Proc. Natil. Acad. Sci. USA Vol. 86, pp. 8818-8822, November 1989 Developmental Biology Temporal expression and location of colony-stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1-regulated placental development (c-fms protooncogene/placenta/uterus) ROBERT J. ARCECI*, FRANCES SHANAHANt, E. RICHARD STANLEYt, AND JEFFREY W. POLLARDtf tDepartment of Developmental Biology and Cancer, Albert Einstein College of Medicine, 1300 Moms Park Avenue, Bronx, NY 10461; and *Pediatric Hematology/Oncology, Dana-Farber Cancer Institute and Children's Hospital, 44 Binney Street, Boston, MA 02115 Communicated by Harry Eagle, July 31, 1989 (receivedfor review March 30, 1989)

ABSTRACT During pregnancy the mouse uterine epithe- expression of CSF-1 and CSF-lR in the uterus and placenta lial synthesis of the mononuclear phagocyte growth factor throughout pregnancy. The data presented are compatible designated colony-stimulating factor 1 (CSF-1) is regulated by with a role for uterine CSF-1 in regulating both macrophage female sex steroids. To study the role of CSF-1 in the pregnant accumulation and the formation and differentiation of the female reproductive tract, the temporal expression and cellular placenta. sites of synthesis of CSF-1 and CSF-1 receptor (CSF-1R) mRNA were determined. CSF-1 mRNA, predominantly the MATERIALS AND METHODS 2.3-kilobase (kb) form, was first detected by in situ hybridiza- tion in uterine epithelium prior to implantation on day 3 and Animals and Cells. Adult female Schneider or C57BL/6 subsequently increased, reaching a peak at days 14-15. Its mice were paired with males. Appearance of a vaginal plug expression was restricted to the uterine epithelium at all stages was designated day 1 of pregnancy. Placental weights were of gestation and was not localized to areas of implantation. determined after dissecting placenta free of fetal membranes CSF-1R mRNA was first detected in maternal decidua at day and uterine tissue. The BAC1.2F5 cell line was grown as 6. It was expressed in the decidua basalis during placentation, described (13). after which its expression declined. At day 7.5, trophectoder- RNA Analysis. Paraffin sections of C57BL/6 mouse gravid mal cells also expressed CSF-1R mRNA; during placentation, uteri were subjected to in situ hybridization (14) by using it was found also in the diploid trophoblasts. The high level of [32P]CTP-labeled sense (control) or antisense (experimental) CSF-1R mRNA expression by trophoblast giant cells was RNA probes transcribed from pGEM2-MCSF53 (15) and independent of their location around the conceptus. There was p755 (16) directed against mouse CSF-1 and mouse c-fms a differential distribution of CSF-1R mRNA expression in the (CSF-1R), respectively. After autoradiography, slides were mature placenta, with expression in the giant trophoblastic stained with hematoxylin/eosin prior to photographic or layer > spongiotrophoblastic layer > labyrinthine layer until grain-count analysis. term. Yolk sac cells also expressed low levels of CSF-1R Total RNA was isolated, and 20-,tg samples were electro- mRNA. The coincidence of uterine CSF-1 mRNA expression phoresed on formaldehyde-agarose gels, blotted onto Hy- and CSF-1 synthesis with both placental growth and CSF-1R bond filters (Amersham), and hybridized as described (6). mRNA expression in decidual cells and trophoblasts strongly Blots were reprobed with aXenopus rRNA probe (pXI1O1A), implicates CSF-1 in the regulation of placental growth and and the level of 18S rRNA was used for normalization of differentiation. RNA loading and transfer. Slot-blot analysis was performed by loading serial dilutions of RNA onto nitrocellulose filters. The growth and differentiation of many cell types are regu- lated by specific polypeptide growth factors (1). The rapid proliferation of both uterine and fetal cells during implanta- RESULTS tion and in the formation ofthe placenta and extra-embryonic CSF-1 mRNA Expression. Fig. la shows the expression of membranes suggests that growth factors play a role in these uterine CSF-1 