Immunolocalization of collagen type I and laminin in the uterus on days 5 to 8 of embryo implantation in the rat.код для вставкиСкачать
THE ANATOMICAL RECORD 237%-20 (1993) Immunolocalization of Collagen Type I and Laminin in the Uterus on Days 5 to 8 of Embryo Implantation in the Rat DAWN E. CLARK, PETER R. HURST, IAN S. McLENNAN, AND DON B. MYERS Department of Anatomy and Structural Biology, University of Otugo, Dunedin, New Zealand ABSTRACT This study investigated the immunohistochemical distribution of collagen type I and laminin during remodeling of the uterine extracellular matrix in response to embryo implantation in the rat. Collagen type I was found to be virtually absent around the embryo on the evening of day 5 of pregnancy. On days 6 to 8 of pregnancy the areas of primary and secondary decidualized tissue contained very little collagen in contrast to the outer nondecidualized stroma and myometrial tissues in which the staining patterns did not appear to alter. Day 8 of pregnancy was also notable for the appearance of collagen type I at the site of the developing placenta. Localization of laminin corresponded to areas of basement membrane and was associated with the redistribution of blood vasculature during implantation. By day 7 laminin staining was diminished in the basal areas of the lumenal epithelium around the implanting embryo. Laminin was also located in a punctate fashion at the margins of the primary decidual cells on day 6 of pregnancy, but by days 7 and 8 this staining pattern was no longer evident. This study has provided further evidence for a decline in a major fibrillar collagen during natural decidualization and also revealed a localized and transient expression of laminin in association with the differentiation of cells during primary decidual formation. 0 1993 Wiley-Liss, Inc. Key words: Implantation, Rat, Uterine remodeling, Collagen, Laminin Embryo implantation in the rat is marked by the transformation of certain stromal cell populations to form uterine decidual cells (Bell, 1983; Vladimirsky et al., 1977). The resultant decidual tissue 'is a transitory tissue and its formation is both rapid and extensive. Embryo implantation in the colony of rats examined was first evident on the evening of day 5 of pregnancy with i.v. Pontamine Blue locating the sites of implantation along the uterine horns. The process of implantation is marked by the development of primary and secondary decidual zones (Enders and Schlafke, 1967; Welsh and Enders, 1991a,b). The primary decidual zone surrounds the lumenal epithelium and is characterised by large cells, some of which display polyploidy, glycogen granules, and an abundance of microfilaments. These cells progressively adhere to one another and display junctional complexes (Parr and Parr, 1986). The collagen family of proteins is an important part of most animal extracellular matrices. At present there are 14 defined collagens (Van Der Rest and Garrone, 1991) with type I being the predominant connective tissue component in vertebrates (Miller and Steffen, 1987). Collagen type I is a large fibrillar protein with structural supportive properties and most commonly found in a heterotrimeric form comprised of two al(1) chains and one a2(I) chain (Miller and Steffen, 1987). Changes in the distribution of the fibrillar compo0 1993 WILEY-LISS, INC nents of the extracellular matrix have been noted in morphological studies of the rat uterus during pregnancy (Enders and Schlafke, 1967; Fainstat, 1963; Welsh and Enders, 1991a). Aniline blue staining suggested the absence of collagen bundles throughout decidual tissue while Wilder's reticulin staining revealed the presence of argyrophilic non-interlacing fragments in the equivalent of day 7 secondary decidual tissue (Fainstat, 1963). Collagen fibrils within the closely packed primary decidual zone take on a mesometriall antimesometrial alignment (Enders and Schlafke, 1967; Parr and Parr, 1986) and tend to be associated with either blood vessels or the subepithelial region; the most notable feature of this zone is, however, the relative absence of collagen fibrils. A TEM study (Welsh and Enders, 1991a) has shown a region rich in collagen fibrils lining the mesometrial chamber on day 8 of pregnancy. These fibrils are found associated with the decidual cells closest to the lumen. Biochemical analysis of total collagen (% collageddry weight tissue) in the rat uterus has revealed that overall collagen Received November 17, 1992; accepted May 6, 1993. Address