Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts

DEVELOPMENTAL

BIOLOGY

116.519-523

(1986)

Fibronectin and Laminin Promote in Vitro Attachment and Outgrowth of Mouse Blastocysts D. RANDALL Department

ARMANT,~ HOWARD

A. KAPLAN, AND WILLIAM J. LENNARZ~

of Biochemistry and Molecular Biology, The University of Texas System Cancer Center, M. D. Anderson Hosp’tal and Tumor Institute, 6723 Bertner Avenue, Houstm, Texas 77030 Received November

15, 1985; accepted in revised farm February

6, 1986

The process of mammalian implantation has been investigated using an in vitro model system wherein the trophoblast cells of mouse blastocysts attach to and outgrow on tissue culture plates containing a complex medium. We now report that two extracellular matrix glycoproteins, fibronectin and laminin, when individually precoated on tissue culture plates promoted in vitro attachment and outgrowth of mouse blastocysts in serum-free medium. The kinetics of attachment and outgrowth processes in the presence of either of these two proteins were identical to that observed in complex, serum-containing medium. In contast, plates containing a collagen matrix or pretreated with a variety of other serum proteins or various lectins failed to support in vitro attachment and outgrowth of blastocysts. Because all components of the culture medium are defined and both fibronectin and laminin are known components of the basement membrane of the endometrium, this in vitro system offers considerable advantages over the serum supplemented system to study in

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iIt@IntatiOn.

0 1986 Academic

Press, Inc.

INTRODUCTION

The molecular basis of mammalian embryo implantation into the uterus is not well understood because of the complexity of the system and its inaccessibility to controlled experimentation at the biochemical level. However, it has been possible to study the development of isolated blastocysts by the use of in vitro culture systems. By the inclusion of glucose, certain amino acids and serum in the medium, blastocysts can be cultured to form trophoblast cells that attach to and spread over the culture plate, forming an outgrowth (Mintz, 1964; Gwatkin, 1966a,b; Spindle and Pederson, 1973). The processesof attachment and subsequent outgrowth in vitro are believed to be analogous to the adhesion and penetration stages of in utero development (Sherman and Atienza-Samols, 1978; Enders et ah, 1981). Our objective has been to use this in vitro model to identify the molecular components mediating attachment and outgrowth by focusing on components of the trophoblast surface. A number of laboratories have previously investigated this question. In a very early study aimed at identifying the functional components of serum, Gwatkin (1966a) found that outgrowth would occur in a defined, serum-free medium supplemented with a preparation of fetuin. Although Rizzino and Sherman (1979) later agreed that fetuin-supplemented medium i Present address: Department of Obstetrics and Gynecology, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, Mass. 02215. ’ To whom correspondence should be addressed. 519

promoted outgrowth, these authors pointed out that other proteins contaminating the fetuin preparation might be the components active in promoting attachment and outgrowth. Given that previous studies have failed to identify in an unequivocal fashion individual components involved in implantation, we have examined the ability of several serum and extracellular matrix proteins to promote mouse embryo attachment and outgrowth in vitro. The results of these studies establish that embryos cultured in a relatively simple medium undergo attachment and outgrowth on tissue culture plates precoated with either fibronectin or laminin. In view of the fact that fibronectin and laminin have been found to be present in the basement membrane of the endometrium (Wartiovaara et al. (1979); Leivo et al. (1980); Grinnell et al. (1982)) and that both have been shown to play a role in cell adhesion in vitro (for reviews see Yamada, 1983; Kleinman et al., 1984), it seems likely that these two proteins are involved in embryo attachment and invasion in utero. MATERIALS

AND METHODS

Materials. CF-1 male and female mice were purchased from Charles River Breeding Laboratories, Kingston, New York. Pregnant mare serum gonadotropin was from Diosynth, Chicago, Illinois. CMRL 1066 medium, Hanks’ balanced salt solution, fetal calf serum (FCS), and fetuin were obtained from Gibco Laboratories, Grand Island, New York; Limulus polyphemus agglutinin and Lotus tetragonolobus lectin were from EY Laboratories, Malvern, Pennsylvania. Ultroser G, a synthetic serum sub0012-1606/86 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