mRNA through gestation and postpartum day processes. During gestation, the uterus and placenta produce 1 in Schneider mice. Major CSF-1 mRNA species were a variety of growth factors (2). Both trophoblasts and decid- approximately 2.3 kb and 4.6 kb, with a minor species of 2.8 ual cells express receptors for some of these growth factors, kb consistently being found during the peak period ofexpres- raising the possibility of autocrine, paracrine or local, non- sion. Since the RNA contained nuclear RNA, this minor systemic stimulation (2, 3). species could be a splice precursor. The 2.3-kb mRNA We recently demonstrated that a growth factor for mono- species, representing 70-90% of the total CSF-1 mRNA, was nuclear phagocytes, colony-stimulating factor 1 (CSF-1; ref. undetectable in nongravid uterus but was strongly induced 4), is synthesized by uterine epithelium during pregnancy in during gestation. It occasionally could be detected on long the mouse (5, 6). Although macrophages accumulate to high exposures of RNA (Northern) blots on day 5 and was numbers in the pregnant uterus (7), the detection ofthe CSF-1 reproducibly present by day 7. Thereafter, it increased receptor (CSF-1R, the c-fms protooncogene product; ref. 8) steadily through day 12 ofgestation, when there was a further mRNA in human and mouse placenta (9-11) and the stimu- 8-fold stimulation to reach a peak on day 15. After day 15 it lation of the proliferation of a mouse placenta-derived cell declined until term (Figs. la and 2A), but it could still be line by CSF-1 (12) suggest another function for uterine detected 1 day postpartum (Fig. la). There was no enhance- CSF-1. In this paper we describe the timing and sites of ment of the 2.3-kb mRNA species in RNA isolated from

The publication costs of this article were defrayed in part by page charge Abbreviations: CSF-1, colony-stimulating factor 1; CSF-1R, CSF-1 payment. This article must therefore be hereby marked "advertisement" receptor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4To whom reprint requests should be addressed.

8818 Downloaded by guest on September 23, 2021 Developmental Biology: Arceci et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8819

a CSF-I C 60 100 140 I _ 80 120 185~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1*il _ 1 | of t < 2.3 100 ,,60. 18S- 80.E 40 0 N l5 51 6 7 71 8I 101 11 12 13 14 15 17 18 Pp 191 I0 DAYS OF PREGNANCY 20 II b CSF-1R 20 0 2 4 6 8 101214 If 5 7 9 11 B 51719 .m -4.5 DAY OF PREGIANCY

18U P FIG. 2. Quantitation ofCSF-1 and CSF-lR mRNAs and placental growth throughout pregnancy. (A) CSF-1 2.3-kb (n) and 4.6-kb (W) UTERUS PLACENTA mRNAs from Northern analysis. (B) CSF-1 mRNA per cell by grain N 5 6 7 9 9 10 11 13 14 16 17 18 191 B counts of autoradiographs. (C) CSF-lR 4.5-kb mRNA in uterus (A) 1 7I 8I 8NI or placenta (A), and placental weight (o). For A and C Northern blots DAYS OF PREGNANCY were quantitated by densitometry and corrected for loading and transfer; to compare between different days and exposures, values FIG. 1. Detection of CSF-1 and CSF-1R mRNA during preg- were expressed in relative terms in each individual experiment as a nancy. Representative autoradiograms ofblots oftotal uterine (a and percentage ofthe level detected on day 15 (A) or day 9 (B). Only times b) or placental (b) RNA derived from individual Schneider mice that had at least three determinations are shown except for the probed with [32P]dCTP-labeled CSF-1 (a) or CSF-1R (b) cDNA uterine CSF-1R mRNA levels (C), which are the results from the levels was insert. Although individual animal variation in mRNA single blot shown in Fig. lb. In A and C, the values shown on days were at times with similar observed, the blots repeated least four 7 and 8 are those determined for the implantation site. In B, the grains patterns. Autoradiograms were exposed for 48 hr (outside dotted per cell are the average number of grains per cell in the sections lines) or 24 hr (inside dotted lines) (a) or for 240 hr (left of the dotted ofthe dotted probed with an antisense CSF-1 cRNA probe minus the number lines), 140 hr (between the dotted lines), and 10 hr (right obtained in a section from the equivalent day of pregnancy probed lines) (b). 