res. Commun. 140: 485, 1986. 108. Petraglia F., Sawchenko PA, Lim ATW., Rivier G., Vale W Localization, secretion, and action of inhibin in human placenta. Science 237: 187, 1987.

99. Forage R.G., Ring J.M., Brown RW., Mclnerney B.V., Cobon G.S., Gregson R.P., Robertson D.M., Morgan F.J., Hearn MTV., Findlay JK., Wettenhall R.E.H., Burger H.G., De Kretser D.M. Cloning and sequence analysis of cD NA species coding for the two subunits of inhibin from bovine follicular fluid. Proc. Nat!. Acad. Sei. USA. 83: 3091, 1986.

109. Petraglia F. Inhibins in human placenta. In: Genazzani AR, Petraglia F., Volpe A., Facchinetti F. (Eds.), Advances in gynecological endocrinology. Casterton, New York, 1988, p. 477.

100. Mason AJ., Hayflick J.S., Ling M., Esch F., Ueno N., Ying S.Y., Guillemin R, Niall H., Seeburg P.H. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-ß. Nature 318: 659, 1985.

110. Petraglia F., Vaughan J., Rivier J., Vale W. A new family of placental protein hormones: inhibinrelated peptides. In: Mochizuki M., Hussa R (Eds.), Placental protein hormones. Excerpta Medica, Amsterdam, 1988, p. 241.

110. Ling N., Ying S.Y., Ueno N., Shimasaki S., Esch F., Hotta M., Guillemin R. Pituitary FSH is released by a heterodimer of the ßsubunits from the two forms of inhibin. Nature 321: 779, 1986.

111. Petraglia F., Vaughan J., Vale W Inhibin and activin modulate the release of GnRH, hCG and progesterone from cultured human placental cells. Proc. Nat!. Acad. Sei. USA 86: 5114, 1989.

102. Vale W, Rivier J, Vaughan J, McClintock R., Corrigan A, Woo W, Karr D., Spiess J. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321: 776, 1986.

112. McLachlan RJ, Heale D.L., Lutjen P.J., Findlay JK, De Kretser D.M., Burger H.G. The maternal ovary is not the source of circulating inhibin levels during human pregnancy. Clin. EndocrinoL 27: 663, 1987.

103. Vale W., Rivier C., Hsueh A, Campen C., Meunier H., Bicsak T., Vaughan J., Corrigan A., Bardin W, Sawchenko P., Petraglia F., Yu J., Plotsky P., Spiess J.,

113. Yohkaichiya T., Fukaya Y., Hoshiai H., Yajima AM., de Kretser D. Inhibin: a new circulating marker of hydatiform mole? Sr. Med. J. 298: 1684, 1989.

Rivier J.

368

Placental hormones

114. Van Whk J.J., Underwood L.E. The somatomedins and their actions. In: Litwack G. (Ed.), Biochemical actions of hormones. Academic Press, New York, 1978, p. 101.

cytotrophoblast by insulin and insulin-like growth factor I. Endocrinology 121: (5): 1845, 1987.

115. Froesch E.R., Schmid C., Schwander J., Zapf J. Actions of insulin-like growth factors. Annu Rev. Physiol. 47: 447, 1985.

126. Cohen S., Carpenter G. Human epidermal growth factor isolation and chemical and biological properties. Proc. Natl. Acad. Sci. USA 72: 1317, 1975.

116. Liu K.S., Wang CY., Mills N., Gyves M., Ilan J. Insulin- related genes expressed in human placenta from normal and diabetic pregnancies. Proc. Natl. Acad. Sci. USA 82: 3868, 1985.

127. Carpenter G., Poliner L., King Jr. L. Protein phosphorilation in human placenta: stimula· tion by epidermal growth factor. Mol. Cell. Endocrinol. 18: 189, 1980.

117. Shen S.J., Wang CY., Nelson K.K., Jansen M., lIan

128. Gospodarowicz O. Epidermal and nerve growth factors in mammalian development. Annu Rev. Physiol. 43: 251, 1981.

J. Expression of insulin-like growth factor II in human placentas from normal and diabetic pregnancies. Proc. Natl. Acad. Sei. USA 83: 9179, 1986.

129. Lai W.H., Guyda H.J. Characterization and regulation of epidermal growth factor receptors in human placental cell cultures. J. Clin. Endocrinol. Metab. 58: (2): 344, 1984.

