Expression of Opioid Receptors and Ligands in Pregnant Mouse Uterus and Placenta¹

^a Department of Neuroscience&Cell Biology, University of Medicine&Dentistry of New Jersey - Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

	ABSTRACT

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The endogenous opioid system has been implicated in the regulation of hormonal secretion, pain perception, and uterine contractility during pregnancy, but there is only limited information about the cellular location of opioid receptor and opioid peptide gene expression in the pregnant rodent uterus and placenta. In this study, we have used in situ hybridization to identify expression sites of mRNAs encoding the delta (), kappa (), and mu (µ) opioid receptors as well as the endogenous opioid peptide precursors proenkephalin (PENK), prodynorphin (PDYN), and proopiomelanocortin (POMC) in pregnant mouse uterus and placenta. Soon after implantation, all three opioid receptor genes as well as POMC and PENK, but not PDYN, were detected in the uterine environment. Each expressed gene exhibited a distinct expression pattern that was generally retained until late gestation. The receptor and POMC were coexpressed in the trophoblast giant cells, which remained the only cells of the placenta/uterus to express these two genes throughout gestation. Cells expressing receptors were absent from the placenta but instead were found in the basal part of the decidualized uterine endometrium. While and µ receptors were transiently expressed in the uterine myometrium (until embryonic day 8.5), substantial levels of PENK were continuously detected in this region until at least embryonic day 18. In addition, complementary expression of the µ receptor and PENK genes in the uterus was detected. Taken together, these results suggest multiple roles for the opioid receptors and opioid peptides in maternal adaptation to pregnancy and in supporting embryo growth.

	INTRODUCTION

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Opiate drugs such as morphine affect the perception of pain, consciousness, motor control, and autonomic function by interacting with specific membrane opioid receptors located throughout the central and peripheral nervous systems. On the basis of radioligand binding and pharmacological experiments, opioid receptors were classically divided into three types: the delta (), kappa (), and mu (µ) opioid receptors. The endogenous ligands of these opioid receptors include more than 20 opioid peptides derived from three precursors: proenkephalin (PENK), prodynorphin (PDYN), and proopiomelanocortin (POMC) [1]. While numerous studies have been conducted to elucidate the functions of the opioid system in analgesia and other physiological functions, relatively little is known about the role of the opioid system during pregnancy.

The mammalian embryo cannot develop without the placenta. During pregnancy, initial developmental decisions set aside three unique extraembryonic lineages that contain precursors of placental cells: the trophoblast, the extraembryonic endoderm, and the extraembryonic mesoderm [2]. During development, the trophoblast attaches the embryo to the uterus (implantation) and forms vascular connections necessary for nutrient transport. In addition, the placenta optimizes maternal endocrine, immune, and metabolic contributions to embryo development [2]. Previous studies have established that at least some opioid receptor and peptides are present in human placenta. For example, a homogeneous population of only opioid receptors was detected in the human placenta [3]. Opioid peptides have also been identified, and shown to be synthesized, in human placental villus tissue. These include ß-endorphin [4], met-enkephalin [5], and multiple dynorphins: 1–11, 1–13 [6], 1–7, 1–8, 1–12, and 1–17 [7]. The physiological role of the opioid receptors and peptides in the placenta, an organ lacking innervation, is not fully understood. Different in vitro systems have been used to show that placental opioid receptors regulate the secretion of acetylcholine [8] and hCG [9] and the release of human placental lactogen [3] from trophoblast tissue.

The pregnant rodent uterus is a dynamic tissue that undergoes morphological and physiological changes in order to accommodate, protect, and nurture the developing embryo. The presence in rat uterus of mRNA and peptide products derived from the opioid peptide precursors POMC [10], PENK [10], and PDYN [11], as well as opioid receptor binding sites [12], has been reported. Both PENK gene expression [10] and opioid receptor binding sites [12] change during rat pregnancy, leading to the suggestion that the opioid system is involved in maternal adaptation to pregnancy and uterine motility in many species including the human. For example, an enhanced threshold to pain has been described during human pregnancy [13]. In pregnant women, plasma concentrations of ß-endorphin rise throughout pregnancy [14] and reach peak values during labor [15, 16]. This increase in opioid peptide concentration during labor correlates in a linear fashion with the number and density of uterine contractions [16]. These data suggest that the opioids could be involved not only in the modulation of analgesia during pregnancy but also in the regulation of uterine motility.