0, ovariectomized; N, nonpregnant, randomly cycling; I, with the sense-strand probe. At least 750 grains were counted in implantation site; NI, Nonimplantation site; B, BAC1.2F5; PP, every section. In the nonpregnant animal, the value with the an- postpartum. The left ordinate shows the position of 28S and 18S increases were rRNA; the right ordinate shows the apparent size in kb of the major tisense probe was -0.14 grains per cell. Significant mRNA bands estimated from rRNA markers. Gestation period is 19 considered to be >0.28 grains per cell. ± 1 (mean ± SD) days. CSF-1R mRNA Expression. An approximately 4.5-kb CSF- implantation sites or interimplantation sites on day 7, being 1R mRNA was detected in placenta (Fig. lb). This mRNA 0.3% of the day 15 level in each case (Fig. la). The 4.6-kb size was comparable to that reported in macrophages (16) and mRNA species, although expressed at a lower level than the was similar to that detected in the mouse macrophage cell line 2.3-kb form at all times during gestation, followed a similar BAC1.2F5 (Fig. lb). There was high CSF-lR mRNA expres- time course of expression (Figs. la and 2A). Essentially an sion in the placenta throughout pregnancy until day 17, when identical pattern was detected during gestation in C57BL/6 the-concentration decreased. There was no statistically sig- mice (data not shown). On day 15 ofgestation, the expression nificant difference in-the concentration between days 9 and of total CSF-1 mRNAs by slot-blot analysis, which detects a 15. The changing pattern of cell types in the placenta may very low level of hybridization in control tissue, was at least explain why levels were somewhat variable between animals. 125-fold greater than that in the nonpregnant uterus (data not After day 17, in at least four different animals, expression was shown). CSF-1 mRNA could not be detected in placental always lower than that during days 9 to 16. The more marked samples by Northern blotting (data not shown). variation on days 16 and 17 (Fig. lb) is probably due to To analyze the uterine expression of CSF-1 mRNA, in situ slightly different durations of gestation between individual hybridization was performed during the pre- and postimplan- mice. Fig. 2C shows that the placenta grew rapidly from day tation periods. CSF-1 mRNA expression was undetectable in 9, the first day it could be removed substantially free of nonpregnant uteri, but it increased steadily in uterine epithe- uterine contamination, through day 15, when it attained a lium from day 2 after mating, with significantly elevated constant weight until term. Northern blots also showed levels detected by day 3 ofgestation (Fig. 2B). This increased expression of CSF-lR mRNA in uterine RNA from day 6 of expression coincided with the first production of progester- pregnancy onwards (Fig. lb). A significant enrichment of this one (17), and it was noticeable that on days 3-4 the epithe- mRNA was found in samples derived from areas of implan- lium displayed a morphology characteristic of progesterone tation (3 times higher on day 7) than from the interimplan- exposure (18), with regularly aligned nuclei, subnuclear vac- tation areas (Fig. lb). uolation, and corrugated luminal cell surfaces. CSF-1 mRNA To determine which cells express CSF-lR mRNA and to expression per cell then continued to increase through the determine the pattern of expression in the placenta, in situ period of implantation to reach a peak ofexpression between hybridization was performed on gravid uteri at different days days 11 and 13 of gestation, followed by a decline on day 15 of pregnancy. On day 7.5, high levels of CSF-1R mRNA (Fig. 2B). CSF-1 mRNA at all stages of pregnancy was only expression were detected in maternal decidua (Fig. 3 e andf). detected in the luminal and glandular epithelium (Fig. 3 a-d This hybridization was highest in the region