118. Wang CY., Oaimon M., Shen S.J., Engelmann G.L., Ilan J. Insulin-like growth factor I mRNA in the developing human placenta and in term placenta of diabetics. Mol. Endocrinol. 2: 217, 1988.

130. Raomani N., Chegini N., Rao Ch. V., Woost P.G. The presence of epidermal growth factor binding sites in the intracellular organelles of term human placenta .. J. Cell Sci. 84: 19, 1986.

119. Massague J., Czech M.P. The subunit structure of two distinct receptors for insulin-like growth factor land II and their relationship to the insulin receptor. J. Biol. Chem. 257: 5038, 1982.

131. Maruo T., Matsuo H., Oishi T., Hayashi M., Nishino R., Mochizuki M. Induction of differentiated trophoblast function by epidermal growth factor: relation of immunohistochemically detected cellular epidermal growth factor receptor levels. J. Clin. Endocrinol. Metab. 64: 744, 1987.

120. Fant M., Hamish M., Moses A.C. An autocrine/ paracrine role for insulin-like growth factor in the regulation of human placental growth. J. Clin. Endocrinol. Metab. 63: 499, 1986. 121. Ritvos 0., Rutanen E.M., Pekonen F., Jalkanen J., Suikkari A.M., Ranto T. Characterization of functional type I insulin- like growth factor receptors from human choriocarcinoma cells. Endocrinology 122: 395, 1988.

132. Brown M.J., Cook C.L., Henry J.L., Schultz G.S. Levels of epidermal growth factor binding in third trimester and term human placentas: elevated binding in term placentas of male fetuses. Am. J. Obstet. Gynecol. 157: 716, 1987. 133. Morrish OW., Bhardwaj 0., Dabbagh L.K., Marusyk H., Siy O. Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J. Clin. Endocrinol. Metab. 65: 1282, 1987.

122. Jonas HA, Harrison L.C. The human placenta contains two distinct binding and immunoreactive species of insulin-like growth factor I receptors. J. Biol. Chem. 260: 2288, 1985. 123. Hochberg Z., Perlman R., Brandes J.M., Benderli A. Insulin regulates placental lactogen and estradiol secretion by cultured human trophoblast. J. Clin. Endocrinol. Metab. 5: 1311, 1983.

134. Magid M., Nanney L.B., Stoscheck C.M., King Jr. E.R. Epidermal growth factor binding and receptor distribution in term human placenta. Placenta 6: 519, 1985.

124. Perlman R., Barzilai 0., Bick T., Hochberg Z. The effect of inhibitors on insulin regulation of hormone secretion by cultured human trophoblast. Mol. Cellvive Endocrinol. 43: 77, 1985.

135. Huot RJ, Foidart J.M., Nardone R.M., Strom berg K. Gonadotropin secretion by epidermal growth factor in normal and malignant placental cultures. J. Clin. Endocrinol. Metab. 53: 1059, 1981.

125. Nestler JE, Wiliams T. Modulation of aromatase and P450 cholesterol sidechain cleavage enzyme activities of human p/acental

136. Wilson EA, Jawad M.J., Vernon MW. Effect epidermal growth factor on hormone secretion

369

F. Petraglia, L. Calza, G.G. Garuti, et al.

by term placenta in organ cuiture. Am. J. Obstet. Gynecol. 149: (5): 579, 1984. 137. Thomas K.A., Rios-Candelore M., Fitzpatrick S. Purification and characterization of acidic fibroblast growth factor from bovine brain. Proc. Natl. Acad Sci. USA 81: 357, 1984. 138. Lemmon S.K., Bradshaw RA Purification and partial characterization of bovine pituitary fibroblast growth factor. J. Cell Biol. 21: 195, 1983.

Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science 238: 511,1987. 148. Flynn A. Placental mononuclear phagocytes as a source of interleukin-1. Science 218: 475,1982. 149. Flynn A., Finke J.H., Loftus M.A. Comparison of interleukin 1 production by adherent cells and tissue pieces from human placenta. Immunopharmacology 9: 19, 1985.

139. Oberbauer A.M., Linkhart TA, Mohan S., Longo LD. Fibroblast growth factor enhances human chorionic gonadotropin synthesis independent of mitogenic stimulation in Jar choriocarcinoma Gell. Endocrinology 123: 2696, 1988.

150. Flynn A. Stimulation of interleukin-1 production from placental monocytes. Lymphokine. Res. 3: 1, 1984. 151. Yagel S., LalaP.K., Powell W.A., Casper R.F. Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast. J. Clin. Endocrinol. Metab. 68: 992, 1989.