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stitute composed of undisclosed bovine serum proteins, growth factors and nutrients, was purchased from LKB Instruments, Gaithersburg, Maryland. Laminin used in this study was obtained from Dr. R. B. Runyan, Department of Biochemistry and Molecular Biology, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas, and gave identical results with laminin purchased from either EY Laboratories (San Mateo, Calif.) or Bethesda Research Laboratories (Gaithersburg, Md.). A highly purified preparation of human plasma fibronectin was a generous gift from Dr. L. B. Chen, Dana Farber Institute, Boston, Massachusetts. All other chemicals used were purchased from Sigma Chemical Co., St. Louis, Missouri, or were of the highest grade commercially available. Embryos and outgrowth culture. Embryos were obtained as previously described (Armant et al., 1986). Blastocysts were cultured on tissue culture plates (Falcon 3001) under mineral oil (Aldrich) in CMRL 1066 medium supplemented with 2.24 mg/ml sodium lactate and 0.06 mg/ml sodium pyruvate, as well as penicillin and streptomycin. Protein was provided in this medium by bovine serum albumin (BSA) (4 mg/ml), FCS (lO%)or Ultroser G (lO%),as described in Results. Embryos were cultured at 37°C in a 5% C02/95% air atmosphere for 2-3 days to obtain trophoblast outgrowths. Following outgrowth, embryos were fixed with 2% paraformaldehyde, rinsed with Hanks’ balanced salt solution and stained with eosin-methylene blue to enhance contrast for photomicrography. Zona-free expanded blastocysts were obtained mechanically by drawing them in and out of a micropipet that had a diameter slightly smaller than that of the embryo.

VOLUME 116. 1986

Pretreatment of tissue culture plates. Samples used for pretreating were prepared at various concentrations in Hanks’ balanced salt solution and placed as individual drops on a platic tissue culture dish. Drops were then covered with mineral oil. After 4 hr at 37”C, the precoating solution was removed and the precoated spot on which the drop had been applied was washed 10 times with Hanks’ balanced salt solution. Finally, culture medium containing BSA was placed on the spot and preequilibrated in the incubator for 2 hr before embryos were introduced. Precoating of plates with collagen gels was done as previously described (Elsdale and Bard, 1972). Quantitative assessment of attachment and outgrowth. Embryo attachment to the substratum was quantified by the method of Sherman and Atienza-Samols (1978), and embryo outgrowth was morphologically identified by light microscopy. RESULTS

Attachment and outgrowth in vitro of mouse blastocysts occurred routinely when embryos were cultured in a complete medium, i.e., CMRL 1066 supplemented with FCS or a commercial serum substitute (Ultroser G) that is composed primarily of bovine serum proteins. Boiling completely eliminated all outgrowth-promoting activity of FCS or Ultroser G. To test the possibility that these medium supplements might contain fibronectin or other proteins that functioned as attachment factor(s), tissue culture plates were pretreated with either FCS or Ultroser G. As shown in Fig. 1, such pretreament with FCS (Fig. 1B) or Ultroser G (Fig. 1C) resulted in normal

FIG. 1. Photomicrographs of blastocysts cultured for 3 days on plates precoated with 4 mg/ml BSA (A), 10% FCS (B), or 10% Ultroser G (C). No outgrowth is apparent in (A), whereas extensive outgrowth is evident in (B) and (C). Embryos were paraformaldehyde-fixed and then stained with eosin-methylene blue to enhance constrast. The bar indicates 100 pm.