immediately and Fig. 4b). It was not confined to sites of implantation and around the implanting blastocyst and declined towards the could be detected at similar levels in both the luminal and outer decidual zone. By day 9.5 of pregnancy, expression of glandular epithelium in uterine areas well away from con- CSF-1R mRNA was detected in the decidua basalis (Fig. 5b), ceptuses (data not shown). However, prior to implantation, but by the time the mature placenta was formed (day 11 CSF-1 mRNA levels were consistently higher in the luminal onwards), little if any expression could be found in either the than in the glandular epithelia (data not shown). Interestingly, decidua capularis or basalis (Fig. 3 g and h; data not shown) when the epithelium converted from a typically columnar to as it became compressed and fibrotic (19). Significant expres- a squamous epithelium (often close to the placenta), CSF-1 sion of CSF-1R mRNA was also found on the invading mRNA expression was lost (Fig. 3 c and d). trophoblasts at day 7.5, although at the resolution of the Downloaded by guest on September 23, 2021 8820 Developmental Biology: Arceci et al. Proc. Nati. Acad. Sci. USA 86 (1989)

FIG. 3. Localization of CSF-1 and CSF-1R in the gravid uterus and placenta by in situ hybridization. Sections were hybridized with an antisense RNA probe to CSF-1 (a-d) or CSF-1R (e-h). (a and b) Transverse section of day 7.5 uterus showing the antimesometrial pole of the uterus with epithelium (E), probably glandular, sandwiched between the myometrium (M) and decidua (D) that have-been slightly torn away in this section. (c and d) Longitudinal section through a day 11 conceptus site showing the giant trophoblast layer (GC), the spongiotrophoblastic (SP) and labryinthine (L) regions of the placenta, the maternal decidua basalis (DB), and a large area of epithelium (E). (e and f) Transverse section through an implantation site at 7.5 days showing invading trophoectoderm (TE), maternal decidua (D), and myometrium (M). (g and h) Uterus and placenta at day 11 of gestation. (a, c, e, and g) Bright-field photomicrographs. (b, d, f, and h) Dark-field photomicrographs. (Bar = 0.1 mm.) dark-field photomicrographs shown (Fig. 3 e andf), this was adjacent large cells ofthe maternal decidua (at high power the not apparent because of the high level of expression on the bright areas in the dark field shown do not correspond with

a EIE ,I a~~~~~~~~~~~~~~~'

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MOM* FIG. 4. In situ localization of CSF-1 and CSF-1R mRNA in ~~ ~ day 11 pregnant uterus. (a) Area of Fig. 3g shown at high power. - b Similar section to i but hy- Of bridized with a CSF-1-specific ^*LYsSDtWt iGC !^antisenseAdz As probe.GC A:(c and d) Yolk sac (YS), which has separated s somewhat91 t^ X\duringen \V~tfixation, and Gg4Ad'*GCi*E z the adjacent giant trophoblast layer (GC) hybridized with CSF- ;i<>*3'41R antisense (c) or sense (d) I. Ax s ^ ! @l ^ As_ by X. a ^^_ probes. (Bar = 0.05 mm.) Downloaded by guest on September 23, 2021 2_.it*>@jL Developmental Biology: Arceci et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8821 sion on the giant cell layer > spongiotrophoblastic layer > labyrinthine layer and in the extraembryonic membranes, once established, persisted throughout gestation.

DISCUSSION Increased concentrations of uterine CSF-1 over nonpregnant uteri are detected within 5 days of copulation, and its concentration continues to increase dramatically through gestation (5). CSF-1 is also found in placenta, amniotic fluid, and at low concentrations in the fetus (5, 20, 21). CSF-1 mRNA is restricted to the luminal and glandular epithelium throughout pregnancy, indicating that the CSF-1 detected in the placenta and amniotic fluid is synthesized in the uterus. During pregnancy, until day 16, there is a close parallel between CSF-1 mRNA levels detected by Northern blotting and the uterine accumulation ofCSF-1. After day 16, both the CSF-1 mRNA and CSF-1R mRNA levels decline, but CSF-1 continues to accumulate. These data suggest