140. Heinrich G., Meyer T.E. Nerve growth factor (NGF) is present in human placenta and sement but undetectable in normal and Paget's disease blood: measurements with an antimouse-NGF enzyme immunoassay using a recombinant human NGF reference. Biochem. Biophys. Res. Commun. 155: (1): 482, 1988.

152. Petraglia F., Garuti G.C., Oe Ramundo B., Angioni S., Genazzani A.R., Bilezikjian L.M. Mechanism of action of interleukin-1 ß in increasing corticotropin-releasing factor and adrenocorticotropin hormone release from cultured human placental cells. Am. J. Obstet. Gynecol. (in press) 1990.

141. Burgos H. Angiogenic factor from human term placenta. Purification and partial characterization. Eur. J. Clin.lnvest. 16: 486,1986.

153. Romero R., Wu Y.K., Brody 0., Oyarzun E., Duff G.w., Durum SK Human decidua: a source of interleukin-1. Obstet. Gynecol. 73: 31, 1989.

142. Frolik CA, Dart L.L., Meyers CA, Smith D.M., Sporn M.B. Purification and initial characterization of a type ß transforming growth factor from human placenta. Proc. Natl. Acad. Sci. USA 80: 3676, 1983.

154. Romero R., Durum S., Dinarello C., Oyarzun E., Hobbins J.C., Mitchell MD. Interleukin-1 stimulates prostagiandin biosynthesis by human amnion. Prostaglandins 37: 13, 1989.

143. Goustin A.S., Betsholtz C., Pfeifer-Ohlsson S., Persson H., Rydnert J., Bywater M., Holmgren G., Heidin C.H., Westermark B., Ohlsson R. Coexpression of the sis and myc protooncogenes in developing human placenta suggests autocrine control of trophoblast growth. Cell 41: 301, 1985.

155. Romero R., Brody D.T., Oyarzun E., Mazor M., Wu Y.K., Hobbins J.C., Durum SK Interleukin-1 A signal for the onset of parturition. Am. J. Obstet. Gynecol. 160: 1117, 1989. 156. Smith K.A., Lachman L.B., Oppenheim J.J., Favata M.F. The functional relationship of the interleukins. J.Ex~ Med. 151: 1551,1980.

144. Hunziker RD., Wegmann T.G. Placental immunoregulation. CRC Crit. Rev. Immunol. 6: 245,1989. 145. Dinarello CA Biology of interleukin-1. FASEB J. 2: 108, 1988.

157. Boehm KD., Kelley M.F., lIan J. The interleukin 2 gene is expressed in the syncytiotrophoblast of the human placenta. Proc. Natl. Acad. Sci. USA 86: 656, 1989.

146. Sapolsky R., Rivier C., Yamamoto G., Plotsky P., ValeW. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 238: 522, 1987.

158. Burke CD. The interferons. Br. Med. Bull. 41: 333, 1985. 159. Chard T., Craig P.H., Menabawey M., Lee C. Alpha interferon in human pregnancy. Br. J. Obstet. Gynaecol. 93: 1145, 1986.

147. Berton E.W., Beach J.E., Holaday J.w., Smallridge R.C., Fein H.G.

370

Placental hormones

Placenta as source of brain and pituitary hormones. Biol. Reprod. 26: 55, 1982.

160. Schlunk T., Schleyer M. Preparative purification of human placental colonystimulating factors. Blut 47: 211, 1983.

164. Siler-khodr T.M. Hypothalamic-like releasing hormones of the placenta. Clin. Perinatol. 10: 553, 1983.

161. Pollard J.w., Bartocci A., Arceci R., Orlofsky A., Ladner M.B., Stanley E.R. Apparent role of the macrophage growth factor, CSF1, in placental development. Nature 330: 484, 1987.

165. Smith R. The neuroendocrinology of the pregnant woman. In: Dennerstien L., Fraser I. (Eds.), Hormones and behaviour. Elsevier Science Publ., Amsterdam, 1986, p. 466.

162. Casey M.L., Cox S.M., Beutler B., Milewich L., MacDonald P.C. Cachetin/Tumor necrosis factor-a formation in human placenta. J. Clin. Invest. 83: 430, 1989.

166. Petraglia F., Volpe A., Genazzani A.R., Rivier J. Sawchenko P.E., Vale W. Neuroendocrinology of the human placenta. Front. Neuroend. 11: 6, 1990.

163. Krieger D.T.

371