The cloning of the , µ, and opioid receptors [17] has provided a unique opportunity to undertake a comprehensive spatial and temporal analysis of the opioid receptor gene expression in the uterus and placenta. In an attempt to correlate expression sites of the opioid receptors with their endogenous ligands, the present study has elucidated mRNA expression of the µ, , and opioid receptors and their endogenous ligand precursors POMC, PENK, and PDYN in the mouse uterus and placenta in sequential pregnancy stages using in situ hybridization.

	MATERIALS AND METHODS

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Tissue Preparation

All studies were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Four uteroplacentas were examined on each of embryonic days 3.5 (e3.5), e7.5, e8.5, e9.5, and e10.5, and two were examined on each of the other days studied (e12.5. e14.5, e16, and e18). Timed-pregnant C57Bl/6J mice were killed by decapitation. Early postimplantation conceptuses were staged according to the paper published by Muntener and Hsu [18], and others (e10.5–e18) by examining embryonic limb morphology based on the method of Wanek et al. [19]. Whole uteri, including the entire embryonic extraembryonic and uterine area in which the embryo resides, were freshly frozen and embedded in Tissue-Tek II OCT embedding compound (Baxter Scientific, McGaw Park, IL) for cryostat sectioning. Sections were cut with a cryostat as described previously, mounted on slides coated with TESPA (Pierce Chemical Company, Rockford, IL), and stored at -70°C until use in the hybridization procedure.

Probes

[³³P]UTP-labeled single-stranded RNA probes were synthesized and purified in vitro from the plasmid vectors harboring the appropriate cDNA sequences. Transcription templates were selected to ensure that hybridizations distinguished distinct expression patterns of the three opioid receptors without cross-hybridization. For the opioid receptor cRNA probe, plasmid DK1B containing the whole mouse cDNA sequence of DOR-1 (a gift from Dr. Christopher Evans, University of California-Los Angeles) was cut with Sac I and transcribed with T3 RNA polymerase to produce a 630-basepair (bp) antisense cRNA probe corresponding to nucleotide (nt) 1206–1835. The same plasmid was linearized with Bgl II, transcribed with T7 RNA polymerase to make a sense probe (669 bp, corresponding to nt 1–668 of the cDNA). For the µ opioid receptor, a 346-bp polymerase chain reaction (PCR) fragment (generated from the 5' primer: aaa gcg cct ccg tgt act tc, and the 3' primer: g ctc aac ttg tcc cac gtt gat) corresponding to nt -237 to 108 of the mouse µ receptor cDNA (a gift from Dr. Christopher Evans) was subcloned into pCRII vector using the TA Cloning kit from Invitrogen (San Diego, CA). To make an antisense probe, the vector was linearized with HindIII (at the 5' end of the insert) and then transcribed with T7 RNA polymerase. To produce a sense probe, the vector was linearized with Xho I (at the 3' end of the insert) and transcribed with SP6 RNA polymerase. For the opioid receptor, a 376-bp Pst I-EcoRI fragment of msl-1 corresponding to nt 172 to 548 of the mouse receptor cDNA was subcloned into pGEM-3Z (entire plasmid was a gift from Dr. Graeme I. Bell, University of Chicago). The receptor antisense probe synthesis involves linearizing with HindIII at the 5' end of the insert and transcribing with T7 RNA polymerase. The sense probe synthesis involves linearizing with EcoRI at the 3' end of the insert and transcribing with SP6 polymerase. The PENK RNA probe was synthesized from a plasmid pSP65, which contains a 520-bp Pst I fragment of rat PENK cDNA corresponding to C-terminal region of proenkephalin transcripts [20]. The POMC probe was synthesized from p10D, which contains a 220-bp rat POMC cDNA (exon 1 and exon 2) [21]. The PDYN probe was synthesized from rat PDYN cDNA (corresponding to nt 292–1900), which was kindly provided by Dr. James Douglas (Vollum Institute, Portland, OR).