ARMANT, KAPLAN, AND LENNARZ

outgrowth when embryos were subsequently cultured in CMRL 1066 medium containing only BSA. As expected, pretreating plates with BSA followed by cultivation in CMRL 1066 medium containing only BSA did not support attachment and outgrowth of embryos (Fig. 1A). The attachment- and outgrowth-promoting factor(s) in Ultroser G was further characterized by enzymatic digestion and gel filtration chromatography. When dishes pretreated with Ultroser G were preincubated with 10 pg/ml trypsin at 22°C for 30 min and then extensively washed, subsequent attachment and outgrowth of blastocysts did not occur. Fractionation of Ultroser G by Sephacryl S-200 chromatography revealed that the excluded fractions (M, > 250,000) contained the majority of the attachment- and outgrowth-promoting activity (data not shown). This result, coupled with the observations on trypsin sensitivity, is consistent with the possibility that the essential factor(s) mediating trophoblast attachment and outgrowth was a high molecular weight protein(s) such as fibronectin. To test the idea that fibronectin might function in mouse embryo attachment and outgrowth, tissue culture plates were pretreated with a highly purified preparation of fibronectin. As shown in Fig. 2A, trophoblast cells outgrew well in medium containing only BSA as a macromolecular component when the plates were pretreated with 25 pg/ml fibronectin. Like fibronectin (Wartiovaara et al, 1979; Grinnell et al, 1982), laminin is also found in the basement membrane of the endometrium (Leivo et al., 1980). When laminin was tested in the in vitro system, it was also found to promote attachment and outgrowth (Fig. 2B). Pretreament of plates with con-

Mouse Trophoblmt Outgrowth

521

centrations of fibronectin or laminin at or above 25 pg/ ml were found to yield consistent results for attachment and outgrowth, i.e., greater than 95% of the embryos both attached and outgrew. In addition, trophoblastic outgrowths obtained on fibronectin and laminin substrata were morphologically indistinguishable from those obtained using FCS (cf. Figs. 1 and 2). Collagen is also present in the uterine extracellular matrix, but gel matrices of types I, III, or IV collagens failed to support in vitro attachment and outgrowth of blastocysts. Plates pretreated with various serum proteins, i.e., fetuin, albumin, transferrin, immunoglobulin G, al-acid glycoprotein, or trophoblast binding lectins, i.e., wheat germ agglutinin, concanavalin A, Limulus polyphemus agglutinin, and Lotus tetragcrnolobus lectin, also failed to support in vitro attachment and outgrowth of blastocysts. However, we cannot exclude the unlikely possibility that these proteins failed to promote embryo attachment and outgrowth because they did not adhere to the tissue culture plate. The ability of blastocysts to attach and outgrow in medium supplemented with serum has been shown to be developmentally regulated and not a mere consequence of the loss of the zona pellucida (Jenkinson and Wilson, 1973; Sherman and Atienza-Samols, 1978). In fact, Sherman and Atienza-Samols (1978) have shown that the kinetics of attachment are similar for enzymatically dezonaed and spontaneously hatched blastocysts. Therefore, it was of interest to determine whether trophoblast cells of zona-free blastocysts cultured on plates precoated with the various components discussed above exhibited similar kinetics of attachment and out-

FIG. 2. Photomicrographs of blastocysts cultured for 3 days in CMRL 1066 containing 4 mg/ml BSA on plates precoated with 25 rg/ml of either fibronectin (A) or laminin (B) showing outgrowth. Embryos were paraformaldehyde-fixed and then stained with eosin-methylene blue to enhance contrast. The bar indicates 100 pm.

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VOLUME 116.1986

growth. The results shown in Fig. 3 indicate that when using plates precoated with fibronectin (Fig. 3B) or laminin (Fig. 3C), the attachment of zona-free blastocysts occurred over the same developmental period observed with zona-free embryos cultured in FCS on untreated plates (Fig. 3A).