reprint requests to Dr. Peter R. Hurst, Department of Anatomy and Structural Biology, University of Otago, Box 913, Dunedin, New Zealand. COLLAGEN I AND LAMININ DURING IMPLANTATION concentration in implantation sites is lower than in non-implantation sites on each day of pregnancy (Myers et al., 1990). Furthermore, on days 6-8 of pregnancy, dissected decidual tissue was found to contain very little collagen when compared with non-implantation sites (Clark et al., 1992). It has also been reported that collagen type VI, an important fibrillar collagen, is lost during the decidualization process of embryo implantation (Mulholland et al., 1992). Laminin is a glycoprotein with a MW of 800 kDa consisting of three polypeptide chains comprising A(400 kDa) and B1/B2 (200-220 kDa) chains, on reducing SDS gels. The multi domain nature of laminin has been recently reviewed (Beck et al., 1990).Laminin has been localized within the basement membranes of non-pregnant uterus (Karkavelas et al., 1988). Laminin is known to have a punctate association with mesenchymal cells during development in the kidney, lung, and small intestine (Ekblom, 1989; Schuger et al., 1991; Simon-Assmann et al., 1990). TEM has revealed the presence of an amorphous intercellular substance at the sites of interdigitation between primary decidual zone cells on day 6 (Enders and Schlafke, 1967) and day 7 of pregnancy (Parr and Parr, 1986; Tung et al., 1986). This substance appears to be basement membrane-like. Glasser et al. (1987) examined the distribution of laminin in decidual and deciduomal tissue (artificially stimulated). Deciduomal tissue revealed a matrix of laminin, not evident in the basal nondecidualized stromal tissue. Decidual cells on day 9 of natural pregnancy (day 1 equals the day after mating) appeared to have a pericellular matrix of laminin. The expression of laminin in relation to the implanting embryo of early natural pregnancy has not been studied. Morphological assessment of the lumenal basement membrane revealed that by day 7 of pregnancy it was disrupted in the region of the implanting embryo and also beneath the mesometrial chamber epithelium (Welsh and Enders, 1991b); it appears that the decidual cells are important in this process (Schlafke et al., 1985). Laminin first appears within the mouse embryo a t the 8-cell stage and later in development is localized to the parietal yolk sac membrane known as Reichert’s membrane. Within the uterine tissue, decidual cells of day 8 pregnant mice were shown to be surrounded by laminin (Wu et al., 1983). The artificially induced deciduoma of day 7 pregnant mice also showed immunoreactivity for laminin (Wewer et al., 19861, however it was not clear whether that was also the case for the decidual cells in close proximity to the embryo. A recent description (Farrar and Carson, 1992) of A, B1, and B2 mRNA chain expression during implantation in the mouse revealed extensive and differential expression of the laminin chains. The protein localization of laminin did not, however, exhibit the expected intensity in relation to the in situ hybridization. This suggests either post translational removal or proteolytic degradation within the extracellular matrix. Immunohistochemical procedures make it possible to investigate the distribution of significant extracellular matrix proteins. Collagen type I and laminin were considered likely to undergo important changes during the uterine response to implantation. Here we report the 9 results of an immunohistochemical study of the distribution of collagen type I and laminin in the sites of implantation over days 5-8 of pregnancy in the rat. MATERIALS AND METHODS Animals Wistar rats maintained on a regular 12 hour day112 hour night cycle were mated on the evening of proestrus. Animals had vaginal smears taken on the following morning and the presence of spermatozoa signified day 1 of pregnancy. Animal procedures were approved by the Committee on Ethics in the Care and Use of Laboratory Animals of the University of Otago. Antibodies and Controls The polyclonal antibodies used were a rabbit anti-rat collagen type I from skin (Chemicon, Temecula, CA; Catalogue No. AB755) and a rabbit anti-mouse laminin from EHS sarcoma (Gibco, Grand Island, NY; Catalogue No. 680-3019). The proteins used for testing the antibodies were collagen type I (Sigma Chemical Co., St. Louis, MO) prepared from rat tail collagen and laminin (Bethesda Research Laboratories, Gaithersburg, MD) from EHS mouse sarcoma. Collagen type I11 