that, although production of CSF-1 is reduced, destruction via the CSF-1R (22) is even more reduced, resulting in a steady increase in the uterine CSF-1 concentration. The peak of uterine CSF-1 mRNA per cell detected by in situ hybridization is observed on day 13, whereas the peak of uterine CSF-1 mRNA detected by Northern blotting is on day 15. The most obvious explanation for this difference is that there is an increase in the proportion of uterine columnar epithelial cells in the uterus at this time. The increased uterine CSF-1 concentrations found in the first half of pregnancy can be mimicked by treatment of ovariectomized mice with progesterone, estradiol-1713, and a decidual stimulus and was associated with an elevation in uterine CSF-1 mRNA (6). Consistent with these observa- tions, by day 3 of pregnancy, when the luminal and glandular FIG. 5. Both mural and polar giant trophoblasts express CSF-1R epithelium first appeared progestational, significantly in- mRNA. Bright-field (a and c) and dark-field (b and d) photomicro- creased epithelial CSF-1 mRNA levels could be detected by graphs of saggital sections through the mesometrial (a and b) and in situ antimesometrial poles (c and d) ofday 9.5 gravid uterus. DC, decidua hybridization. During pregnancy, the increase in capsularis, V, maternal vascular space, R; Reichert's membrane, epithelial CSF-1 mRNA level was not confined to the sites of EC; exocoelom. E' -* points to the position of the embryo. Other implantation or at later times to the areas of the conceptus. abbreviations are as in Figs. 3 and 4. (Bar = 0.1 mm.) Together these observations strongly suggest that uterine CSF-1 synthesis during gestation is under systemic control the giant cells of the trophectoderm). Once the differentiated involving ovarian sex steroids. placental elements could be identified, however, the major The cell types expressing CSF-1R mRNA change during expression of the CSF-1R mRNA was in giant trophoblasts. pregnancy. Initially, during decidualization, there is expres- This high level of expression continued on these giant cells sion by maternal decidual cells, particularly those in the until term (Fig. 3 g and h, Fig. 4 a and c, Fig. 5 a-d and data periimplantation region. These cells are unlikely to be mac- not shown). No differences could be detected between mural rophages, which are known to accumulate in the uterus and polar giant trophoblasts (Fig. 5 b and d). In some cases during pregnancy (7), because the cells expressing CSF-1R in both the developing and mature placenta, the epithelial mRNA are not macrophage-like. However, the possibility cells producing CSF-1 abutted against the giant trophoblasts that some of the cells detected in the decidual layer are expressing CSF-1R mRNA (Fig. 3 c, d, g and h and Fig. 4 a macrophages cannot be excluded. Invading trophoectoder- and b). mal cells on day 7.5 also express CSF-1R mRNA but at a CSF-1R mRNA expression could also be shown in diploid lower level than decidual cells. Transformation of the tro- trophoblasts during the formation of the placenta, when the phoblasts into giant trophoblasts is associated with a dra- spongiotrophoblastic and labyrinthine layers had not yet matic enhancement of CSF-1R mRNA expression, indepen- become established (Fig. 5 a and b). In mature placenta, dent of their location around the conceptus. Diploid spon- when the different cell layers could be easily identified, giotrophoblasts also express CSF-1R mRNA, and when the CSF-1R mRNA expression was found at high levels through- definitive layers of the placenta are formed, a lower level of out the spongiotrophoblastic layer and also at lower levels in expression can also be detected in the labyrinthine layer. the labyrinthine region (Fig. 3 g and h). At late stages of Visceral yolk sac cells and macrophages also express CSF-1R gestation, the relative increase in the proportion of the mRNA. Previous studies have shown that yolk sac- labyrinthine layer (19) with its lower level of CSF-1R mRNA conditioned medium contains CSF-1 (23). The