temporal differentiation of the trophectoderm into trophoblast. During in utero implantation, the trophoblast and uterine epithelium are initially in contact through extensive interdigitation of their microvilli (Potts, 1968). This clearly cannot occur in the in vitro outgrowth model system. Indeed, coculture of blastocysts and uterine epDISCUSSION ithelial cells results in a clearing of the epithelial cells These studies have established that fibronectin, a in the vicinity of the embryo, presumably due to withknown cell attachment molecule (Yamada, 1983), very drawal of these cells immediately after contacting the effectively promotes attachment of blastocysts and out- trophoblast (Glass et aZ.,1979). However, in this context growth of the trophoblast cells in vitro. Fibronectin is it should be noted that in mice or other species where found not only in serum but as a component of the base- implantation is either partially or completely interstitial, the uterine epithelial cells detach from the underment membrane of the endometrium and interstitial matrix of the uterine stroma (Wartiovaara et al., 1979; lying basement membrane during decidualization and the trophoblasts become imbedded in the underlying Grinnell et aZ., 1982). Laminin, another glycoprotein stroma (Enders et al, 1981; Chavez, 1984). In view of present in the basement membrane of the endometrium this, it is most likely that trophoblast outgrowth in vitro (Leivo et al, 1980) was also found to provide a suitable is a model for the invasive phase of implantation wherein substratum for in vitro attachment and outgrowth of trophoblast cells attach to and degrade the fibronectinmouse blastocysts. As in the in vitro system using meand laminin-containing basement membrane that undium supplemented with serum, and as observed during derlies the epithelium and pervades the stroma. in utero implantation, attachment, and outgrowth on Both fibronectin and laminin interact with a variety either fibronectin or laminin coated plates required the of molecules in the extracellular matrix, such as collagens and glycosaminoglycans (Yamada, 1983; Kleinman A et al, 1984). In view of this fact, one possible mechanism for temporal control of the onset of attachment competence as the trophectoderm differentiates into trophoblast involves the synthesis and secretion of molecules capable of linking the cell surface of the trophoblasts to either fibronectin or laminin bound to the tissue culture plate. Alternatively, attachment and/or out6 growth could involve a developmentally regulated receptor for fibronectin and/or laminin. Plasma membrane receptors on cultured cells have been reported for both fibronectin (Pytela et aZ.,1985; Brown and Juliano, 1985) and laminin (Rao et al, 1983; Malinoff and Wicha, 1983; Lesot et al., 1983). Indeed, it was recently reported that -Jr an antiserum raised against a group of glycoproteins, C including one of 140 kDa, prevented the attachment of mouse blastocysts cultured in the presence of serum (Richa et aL, 1985); perhaps this antiserum acts by binding to the cell surface receptor for fibronectin. Clearly, based on these observations and the results in the cur--&lf! rent study, the possibility that receptors for fibronectin 0 20 40 60 and laminin are involved in the developmentally reguDevelopmental Time lated acquisition of the attachment competency of the (hr in culture) blastocyst cell surface warrants further study. FIG. 3. Kinetics of attachment and outgrowth of zone-free blastocysts

-AC

on nonprecoated or precoated plates. In (A) attachment (0) and outgrowth (0) are shown using plates that were not precoated with FCS, although the latter was present in the culture medium. In (B) and (C) the plates were precoated with fibronectin or laminin, respectively. No FCS was present in the culture medium. The in vitro developmental time is shown beginning with blastocysts flushed from the uterus on the fourth day of pregnancy (90 hr post-hCG). Each point represents the mean of 4 analyses; reproducibility was within flO%.

We are indebted to Mr. Glenn Decker for expert preparation of photomicrographs. We appreciate the technical assistance of Mss. J. Greer, M. Rivero, H. Park, and F. Tan and the editorial assistance of Ms. D. Welch. We are also grateful to Dr. R. B. Runyan, Dr. E. Ruoslahti, MS. J. L. Roe, Dr. N. Ruin-Bravo, Dr. D. D. Carson, and Dr. B. D. Shur for helpful discussions. This work was supported by grants from the National Institutes of Health (HD 18600) and The March of Dimes Foun-

ARMANT, KAPLAN, AND LENNARZ dation (MOD l-867) to W.J.L. and a National Research Service Award GM-07965 and a grant from the National Institutes of Health (HD 19977) to D.R.A. Dr. William J. Lennarz, who is a Robert A. Welch Professor of Chemistry, gratefully acknowledges the Robert A. Welch Foundation.