protein (Sigma) and collagen type IV protein (Sigma) were from human placenta. Sections of rat kidney were used as positive controls. Heat inactivated rabbit serum, rabbit IgG, and an antibody to myelin basic protein were used as negative controls. lrnmunoabsorption Laminin and collagen type I antibodies were absorbed against their respective proteins. Immunoabsorption was conducted in eppendorfs coated with silicone (Coatasil, Ajax Chemicals, Auburn, Australia). Small pieces of immobilon (5 mm2) were incubated in either the laminin or collagen type I protein or an equivalent concentration of BSA control; the protein was present at a concentration in excess of the immobilon’s binding capacity. Additional blocking of the immobilon with BSA was conducted before it was incubated in the collagen type I or laminin antibody. The immobilon was removed and the remaining solution serially diluted and used immediately for either immunohistochemistry or slotblotting. A slot blot was conducted (Schleicher and Schuell Minifold I1 apparatus) to confirm that the absorption of the laminin antibody had been successful. Laminin protein was loaded onto nitrocellulose in duplicates of 100, 50, and 10 ng of protein. These were then reacted with the laminin antibody, absorbed antibody, BSA absorbed antibody, or in the absence of the primary antibody. Blots were conducted as outlined in Bio-Rad (Richmond, CAI instructions and detected using a biotin-streptavidin-horseradish peroxidase system and developed with 3,3-diamino benzidine (Sigma) using the method outlined by Amersham (Arlington Heights, IL; product information). Gel Electrophoresis and lrnrnunoblotting The most common contaminant of laminin is collagen type IV. Immunoblotting of the laminin antibody against collagen type IV and laminin was conducted. The collagen type I antibody was tested against collagens I11 and IV. Gels were run in a mini gel system 10 D.E. CLARK ET AL. (Mini-Protean I1 cell, Bio-Rad) with a 3% stacking and a 6% separating polyacrylamide gel according to Miller and Rhodes (1982). Electrophoresis was conducted a t 50 V for 30 minutes or until the bromophenol blue entered the separating gel a t which time the gel was reduced in situ for 30 minutes with 2-mercaptoethanol (BDH).Electrophoresis was continued a t 60 V until the bromophenol blue ran off the gel. The proteins were transferred onto Immobilon-P (Millipore, Bedford, MA) at 15 V for 65 minutes using a Transblot Semi-Dry Transfer Cell (Bio-Rad). Membranes were blocked a t 37°C for 90 minutes and incubated in the primary antibody for 2 hours at room temperature followed by the biotin-streptavidin-horseradishperoxidase system and development with 3,3-diamino benzidine with nickel enhancement (Frigo et al., 1991). Tissue Preparation Animals were anaesthetized with Nembutal (60 mgkg intraperitoneal) at 10 P.M. on the evening of day 5 and between 10.30 A.M. and 11.30 A.M. on days 6-8 of pregnancy. Pontamine Blue (0.5 ml) was intravenously injected and after 20 minutes the animals were perfused via the abdominal aorta with PBS (Phosphate Buffered Saline) containing heparin, followed by 4% paraformaldehyde in PBS. The uterus was removed and sliced into implantation and non-implantation sites, as determined by the Pontamine Blue localization within the edematous implantation sites. Tissue blocks were further fixed in 4% formaldehyde/lM sucrose (1.5 hours) and briefly washed in PBS/lM sucrose before being placed in Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, IN) and frozen in liquid nitrogen cooled isopentane. Frozen tissue was stored at -70°C. lmrnunohistochemistry Uterine tissue blocks were warmed to -26°C and transverse 5 pm cryostat sections obtained through the embryo. Sections were air dried onto ATS (3-aminopropyltriethoxyxlane) coated slides for 2 hours and left overnight at 37°C. For immunohistochemical detection the sections were washed in PBS. Collagen type I sections were treated for 30 minutes a t room temperature with hyaluronidase (1 mg/ml in 0.1 M sodium acetate buffer, pH 5.5) and laminin sections were treated for 5 minutes with Proteinase K (Sigma, 20 mg/ml in 50 mM Tris with 5 mM EDTA at pH 7.0). Sections were subsequently washed in PBS with 0.1 M glycine. Incubation in PBS containing 1%BSA/3% goat serum (heat inactivated) for 30 minutes was followed by an overnight incubation with primary antibody a t 4°C. Antibodies to collagen type I and laminin were used at a dilution of 1:400 and 1:1,500, respectively. Sections were then