Immunochemical localization of vitamin D-dependent calcium-binding protein in mouse placenta and yolk sac.код для вставкиСкачать
THE ANATOMICAL RECORD 213514-517 (1985) lmmunochemical Localization of Vitamin DDependent Calcium-Binding Protein in Mouse Placenta and Yo1k Sac M. ELIZABETH H. BRUNS, ELLEN KLEEMAN, STACEY E. MILLS, DAVID E. BRUNS, AND JOHN C. HERR Departments of Pathology (M.E.H. B., E.K., S.E. M., D.E.B.), Biochemistry (M.E. H. R.), and An,atomy (J.C.H.), University of Virginia Medical Center, Charlottesuille, VA 22908 ABSTRACT A 10,000-dalton calcium-binding protein (10-kd CaBP) has been described in the placentae and yolk sacs of rats and mice. This protein is similar or identical to vitamin D-dependent intestinal CaBP and these proteins have been implicated in the molecular mechanisms of intestinal calcium absorption and transplacental calcium transport. Using a n antiserum to the purified 10-kd rat intestinal CaBP, the localization of CaBP in the 16-17-day mouse yolk sac and placenta was studied immunocytochemically with peroxidase-antiperoxidase labelling and quantified by radial immunodifussion. A high concentration of immunolabelling was observed in the endodermal cells of the intraplacental yolk sac lining the sinuses of Duval. The columnar endodermal cells lining one side of the endodermal sinuses adjacent to fetal vessels contained most of the immunoreactive label. Quantitation by radial immunodiffusion demonstrated a 5.5-fold higher concentration of CaBP in the portion of the placenta containing most of the intraplacental yolk sac than in the maternal half of the placenta. This demonstration of a 10-kd CaBP within the intraplacental yolk sac suggests this protein functions to facilitate placental calcium transport and is the first study which directly supports the hypothesis of a functional role for the sinuses of Duval in calcium transport. The placentae and yolk sacs of mice and rats contain high concentrations of a 10-kilodalton (kd) vitamin Ddependent calcium-binding protein (CaBP) (Bruns et al., 1978, 1981; Marche et al., 1978; Delorme et al., 1979, 1982). The steady-state concentration of the vitamin Ddependent CaBP has proven to be a consistent marker of vitamin D metabolite action on calcium transport in the small intestine (Wasserman and Fullmer, 1982). A similar role of CaBP in placental calcium transfer is suggested by several findings, including evidence that rat yolk sac and placenta contain 1,25-dihydroxycholecalciferol (Stumpf et al., 1983) as well as its receptors (Pike et al., 1980; Danan et al., 1981). A role for CaBP in placental calcium transfer is suggested by the parallel gestational changes in placental CaBP and fetal growth (Bruns et al., 1978), which in turn reflect the fetal accumulation of calcium (Comar, 1956). The present studies were undertaken to investigate the cellular localization of CaBP in placenta in order to understand how this protein may function in the transfer of calcium from mother to fetus. As reported by Delorme et al. (19831, CaBP in the yolk sac is localized to columnar epithelial cells which resemble absorptive intestinal cells. We have found CaBP to be concentrated additionally in columnar cells of visceral yolk sac endoderm which penetrate the placental labyrinth in the region bordering the attachment of the umbilical cord. As detailed below, these cells line the endodermal sinuses or pits, or sinuses of Duval (Mossman, 1937; Amo0 1985 ALAN R. LISS, INC. roso, 1952).These findings are of anatomical importance because CaBP provides a unique marker for intraplacental endodermal cells that will be useful in examining their distribution and appearance during gestation. Moreover, the finding of CaBP within the intraplacental yolk sac cells suggests that this region is a site for active calcium transport from mother to fetus. MATERIALS AND METHODS Maferials Glutaraldehyde and 3,3-diaminobenzidine tetrahydrochloride were from Sigma. Antirabbit IgG and peroxidase were obtained from Cappell or Miles. Acrolein was purchased from Polysciences Inc. Animals Pregnant ICR mice were obtained from Charles River. The day of mating was considered day 0 of gestation. Term was observed at 19 days of pregnancy. The animals were fed normal rat chow ad libitum and studied on days 16-17. CaBP and Antiserum The rat intestinal CaBP was purified as described (Bruns et al., 1977) with a final preparative slab gel Received February 28, 1985; accepted June 4, 1985. CABPIN MOUSE PLACENTA AND YOLK SAC 5 15 electrophoresis using 0.1 M EDTA added to buffer. The ta’s fetal surface surrounding the umbilical attachment production and validation of rabbit antisera which cross- and 2) the placenta minus intercept (180-290 mg wet react with both rat and mouse CaBP were performed as weight) which was comprised of peripheral portions of the labyrinth. All tissues were weighed and then homogoutlined (Bruns et al., 1978). enized, using a Tissuemizer, in a n equal amount (1 ml Tissue Fixation per gram of tissue) of buffer containing 10 mM Tris-HC1, A freeze-substitution method for tissue fixation (Tay- 5 mM