In Situ Hybridization

In situ hybridization was performed according to the protocols described [22]. Autoradiography was carried out at 4°C using a 1:1 dilution of Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) for 4 wk. In all cases, hybridization with control (sense) RNA in adjacent sections yielded only low background.

	RESULTS

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In this study, we identified sites of mRNA expression for the , , and µ opioid receptors and their endogenous peptide ligands in both extraembryonic cells of the fetus and the maternal uterus. No mRNA expression for any of the opioid receptor or peptide genes was detected in the uterus at e3.5, before implantation of the embryo (data not shown). After implantation, however, the three opioid receptors as well as POMC and PENK were all detected in the uterine environment, but each had a distinct expression pattern that was generally retained until late gestation.

Receptor and POMC Were Both Expressed in the Trophoblast Giant Cells (Fig. 1)

At all ages examined after implantation, the trophoblast giant cells were the only cells expressing receptor and POMC mRNAs. Giant cells are large, trophoblast-derived cells that are associated with the mammalian placenta. Soon after implantation, the giant cells cease mitosis but endoreduplicate their DNA and thus contain more than the diploid quantity (up to 1024 times the haploid value) of DNA [23]. The giant cells are formed from both mural trophectoderm (trophoblast cells surrounding the blastocoel cavity) and polar trophectoderm (trophoblast cells that are in contact with the inner cell mass) [24]. At e7.5, the earliest stage examined after implantation, receptor mRNA was detected in trophoblast giant cells (Fig. 1A). The expression was detected in only one or two giant cells on each section at this stage, suggesting that expression probably begins near that time. Interestingly, POMC expression was also detected in the giant cells at this age (Fig. 1E). During early development, both receptor and POMC transcripts were confined to the mural trophoblast giant cell layer (Fig. 1, B, C, F, and G). Later, both transcripts were abundant in the giant cells of the developing placenta at e10.5 and e12.5 (Fig. 1, D, H, I, and M) and the mature placenta at e14.5 and e16 (Fig. 1, J, K, N, and O). Expression of the receptor and POMC in the giant cells continued until at least e18 (Fig. 1, L and P). At the time of peak expression (e9.5–e10.5), hybridization of both and POMC probes was detected in at least 75% of the giant cells on near-adjacent sections.

View larger version (116K): [in this window] [in a new window]   FIG. 1. Receptors and POMC were both expressed in trophoblast giant cells. All panels are high-power (x200 magnification) epipolarization/bright field (EP/BF) photographs except C, G, L, and P, which are low-power (x80) darkfield (DF) photographs. POMC and receptors were expressed in trophoblast giant cells (arrowheads) on adjacent sections at multiple ages (e7.5, e8.5, e9.5, e10.5, e12.5, e14.5, e16, and e18) throughout gestation. Note expression of the receptor (C) and POMC (G) in mural trophoblast giant cells (m) but not polar trophoblast giant cells (p) at e9.5. db, Decidual basalis; em, embryo; pl, placenta.

Receptor and PENK Were Expressed in the Decidual Basalis (Fig. 2)

The uterus is a dynamic organ comprising two muscular layers surrounding a highly vascularized steroid-sensitive endometrium [25]. Blastocyst attachment stimulates the uterine stroma to form the spongy mass of cells known as the decidua [26]. Numerous growth factors such as insulin-like growth factors [27] and proteases such as PACE4 [28] are expressed in this tissue. At e7.5, receptor mRNA, while not expressed in the fetal cells, was instead detected in the basal part of the decidualized uterine endometrium, e.g., the decidual basalis, as it first formed (Fig. 2E). receptors continued to be expressed by the decidual basalis until at least e18 (Fig. 2, F–K). Expression of receptor mRNA in the myometrium was also apparent (Fig. 2F) and is discussed below.