role of uterine probably accounts for the decline in CSF-1R mRNA levels CSF-1 in the regulation of yolk sac cells is not clear. detected on Northern blots. Significant but low levels of During the preparation of this manuscript, Regenstreif and CSF-1R mRNA expression were also found in the visceral Rossant (24) communicated to us their then unpublished yolk sac on day 11 (Fig. 4 c and d) as well as in occasional results of a similar qualitative in situ hybridization study. mononuclear cells, particularly in the fetal liver (data not Their data describing the localization of the CSF-1 and shown), although a systematic study was not performed on CSF-1R mRNAs agree with those reported here. However, the fetus. No expression could be detected in the allantois or we have detected CSF-1 and CSF-1R mRNA at earlier stages amnion. This expression pattern in the placenta with expres- than they reported and have been able to quantitate the Downloaded by guest on September 23, 2021 8822 Developmental Biology: Arceci et al. Proc. Natl. Acad. Sci. USA 86 (1989) changing levels of these mRNAs. While CSF-1 mRNA has 1. Mercola, M. & Stiles, C. D. (1988) Devlelopment 102, 451-460. been detected in placental samples by Northern blotting (6, 2. Brigstock, D. R., Heap, R. B. & Brown, K. D. (1989) J. 20), both in situ studies failed to detect significant placental Reprod. Fertil. 85, 747-758. CSF-1 mRNA. This suggests that the detection of "placen- 3. Pollard, J. W. (1989) J. Reprod. Fertil., in press. tal" CSF-1 mRNA by Northern was due to uterine 4. Stanley, E. R., Guilbert, L. T., Tushinski, R. J. & Bartelmez, blotting S. H. (1983) J. Cell. Biochem. 21, 151-159. contamination of placental samples. 5. Bartocci, A., Pollard, J. W. & Stanley, E. R. (1986) J. Erp. CSF-1 has been shown to be a chemoattractant for mac- Med. 164, 956-961. rophages (25); thus, uterine CSF-1 may have a role in uterine 6. Pollard, J. W., Bartocci, A., Arceci, R., Orlofsky, A., Ladner, macrophage recruitment and/or proliferation. Uterine mac- M. B. & Stanley, E. R. (1987) Nature (London) 330, 484-486. rophages immunosuppress the host response to the semial- 7. Hunt, J. S., Manning, L. S., Mitchell, D., Selanders, J. R. & logenic fetus (26) and produce a variety of cytokines (27). Wood, G. W. (1985) J. Leuk. Biol. 38, 255-265. These cytokines may act directly on cells of the uteropla- 8. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., cental unit (27, 28) or be involved in regulatory networks (12, Look, A. T. & Stanley, E. R. (1985) Cell 41, 665-676. 28) with other blood cells localized at the 9. Muller, R., Slamon, D. J., Adamson, E. D., Tremblay, J. M., uteroplacental Muller, D., Cline, M. J. & Verma, I. M. (1983) Mol. Cell. Biol. interface. Although CSF-1 does not activate macrophages in 3, 1062-1069. a classical sense (reviewed in ref. 29), such an immunological 10. Muller, R., Verma, I. M. & Adamson, E. D. (1983) EMBO J. role for uterine CSF-1 cannot be ruled out. 2, 679-684. The expression of uterine CSF-1 mRNA and trophoblastic 11. Hoshina, M., Nishio, A., Bo, M., Boime, I. & Mochizuki, M. CSF-1R mRNA is correlated with the period of maximal (1985) Acta Obstet. Gynaecol. Jpn. 37, 2791-2798. proliferation of trophoblasts and placental growth (19), con- 12. Athanassakis, I., Bleackley, C. R., Paetkau, V., Guilbert, L., sistent with the regulation of trophoblast growth and devel- Barr, P. J. & Wegmann, T. G. (1987) J. Immunol. 138, 37-44. opment by uterine CSF-1. The CSF-1-stimulated prolifera- 13. Morgan, C. J., Pollard, J. W. & Stanley, E. R. (1987) J. Cell tion Physiol. 130, 420-427. of primary mouse placental cells and mouse placental 14. Arceci, R. J., Croop, J., Horwitz, S. & Housman, D. (1988) cell lines (12) provides further evidence supporting this Proc. Natl. Acad. Sci. USA 85, 4350-4354. regulatory role for CSF-1. Very high levels ofCSF-1R mRNA 15. Ladner, M. B., Martin. G. A., Noble, J. A., Wittman, V. P.. expression were also detected in trophoblastic giant cells, Warren, M. K., McGrogan, M. & Stanley, E. R. (1988) Proc. which are unable to proliferate but endoreduplicate DNA Natl. Acad. Sci. USA 85, 6706-6710. (30). Interestingly, maternal decidual cells, which also ex- 16. Rothwell, V. M. & Rohrschneider, L. R. (1987) Oncogene Res. press CSF-1R mRNA, similarly undergo polyploidization 1, 311-324. (31). Therefore, it is possible that endoreduplication of DNA 17. Finn, C. A. & Martin, L. (1971) J. Reprod. Fertil. 