washed for 1 hour with PBS supplemented with 1%non-fat milk powder and 0.1% Tween-20. Sections studied for the distribution of collagen type I were treated with the biotinylated anti-rabbit IgG (Amersham) for 1hour a t room temperature. Washing for 30 minutes (PBS, milk powder, Tween-20) was followed by a 10 minute treatment with 0.3% H20, in methanol to destroy endogenous peroxidases. After washing in PBS the sections were incubated with streptavidin-HRP (Amersham) for 1hour a t room tem- perature. Sections were washed in PBS for 30 minutes before being developed with AEC (3-amino-9-ethylcarbazole) for 4 minutes. After washing with dH20, sections were coverslipped with 90% glycerol in PBS and photographed with Pan F 50 using a green filter. Laminin sections were incubated in the dark with an anti-rabbit FITC (Silenus, Hawthorn, Australia), washed for 30 minutes, and coverslipped with an antifadent containing p-phenylenediamine. Photographs were taken on Ektachrome 400 ASA film a t 800 ASA. After photography the coverslips were removed by soaking in acetone for 30 minutes and sections stained with haematoxylin and eosin for morphological comparison. RESULTS Testing of the collagen type I antibody was undertaken to ensure its specificity. Absorbed collagen type I antibody on tissue sections gave no staining while those sections treated with the absorption control (BSA) antibody showed strong immunoreactivity. An immunoblot revealed specific staining of the 01 and p chains of collagen type I and no immunoreactivity with collagen types I11 and IV. Results from the controls conducted during immunohistochemistry were as follows. The antibody to myelin basic protein was used as a negative control and no staining was observed. Heat-treated rabbit serum gave only light intracellular staining at comparable dilutions to the collagen antibody. The IgG controls as represented by Figure 6 revealed some light intracellular background and suggested that some localization in the region of the epithelial basement membrane may be due to the binding of IgG. This IgG staining receded mesometrially as implantation proceeded. The embryo was located in apposition to the antimesometrial lumenal epithelial cells on the evening of day 5 (Fig. 1)of pregnancy. Immunohistochemical localization revealed low levels of collagen type I in the subepithelial tissue around the embryo. This area correlates with the region in which decidualization has been initiated. In the antimesometrial nondecidualized stroma there is a matrix rich in collagen type I. The embryo appears to have several spots of immunoreactivity, when stained with the collagen type I antibody, which are likely to be non-specific and were not seen in the day 6 embryo. Also with respect to this, Leivo et al. (1980) has shown that in the mouse collagen type I is not present until day 7 (a 3-4 somite embryo) and that very early mouse embryos are adhesive as shown by their affinity for collagen type 11. Immunolocalization in the region of the lumenal basement membrane is more difficult to interpret as the IgG controls revealed that there was background localization in this region (Fig. 6). Considering the intensity of the staining and its removal after immunoabsorption it is probable that there is some collagen type I in this region. The tissue surrounding the upper quarter (mesometrial) of the lumen has a moderate distribution of collagen type I (Fig. 1). By the morning of day 6 (Fig. 2) the primary decidual region is essentially devoid of collagen type I with the exception of the lumenal basement membrane region which stained for collagen type I. The region with min- COLLAGEN I AND LAMININ DURING IMPLANTATION Figs. 1-4. Immunolocalizationof collagen type I in transverse sections through the embryo. In all photographs the mesentry is at the top. D, decidualized tissue; N, nondecidualizedtissue; P, primary decidual zone; S, secondary decidual zone; arrowhead, embryo. 