View larger version (116K): [in this window] [in a new window]   FIG. 2. Receptors and PENK were expressed in the decidual basalis, but not in the placenta. Low-power (x80) DF photographs show PENK and receptor hybridization over the decidual basalis (db) at various ages. A) The expression of PENK (arrows) in the glycogen-rich region (g) of the decidual basalis at e7.5. As decidualization proceeded at e8.5 (B), decidual PENK expression levels decreased and the expression was restricted to a more lateral region close to the myometrium (arrows). At e9.5 (C), transcription of PENK was limited to a sparse number of decidual cells (arrows). At e10.5 (D), this expression pattern (arrows) remained the same. Transcription of the receptors also began at time of initial formation of the decidual basalis (E), and continued until late gestation (K). em, Embryo; pl, placenta.

By e7.5, high levels of PENK were expressed in the glycogen-rich region of the decidual basalis (Fig. 2A). As decidualization proceeded (e.g., e8.5), decidual PENK levels decreased, and the expression was restricted to a more lateral region close to the myometrium (Fig. 2B). Interestingly, by e9.5, when the decidua is fully developed [29], transcription of PENK was limited to a sparse number of decidual cells (Fig. 2C). At e10.5, this expression pattern remained the same (Fig. 2D). The intense expression of PENK in the myometrium (Fig. 2, A–D) is described in detail below.

, µ, and PENK Were Expressed in the Myometrium and Adjacent Regions of the Uterus (Fig. 3)

At e7.5, both (Fig. 3A) and µ (Fig. 3B) receptor mRNAs were detected in a few scattered nondecidualized endometrial stroma cells adjacent to the inner circular layer of the myometrium. PENK was also detected in this region, in addition to the inner circular layer of the myometrium, at much higher levels (Fig. 3C).

View larger version (132K): [in this window] [in a new window]   FIG. 3. Expression of and µ receptors in the uterus overlapped with PENK expression. A, B, E, F) High-power (x200) EP/BF photographs; C, D, G, H) EP/BF video micrographs (x400). At e7.5, a few scattered stromal cells expressing (A, arrows) and µ (B, arrows) receptors were located in the nondecidualized endometrium (en) adjacent to the inner circular layer of the myometrium (cm). At the same age, high levels of PENK expression (C, arrow) were detected in the same region, although the expression also covered part of the circular layer of the myometrium. At e8.5, (E, arrow) and µ (F, arrow) receptor transcriptions were primarily detected in the circular layer of the myometrium, while the PENK expression pattern (G, arrow) remained the same as that of e7.5. At e9.5 (D, arrow) and e10.5 (H, arrow), the expression of PENK was detected principally in the circular muscle layer and some cells in the longitudinal muscle layer (lm).

By e8.5, (Fig. 3E) and µ (Fig. 3F) receptor transcriptions occurred closer to the myometrium and were principally detected in the circular layer of the myometrium instead of the endometrium, as well as in some scattered cells in the longitudinal layer. Compared to the previous age, both and µ receptor expression levels increased at e8.5. At this age, the expression pattern of PENK remained the same as at the previous age, e.g., expression covered the inner circular muscle layer and adjacent endometrium (Fig. 3G).

Interestingly, at e9.5, the uterine expression of both and µ receptors suddenly decreased and became almost undetectable (data not shown). However, PENK continued to be expressed by the myometrium. At e9.5 (Fig. 3D) and e10.5 (Fig. 3H), PENK expression was mainly detected in the circular muscle layer, as well as in some cells in the longitudinal muscle layer.

Complementary Expression Pattern of µ Receptor and PENK in the Uterus (Fig. 4)

At both e12.5 (Fig.4, B and D) and e14.5 (Fig. 4F), substantial levels of PENK mRNA were expressed in both the circular layer and the longitudinal layer of the myometrium, as well as in nondecidualized endometrium stromal cells between the myometrium and uterine luminal epithelium, but not in the uterine luminal epithelium, which is the major secretory component of the rodent uterus. Interestingly, cells expressing µ receptor mRNA were restricted to the uterine luminal epithelium (Fig. 4, A, C, and E), the only site expressing µ receptors at these ages. This complementary expression pattern of the µ receptor and PENK continued through late gestation (data not shown).