25, 299-300. in these two different cell types is regulated by CSF-1. 18. Martin, L., Finn, C. A. & Trinder, G. (1973) J. Endocrinol. 56, 303-307. However, CSF-1 probably has roles other than the stimula- 19. Davies, J. & Glasser, S. R. (1968) Acta Anat. 69, 542-608. tion of trophoblast DNA synthesis and cell proliferation 20. Azoulay, M., Webb, C. G. & Sachs, L. (1987) Mol. Cell. Biol. because CSF-1 and CSF-1R mRNA expression could still be 7, 3361-3363. detected in the placenta after it had ceased to grow. In this 21. Pollard, J. W., Arceci, R. J., Bartocci, A. & Stanley, E. R. context, it is relevant to note that CSF-1 regulates the (1989) in The Molecular and Cellular Immunobiology of the survival and differentiation of mononuclear phagocytes as Maternal Fetal Interface, eds. Wegmann, T. G., Gill, T., 1II, & well as their proliferation (4). The diverse endocrine func- -Nisbet-Brown, E. (Oxford Univ. Press, Oxford), in press. tions of giant trophoblasts (32) may also be regulated by 22. Bartocci, A., Mastrogiannis, D. S., Migliorati, G., Stockert, CSF-1, which stimulates macrophages to produce several R. J., Wolkoff, A. W. & Stanley, E. R. (1987) Proc. Natl. Acad. Sci. USA 84, 6179-6183. biologically active compounds including cytokines (33). 23. Johnson, G. R. & Burgess, A. W. (1988) J. Cell. Biol. 77, From a mechanistic standpoint, it is also interesting that 35-47. CSF-1 can be expressed in both secreted and cell surface 24. Regenstreif, L. J. & Rossant, J. (1989) Dev'. Biol. 133, 284-294. forms (34). If blastocysts express the CSF-1R in the trophec- 25. Wang, J. M., Griffin, J. D., Rambaldi, A., Chen, G. & Manto- toderm prior to implantation, cell-to-cell interaction may play vani, A. (1988) J. Immunol. 141, 575-579. a role in ovoreceptivity or trophectoderm invasion once the 26. Hunt, J. S., Manning, L. S. & Wood, G. W. (1984) Cell hln- blastocyst is correctly positioned at the antimesometrial munol. 85, 499-510. uterine lunimal epithelial surface. Similarly, the later appo- 27. Hunt, J. (1989) J. Reprod. Immunol., in press. sition between uterine epithelial cells and trophoblast giant 28. Wegmann, T. G. (1988) Immunol. Lett. 17, 297-302. a 29. Stanley, E. R. (1989) in The Regulation of Proliferation and cells could also involve CSF-1/CSF-1R-mediated cell- Differentiation in Normal and Neoplastic Cells, Proceedings of to-cell interaction. The diversity ofthe CSF-1 functions in the the 10th Annual Bristol-Myers Symposium on Cancer Re- uterus and placenta and the mechanisms of its actions remain search, 1987, ed. Frei, E., 111 (Academic, New York), in press. to be elucidated. 30. Barlow, P. W. & Sherman, M. 1. (1972) J. Emblyol. Erp. Morphol. 27, 447-465. We thank Dr. L. Rohrschneider for the mouse c-fms cDNA probe, 31. Das, R. M. & Martin, L. (1978) J. Reprod. Fertil. 53, 125-128. Dr. B. Sollner-Webb for the rRNA probe, and Dr. J. Rossant for 32. Sherman, M. 1. (1983) in Biology of Trophoblasts, eds. Loke, sharing unpublished results. This work was supported by National W. Y. & Whyte, A. (Elsevier, Amsterdam), pp. 401-465. Institutes of Health Grants, IGM38156-01 to R.J.A., CA26504 and 33. Sherr, C. J. & Stanley, E. R. (1989) in Peptide Growth Factors CA32551 to E.R.S., and HD25074-01 to J.W.P.; The Albert Einstein and their Receptors, eds. Sporn, M. B. & Roberts, A. B. Core Cancer Grant P30-CA1330 to J.W.P. and E.R.S.; the Smith (Springer, Heidelberg), in press. Kline Foundation to J.W.P; and the Lucille P. Markey Charitable 34. Rettenmier, C. W., Roussel, M. F., Ashmum, R. A., Ralph, Trust to E.R.S. P., Price, K. & Sherr, C. J. (1987) Mol. Cell. Biol. 7, 2378-2387. Downloaded by guest on September 23, 2021