11 Fig. 1 . On the evening of day 5 (D5) of pregnancy the embryo is located antimesometrially in the lumen. The subepithelial zone has decidualized while the outer tissue is nondecidualized. x 85. Fig. 2. Day 6 (D6) showing the region of the primary and secondary decidual zones. x 85. imal amounts of collagen type I corresponds to the differentiating tissue of the secondary decidual zone. This region extends around the mesometrial aspect of the uterine lumen and incorporates an area of marked edema antimesometrially. The outer region with a rich matrix of collagen type I correlates with the nondecidualized stromal tissue of the basal zone. On day 7 (Figs. 3,4) the demarcation between the decidualized tissue and the nondecidualized stromal tissue is marked with regard to the distribution of the collagen type I. The primary decidual zone was devoid of collagen and the region of the secondary decidual tissue contained only minimal amounts of collagen type I. Immunoreactivity was evident aligning the epithelial basement membrane and was most marked a t the mesometrial end (Fig. 4). On day 8 (Fig. 5A) of pregnancy the primary decidual zone consisted of an area located centrally in the tissue and a canal extending into the antimesometrial tissue; both of these areas were devoid of collagen type I. The secondary decidual tissue, including the mesometrial glycogen wings (Bell, 1983; Krehbiel, 1937), contained only moderate amounts of collagen type I (Fig. 5B). The mesometrial placental region surrounding the uterine lumen appeared to have more type I collagen than on day 7. Testing of the laminin antibody was conducted to ensure its specificity. Immunoabsorption with laminin protein was undertaken and the resulting solutions tested on a slotblot (Fig. 7) and tissue sections. In both cases the absorbed laminin antibody showed an absence of immunoreactivity while the control (BSA) absorption showed intense staining. Immunoblotting confirmed that the laminin antibody recognized the laminin chains but did not crossreact with collagen type IV (Fig. 8). Although there remained the possibility that the laminin antibody crossreacted with fibronectin, this seems improbable as initial immunolocalization studies with fibronectin revealed a markedly different tissue distribution than that seen with laminin. The IgG binding seen during AEC immunolocalization was not evident when IgG controls of 1:1,000 were used with FITC detection. In the kidney sections the laminin antibody was located in the basement membrane regions. On the evening of day 5 of pregnancy (Fig. 9) laminin was localized in the basement membranes of the lumenal epithelium and numerous stromal blood vessels. Small blood vessels were also evident in the decidualizing tissue close to the embryo. Several glands were also present within the nondecidualized stroma. The epithelial cells located antimesometrially to the embryo were distinctive even a t this early stage of implantation, and were retained until day 8. 12 D.E. CLARK ET AL. By day 6 (Fig. 10) the embryo was expressing laminin in the region of the parietal endodermal basement membrane, later to be called Reichert’s membrane. The scattered blood vessels of the secondary decidual zone had abundant laminin in their basement membranes. Laminin was found to localize, in a punctate fashion, to the margins of cells in the primary decidual zone. This punctate localization was not seen elsewhere in the uterus. Laminin of pregnancy was present underlying the epithelial cells in the region of the uterine lumen but not along the implantation chamber on day 7 (Fig. 11). A thin band of primary decidual zone cells aligning the implantation chamber contained no recognisable blood vessels and no detectable laminin. Blood vessels in the region of the uterine lumen tended to orientate out from the lumen while in the region of the implantation chamber took on a parallel alignment. On day 8 of pregnancy (Fig. 12) the embryo was located centrally within the uterus. Antimesometrially to the embryo was an area of epithelium (Fig. 12) Fig. 3. Immunolocalization of collagen type I on day 7 (D7) of pregnancy. ~ 2 5 . Fig. 3. Fig. 4A.B. Higher magnification of collagen type I distribution on day 7 and morphology of the same section showing the primary and secondary decidual zones around the embryo. x 85. Figs. 4A,B. COLLAGEN I AND LAMININ DURING IMPLANTATION 13 Figs. 5-6. Collagen type I on day 8 of pregnancy and IgG control. P, primary decidual zone; S, secondary decidual zone; arrowhead, embryo; asterisk, mesometrial placental region; arrow, basement membrane region. Fig. 5A,B. Day 8 (D8) of pregnancy with the embryo and developing mesometrial placental region (asterisk). A primary decidual zone is visible extending antimesometrially (A). The secondary decidual zone contains scattered collagen type I. X 25. B is the region of insert from A showing the sparse collagen of the secondary decidual zone. x 85. which was devoid of laminin in the region of its basement membrane (not shown). The embryo (Fig. 13)was closely apposed to the decidual