View larger version (122K): [in this window] [in a new window]   FIG. 4. Complementary expression pattern of the µ receptor and PENK in the uterus. A, B, E, F) Low-power (x80) DF photographs; C, D) high-power (x400) EP/BF video micrographs corresponding to the regions outlined in A and B, respectively. In A, B, E, and F, the uterine wall is inverted away from the placenta (pl). At e12.5, while the µ receptor was expressed in the uterine luminal epithelium (ule) (A and C), PENK was located in both longitudinal muscle layer (lm) and circular muscle layer (cm), as well as the nondecidualized endometrium (en) between myometrium and uterine luminal epithelium, but not in the uterine luminal epithelium (B and D). At e14.5, the complementary expression pattern of the µ receptor (E) and PENK (F) remained.

	DISCUSSION

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In this study, we used in situ hybridization to examine mouse uteroplacental mRNA expression of the , , µ opioid receptors, as well as the POMC, PENK, PDYN peptide ligands through pregnancy. The results clearly indicate that expression of the opioid system in these tissues of the mouse begins soon after implantation and continues until at least late gestation. No direct localization of proteins encoded by these genes has been made in the present study, but these observations nonetheless serve to identify likely sites of synthesis for receptor binding sites previously identified in the pregnant rodent uterus [12]. The uteroplacental expression of opioid system components in the mouse is clearly different from that of human. For example, the receptor, rather than the receptor [3], was the only opioid receptor that could be detected in the mouse placenta, and its expression was restricted to the trophoblast giant cell, which interestingly was also the only site of uteroplacental POMC expression during pregnancy. The identification of POMC-expressing cells in this tissue extends previous findings showing that POMC mRNA was present in rodent placenta [30]. A second clear difference between the human and the mouse is that expression of PDYN, which encodes some of the opioid peptides present in human placenta [6, 7], could not be detected in either the uterus or placenta in the mouse at any age, although its expression in several regions of the embryo was readily detected. Several examples of possible autocrine/paracrine loops of opioid peptide and receptor interaction were also identified, with the possible functional significance of opioid system expression briefly discussed in the following paragraphs.

Opioid System Components in Trophoblast Function

Trophoblast maturation is essential for both implantation and postimplantation development. The expression of the receptor and POMC in the trophoblast giant cells suggests that these components of the opioid system may regulate at least some trophoblast-specific functions. One major function of the trophoblast giant cells is phagocytosis of degenerating decidual cells to create the space slowly occupied by the growing embryo [31]. Previous study of a somewhat analogous system has shown that macrophages migrate rapidly and specifically to sites of cell death in the nervous system, presumably by following chemotactic factors [32]. It has been suggested that opioids constitute the chemotactic factors that initiate this specific macrophage response, which is thought to be mediated by receptors [33]. Therefore, the expression of the receptor in the trophoblast giant cells seen here suggests that the receptor may participate in phagocytic activity of the giant cells towards dead or dying decidual cells.

Other aspects of trophoblast physiology may also be regulated by the opioid system. For example, expression of the receptor and POMC in the giant cells provides a molecular correlate for the finding that the opioid peptides modulate hormone release from trophoblast tissue [3, 9]. In addition, expression of POMC adds to several previously identified hormones and peptides present in this cell type. These include progesterone, which is known to be produced and secreted by the trophoblast giant cells in rodents [34], as well as peptides of the prolactin growth hormone family, including two placental lactogens and proliferin [35–37]. Moreover, at peak times of expression, and POMC hybridization was seen in at least 75% of trophoblast cells. At these stages, it seems likely that POMC and receptor are coexpressed, which suggests that the opioid system may act at least partially by an autocrine mechanism, although direct coexpression was not established by our studies.

Finally, an additional function of the giant cell opioid components is suggested by the location of these cells at the interface between the embryo and the uterus. Giant cell processes extend to blood vessels outside the primary decidua, penetrate between the endothelial cells, and ultimately replace some of these endothelial cells [38]. Thus, receptors expressed in the giant cells may potentially bind and prevent action of maternally derived opioid peptides that could otherwise enter the fetal circulation.

