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.