cells. The embryo’s basement membrane was observed to consist of Reichert’s membrane which aligned the parietal endodermal cells, and the visceral endodermal basement membrane surrounding the egg cylinder. The primary decidual zone region had no detectable laminin while the numerous vessels of the secondary decidual zone were lightly stained. The uterine lumenal epithelium (Fig. 14) was retained on day 8 and had a small amount of associated laminin. Laminin localized strongly to the vessels of this region as well as to the more mesometrial vessels (not shown). As on day 7, the implantation chamber (Fig. 14) was devoid of epithelial basement membrane laminin and now also of epithelial cells. Vessels along the implantation chamber and in the region of the developing venous sinusoids (in a wing out from the lumen, in the region of the upper implantation chamber) had little if any detectable laminin. Just outside the boundary of the decidual tissue (Fig. 15)’ laminin was found to be expressed in a punctate manner within the stromal tissue. This staining pattern ran around the antimesometrial decidual tissue. This punctate expression can also be viewed in Figure 16A just below the developing capsule. Antimesometrially to the embryo and retained epithelial (Fig. 12) a chamber of primary decidual-like cells extends towards the capsule (Fig. 16). This chamber was devoid of laminin even though it contained small blood vessels. Conversely blood vessels outside this chamber had a laminin containing basement membrane (Fig. 16). Smooth muscle cells, seen a t the bottom of Figure 16, had a pericellular basement membrane. DISCUSSION This study has found that the removal of the fibrillar collagen type I from around the embryo was closely related to the progressive uterine decidualization of implantation. This removal was evident on the evening of day 5 and raises the possibility that matrix changes may play an inductive role in regulating cellular phenotype (Bissell et al., 1982) and consequently in the process of decidualization (Aplin, 1989). Conversely, transformed cells have been associated with reduced expression of collagen (Kleinman et al., 1981) and thus 14 D.E. CLARK ET AL. 200 97b 66 M C L CB LB Fig. 8. Western gels and immunoblotting of laminin and collagen type IV after in situ reduction. Molecular weight markers (M). The Coomassie stain of collagen IV (C) shows the upper a1 (IV)chain at 140 kDa and the a 2 (IV) chain a t 100 kDa. The Coomassie stain of laminin (L) reveals the A chain (400kDa) and B chains (-200 kDa). There are additional bands at -75 kDa and 200 kDa which are unlikely to be collagen type IV and which were not detected with the laminin antibody (lane CB). Immunoblotting the laminin antibody with collagen IV (CB) and laminin (LB) proteins reveals the lack of crossreactivity with collagen IV. Fig. 6. IgG (1:400dilution) control on day 6 of pregnancy. There is light intracellular staining and some IgG localization in the basement membrane region (arrow). X 85. 1986; Tung et al., 1986) protecting the embryo from the mother’s immune response. If such a concept is correct the removal of the fibrillar collagen type I from throughout the decidua should allow closer cellular adhesion. The differing distribution within the primary and secondary decidual tissue may reflect either the 100 degree of decidualization within each area or distinct functional roles. The absence of collagen type I in the decidual tissue during implantation may allow for the necessary re50 modeling needed for establishment of the decidual tissue and the subsequent placentation. On day 8 of pregnancy some collagen I was apparent within the secondary decidual zone and this probably signifies a slight increase within this region. Most notable on day 10 8 was the appearance of collagen type I in the mesometrial placental region. Although collagen type I has been shown to be capable of rapidly stimulating vascu2 4 1 lar tube formation in vitro (Jackson and Jenkins, 1991) its role in the formation of the placental vessels is Fig. 7. Slotblot of immunoabsorbed laminin. Numbers down the left represent the amount in nanograms of laminin protein loaded to each likely to be limited because of a restricted distribution duplicate slot. Lane 1, laminin antibody; lane 2, laminin antibody up until day 8 of pregnancy. Collagen type I is, howabsorbed against laminin protein; lane 3, control (BSA) for absorp- ever, likely to be important for support of the placental tion; lane 4, no laminin antibody