Uterine Growth and Contractibility

Opioid system expression is collectively observed in both endometrial and myometrial regions of the uterus. The uterus accommodates the products of conception by progressive growth from early pregnancy to its end. Both hypertrophy and hyperplasia take place during pregnancy and contribute to uterine enlargement [25]. Since opioid antagonists, opioid peptides, and opiate drugs have all been shown to affect cell proliferation and differentiation in different regions of the nervous system [39, 40], the expression reported here of the opioid system in the uterus suggests possible involvement in growth of the uterus, as well as possible analgesic functions.

In mice, decidualization begins soon after implantation, at around e5, and reaches its maximum level by e7.5 [29]. During decidualization, stromal cells of uterus endometrium proliferate, increase in size, establish numerous tight junctional complexes with their neighbors, and eventually differentiate into decidual cells [26]. Subsequent growth of the decidua results from hypertrophy and proliferation of existing decidual cells as well as by addition of newly transformed decidual cells at the decidual periphery. The expression of receptors in the decidual basalis throughout gestation suggests that this receptor class in particular may contribute to development and maintenance of the decidua. More interestingly, high levels of PENK were expressed in the glycogen-rich region of the decidual basalis at e7.5, the peak time of decidualization. By e8.5, the expression was already reduced and confined to a more lateral region close to the myometrium, and by e9.5, when decidualization is essentially completed [29], PENK expression remained in only scattered cells in the decidual basalis. Since the direction of decidual formation is from the lumen, where the embryo implants, towards the myometrium [29], both the spatial and temporal changes of the PENK expression pattern suggest a prospective role of the enkephalin-derived peptides in decidualization.

Like the endometrium, the myometrium also undergoes a program of growth during pregnancy, with hypertrophy of the smooth muscle being a major factor in enlargement of the uterus [25]. Our results show significant PENK expression as early as e7.5 in the mouse myometrium and indicate that this expression continued through midgestation, suggesting the enkephalins may be involved in the regulation of hypertrophy of the uterine smooth muscle. The transient expression of both and µ receptors in the myometrium and adjacent regions, as well as the complementary expression of the µ receptor in the uterine luminal epithelium, suggests that the action of the enkephalins may be mediated by local expression of the opioid receptors.

Finally, in addition to the growth of the uterus, the opioid system may also affect uterine contractility during pregnancy. Met-enkephalin concentrations increase during pregnancy and labor in the hypothalamus and pituitary of pregnant rats [41] as well as in human placenta during labor [5]. Multiple opioid receptors as well as enkephalinase are present in pregnant rat uterus [12, 42], and the results here show receptor expression in smooth muscle cells themselves. Several studies have been conducted to determine whether opioids might regulate myometrial motility. One recent study shows that both Met-enkephalin and enkephalinase inhibitors stimulate the duration and, to a lesser extent, the amplitude of spontaneous uterine contractions in rats [43]. The result is in accord with the action of enkephalin upon contractions of rat uteri at metestrus [44]. Our findings showing endogenous PENK expression in the myometrium and adjacent regions throughout gestation, as well as the expression of cognate receptors, support the notion that the endogenous enkephalins may play a role in modulating myometrial activity during pregnancy.

Taken together, our results suggest multiple roles for the opioid receptors and opioid peptides in both maternal adaptation to pregnancy and in supporting embryonic and fetal growth. In addition, several potential autocrine or paracrine loops of opioid action have been identified. These observations suggest that genetic approaches will reveal required roles for at least some opioid system components in uteroplacental biology.

	ACKNOWLEDGMENTS

 
We are grateful to Joseph Cerro for helpful discussions on uterine anatomy and to Ming-Sing Hsu for expert technical assistance. We are also pleased to thank Dr. Christopher J. Evans for providing the and µ receptor cDNA clones and Dr. Graeme I. Bell for providing the receptor probe.

	FOOTNOTES

 
¹ This work was supported by research grant DA-09040 from the National Institute on Drug Abuse to J.E.P.

² Correspondence: John E. Pintar, Department of Neuroscience&Cell Biology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, CABM 326, 679 Hoes Lane, Piscataway, NJ 08854. FAX: 732 235 4990; pintar{at}mbcl.rutgers.edu

Accepted: June 10, 1998.

Received: April 8, 1998.

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