REFERENCES ARMANT, D. R., KAPLAN, H. A., and LENNARZ, W. J. (1986). N-Linked glycoprotein biosynthesis in the developing mouse embryo. Dev. BioL 113,228-237. BROWN, P. J., and JULIANO, R. L. (1985). Selective inhibition of fibronectin-mediated cell adhesion by monoclonal antibodies to a cellsurface glycoprotein. Science 228,1448-1451. CHAVEZ, D. J. (1984). Cellular aspects of implantation. In “Ultrastructure of Reproduction” (J. van Blerkom and P. M. Motta, eds.), pp. 247-259. Nijhoff, Netherlands. ELSDALE, T., and BARD, J. (1972). Collagen substrata for studies on cell behavior. J. Cell. BioL 54, 626-637. ENDERS, A. C., CHAVEZ, D. J., AND SCHLAFKE, S. (1981). Comparison of implantation in utero and in vitro. In “Cellular and Molecular Aspects of Implantation” (S. R. Glasser and D. W. Bullock, eds.), pp. 365-382. Plenum, New York. GLASS, R. H., SPINDLE, A. I., and PEDERSEN, R. A. (1979). Mouse embryo attachment to substratum and interaction of trophoblast with cultured cells. J. Exp. ZooL 208, 327-335. GRINNELL, F., HEAD, J. R., and HOFFPAUIR, J. (1982). Fibronectin and cell shape in viva: Studies on the endometrium during pregnancy. J. Cell Biol. 94,597-606. GWATKIN, R. B. L. (1966a). Defined media and the development of mammalian eggs in vitro. Ann. N. Y. Acd Sci 139,79-90. GWATKIN, R. B. L. (1966b). Amino acid requirements for attachment and outgrowth of the mouse blastocyst in vitro. J. Cell PhysioL 68, 335-344. JENKINSON, E. J., and WILSON, I. B. (1973). In vitro studies on the control of trophoblast outgrowth in the mouse. J. EmZnyol. Exp. Morphol. 30,21-30. KLEINMAN, H. K., MCGARVEY, M. L., HASSELL, J. R., MARTIN, G. R., VAN EVERCOOREN, and DUBOIS-DALCQ, M. (1984). The role of laminin in basement membranes and in the growth, adhesion and differ-

Mouse

Trophoblast

Outgrowth

523

entiation of cells. In “The Role of Extracellular Matrix in Development” (R. L. Trelstad, ed.), pp. 123-143. Liss, New York. LEIVO, I., VAHERI, A., TIMPL, R., and WARTIOVAARA, (1980). Appearance and distribution of collagens and laminin in the early mouse embryo. Dev. Biol. 76,100-114. LESOT, H., KUHL, U., and VON DER MARK, K. (1983). Isolation of a laminin-binding protein from muscle cell membranes. EMBOJ. 2,861865. MALINOFF, H. L., and WICHA, M. S. (1983). Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells. J. Cell Biol 96,1475-1479. MINTZ, B. (1964). Formation of genetically mosaic embryos and early development of lethal (t12/t’2)-normal mosaics. J. Exp. Zoo1 157,273292. POTTS, D. M. (1968). The ultrastructure of implantation in the mouse. J. Anat. 103,77-90. PYTELA, R., PIERSCHBACHER, M. D., and RUOSLAHTI, E. (1985). Identification and isolation of a 140 kDa cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 40,191-198. RAO, N. C., BARSKY, S. H., TERRANOVA, V.P., and LIOTI‘A, L. A. (1983). Isolation of a tumor cell laminin receptor. B&hem. Biophys. Res. Commun. 111,804-808. RICHA, J., DAMSKY, C. H., BUCK, C. A., KNOWLES, B. B., and SOLTER, D. (1985). Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development. Dev. BioL 108,513-521. RIZZINO, A., and SHERMAN, M. I. (1979). Development and differentiation of mouse blastocysts in serum-free medium. Exp. Cell Res. 121,221233. SHERMAN, M. I., and ATIENZA-SAMOLS, S. B. (1978). In vitro studies on the surface adhesiveness of mouse blastocysts. In “Human Fertilization” (H. Ludwig and P. Tauber, eds.), pp. 179-183. PGS, Boston, Mass. SPINDLE, A. I., and PEDERSON, R. A. (1973). Hatching, attaching and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements. J. Exp. 2001. 186,305-318. WARTIOVAARA, J., LEIVO, I., and VAHERI, A. (1979). Expression of the cell-surface associated glycoprotein, fibronectin, in the early mouse embryo. Dev. BioL 69,247-257. YAMADA, K. M. (1983). Cell surface interactions with extracellular materials. Annu. Rev. B&hem 52, 761-799.