on slotblot. vessels which will begin functioning around day 8. In this study immunoreactivity aligning the uterine epithelial cells is somewhat confusing because of the IgG control localization to this region. Morphological evidence (Welsh and Enders, 1991a,b) and the relative the decidualizing cells may, as a result of differentia- density of the collagen immunolocalization would, however, support the supposition that there is some tion, alter their collagen metabolism. The primary decidual zone has been described as collagen type I in this region. The punctate expression of laminin, only on day 6, forming an immunological barrier (Pam and Pan-, 3 COLLAGEN I AND LAMININ DURING IMPLANTATION Figs. 9-1 0. Immunolocalization of laminin (9AJOA) with the same sections stained for morphology (9BJOB) on day 5 (D5) and day 6 (D6) of pregnancy. Sections were taken transversely through the embryo with the mesentry orientated to the top of the page. D, decidualised tissue; N, nondecidualized tissue; arrows, primary decidual zone; S, secondary decidual zone; arrowhead, embryo. X 175. 15 the basement membranes of the uterine epithelial cells, glands, and blood vessels. Fig. 10A.B. On D6 of pregnancy laminin is found within the embryo. The lumenal basement membrane and blood vessels are labelled for laminin. A punctate localization of laminin was found at the margins of the primary decidual cells. Fig. 9A.B. The embryo on the evening of D5 of pregnancy is located antimesometrially within the uterine lumen. Laminin is localized to may correlate with the TEM reports of an amorphous intercellular substance dispersed around only the primary decidual zone cells on days 6 and 7 of pregnancy (Enders and Schlafke, 1967; Parr and Parr, 1986; Tung et al., 1986). The absence of laminin staining on day 7 of pregnancy in our study may relate to differences between animal colonies or the timing of experiments. The possibility also exists that the antigenicity of the transient laminin alters before final removal on day 7. TEM immunolocalization would be one way of confirming whether the amorphous substance is completely, or in part, laminin. Punctate expression of laminin has been reported within embryonic tissues such as the mesenchymal cells in the subepithelial region of the developing small intestine (Simon-Assmann et al., 1990) and lung (Schuger et al., 1991) and is associated with the proliferation of epithelial cells during organogenesis. The transient expression of punctate laminin during kidney development is associated with the conversion of mesenchyme to epithelium (Ekblom, 1989). This process is marked by progressive adhesion of mesenchyma1 cells to form tissue condensations. This coincides with the expression of the laminin B chain and the disappearance of collagen types I and I11 from the condensations. Implantation results in the production of 16 D.E. CLARK ET AL. Fig. 11. Immunolocalization of laminin (A) and the same section stained for morphology (B).Laminin is present beneath the epithelial cells of the uterine lumen but not the implantation chamber on day 7 (D7) of pregnancy. The embryo and its associated basement mem- brane have been slightly disrupted during processing. Arrowhead, embryo; arrows, primary decidual zone; UL, region of the uterine lumen; IC, implantation chamber. x 175. the primary decidual cells which are closely packed, exclude collagen I, and have a transient expression of laminin. While not forming an epithelium, the epithelioid-like primary decidual zone does form a type of barrier or membrane around the embryo (Damjanov, 1985; Parr and Parr, 1986). The primary decidual zone alignment along the uterine epithelium also strongly suggests some form of epithelial cell interaction or induction. The possibility thus exists that similar mechanisms exist between these systems. The punctate appearance of laminin in the antimesometrial stromal tissue on day 8 is also unusual (Figs. 15,16). It does, however, lie at the extremity of the decidualizing tissue and a t the position of the future COLLAGEN I AND LAMININ DURING IMPLANTATION 17 Fig. 12.Haematoxylin and eosin stained section of the day 8 (DB) implantation site shown in Figures 13-16. Inserts are for Figures 15 and 16.A small triangle of retained epithelium is visible antimesometrially to the embryo. Arrowhead, embryo; E, epithelium. x 22. antimesometrial lumen (Welsh and Enders, 1983). The function of the day 8 antimesometrial primary decidual-like cell extension, which was devoid of both laminin and collagen type I, remains unclear (Fig. 16). Changes to the epithelial and blood vessel basement membranes on days 7 and 8 of pregnancy are probably related to the development of the placenta. The successful maintenance of pregnancy requires the establishment of a nutrient supply via a yolk sac placenta which begins to function on day 8 to 9 of pregnancy and later via a chorioallantoic placenta (Christofferson and Nilsson, 1989; Welsh and Enders, 1991b).The placenta has an arterial supply via the mesenteric triangle that has the same orientation as the implantation chamber and which communicates with the subepithelial capillary plexus a t the most mesometrial aspect of the uterine lumen. This correlates on day 8 with the immunolocalization of collagen type I in the mesometrial placental region, along with intense laminin staining in the vessels close to the uterine lumen. The mesometrial sinusoids radiating out from the mesometrial aspect of the implantation chamber act as a venous system (Christofferson and Nilsson, 1989; Takemori et al., 1984; Welsh and Enders, 1991b). It has been reported that establishment of the mesometrial venous sinusoids involves changes to or removal of the basement membrane structures (Bell, 1983; Welsh and Enders, 1991b). Fig. 13.Immunolocalization of laminin (A) with the same section stained for morphology (B) on day 8 of pregnancy. Within the embryo Reichert’s membrane of the parietal endodermal cells is continuous with the basement membrane of the visceral endodermal cells found around the growing egg cylinder. The primary decidual zone forms a n area around the embryo in which there is no laminin. Within the secondary decidual zone laminin is found in association with the numerous small blood vessels. Arrows, primary decidual zone; S, secondary decidual zone; R, Reichert’s membrane; VE, visceral endoderm. x 175. This study has demonstrated that the extracellular matrix proteins collagen type I and laminin have altered spatial distributions that are indicative of significant changes during uterine remodeling in response to implantation and early pregnancy. ACKNOWLEDGMENTS We would like to thank Rochelle Gibbs for her technical assistance. Dawn Clark is a Postgraduate Scholar and Don Myers a Senior Research Fellow supported by the Health Research Council of New Zealand. The work was supported by a project grant from the Health Research Council of New Zealand. 18 D.E. CLARK ET AL. Fig. 14. Laminin immunolocalization (A) and tissue morphology (B) on day 8. Underlying the epithelial cells (E)in the region of the uterine lumen a small amount of laminin remains and laminin localizes strongly to the blood vessels in this region. Along the lumen of the implantation chamber no laminin is found. Vessels (V) found aligning the implantation chamber have very little laminin. The top of the embryo is just visible (arrowhead). E, epithelium; V, vessels; UL, region of the uterine lumen; IC, implantation chamber; arrowhead, embryo. x 175. COLLAGEN I AND LAMININ DURING IMPLANTATION Figs. 15,16. Laminin immunolocalization (15A,16A) and corresponding morphology (15B,16B) on day 8. These regions are shown by the inserts in Figure 12. Arrows, primary decidual-like zone; 0,punctate laminin; C, capsule. x 175. 19 Fig. 15A.B. This is part of a band of stromal cells running around the antimesometrial decidual tissue which display a punctate laminin staining. Fig. 16A,B. The antimesometrial primary decidual-like zone is devoid of laminin. In the region of the developing capsule laminin is localized to blood vessels. A thin stromal zone with punctate laminin is present next to the capsule (0).Laminin was also found associated with the pericellular basement membranes of smooth muscle cells as seen at the bottom of the photograph. 20 D.E. CLARK ET AL. LITERATURE CITED Aplin, J.D. 1989 Cellular biochemistry of the endometrium. In: Biology of the Uterus. R.M. Wynn and W.P. Jollie, eds. Plenum, New York, pp. 89-129. Beck, K.. I. Hunter, and J . Enml 1990 Structure and function of laminin: Anatomy of a mukidomain glycoprotein. FASEB J . , 4t148-160. Bell, S.C. 1983 Decidualization: Regional differentiation and associated function. Oxf. Rev. Reprod. Biol., 5.220-271. Bissell, M.J., H. Glenn Hall, and G . Parry 1982 How does the extracellular matrix direct gene expression? J . Theor. Biol., 99:31-68. Christofferson, R., and 0. Nilsson 1989 Placentation in the r a t A SEM study of microvascular casts. 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