benzamidine, 1 mM 2-mercaptoethanol, pH 7.2, lor, 1981)was used first for immumohistochemical stain- plus 2 mM phenylmethylsulfonyl fluoride and 0.2 tryping of placental calcium binding protein by the per- sin inhibitor unit/ml of aprotinin. The homogenate was oxidase-antiperioxidase technique (PAP) (Sternberger et centrifuged a t 40,OOOg for 20 minutes; the amount of al., 1970). Taylor (1981) has shown that freeze substitu- immunoreactive CaBP in the supernatant was meation provides accurate localization of CaBP in the chick sured by radial immunodiffusion as described (Bruns et intestine. Using this fixation method with mouse intes- al., 1978). tine, we observed the expected localization of CaBP in (Color Figures 2 and 3 were prepared and appear elsethe absorptive cells of intestinal villi, with acceptable where in this issue. Please see pp. 532-535 for these preservation of morphology. In placenta, we found stain- figures and their accompanying legends.) ing in the placental hilum, in the region of the sinuses RESULTS of Duval. Controls using absorbed antiserum were negTo better explain the results of the immunochemical ative. However, the cellular structures were poorly preserved and positive identification of stained types was staining of the 10-kd CaBP in placenta and yolk sac, a not possible. To achieve better preservation of placental drawing depicting the relationships of the fetal membranes to the mouse fetus and placenta at 16-17 days cell morphology, a perfusion method was used. The pregnant mouse was anesthetized using ether, a gestation is shown in Figure 1; further details may be laparotomy was performed, and the heart was exposed. found in the references (Mossman, 1937; Amoroso, 1952; A needle was placed in the left ventricle and 10 ml of Wislocki and Padykula, 1953; Brambell and Halliday, buffered saline was perfused a t a rate of 8 ml/minute. 1956; Padykula and Richardson, 1963). Padykula and The intercostal arteries cut during exposure of the heart Richardson divided the rat yolk sac into three distinct served as the outflow for the perfusate. After the liver parts: 1) the outer parietal wall (bilaminar omphalocleared, the femoral artery was cut and 50 ml of fixative pleure), which we divided into a) the juxtauterine yolk was perfused (2% acrolein in 0.1 M Na2HP04 buffer, pH sac and b) the periplacental yolk sac, which is the contin7.4, containing 0.25% glutaraldehyde). Tissues were col- uation of bilaminar omphalopleure over the fetal surface lected and immersed in the same fixative for 2-4 hours of the placenta; 2) a n inner visceral wall (vascular splanand rinsed five times in phosphate-buffered saline. The chonopleure) which may be subdivided into a) villous samples were dehydrated with graded ethanol series yolk sac near the placenta and b) a smooth visceral and cleared in toluene for 2-4 hours and embedded in surface over much of the remaining yolk sac; and 3) the placenta’s endodermal sinuses (sinuses of Duval), which paraffin after infiltration for 1hour. we term intraplacentd yoZk sac.The intraplacental yolk lmmunoperoxidase Staining sac endoderm is polarized, consisting of a high to low The peroxidase-antiperoxidase technique of Sternber- columnar epithelium adjacent to fetal vessels and a ger was used (Sternberger et al., 1970). The tissue ob- squamous epithelium adjacent to maternal blood spaces. For the study of CaBP localization in mouse placenta, tained by perfusion was deparafinized using toluene and a graded ethanol series. Nonspecific endogenous peroxi- the complete fetal unit was processed en bloc, including dase was removed using methanol with 0.3% HzOz for placenta, yolk sac, uterus, and fetus, through fixation, 30 minutes. Background staining was reduced by incu- embedding, and immunoperoxidase staining. Fixing the bating sections in 1%cold sheep serum in 0.05 M Tris- whole unit together proved to be a surprisingly valuable buffered saline (TBS), pH 7.6, for 30 minutes. The pri- maneuver. Within the 16-day placenta, immunoreactive mary antiserum (anti-CaBPto rat intestinal CaBP, 150- CaBP was localized to the endodermal cells of both the 1:500 in TBS) was applied for 30 minutes a t room tem- villous yolk sac and the intraplacental yolk sac (Fig. 2). perature. The slides were rinsed in 1% sheep serum; Immunoreactive CaBP is present in the endodermal cells sheep antirabbit IgG (heavy and light chains, Cappell of the villous portion of the yolk sac as indicated by Labs), 1:200 in TBS, was applied for 15 minutes. The moderate brown staining. An abrupt transition from slides were rinsed in 1% sheep serum; PAP (rabbit, Cap- moderate to intense staining was readily observed a t pell, in TBS 1:200) was applied for 30 minutes. The the junction of the villous and intraplacental portions of slides were rinsed in TBS and 3,3-diaminobenzidene was the yolk sac (Fig. 2). The immunoreactive intraplacental applied for 15 minutes to yield a brown reaction product. yolk sac cells ranged from high to low columnar and The slides were rinsed in TBS, counterstained in hema- could readily be distinguished from the squamous entoxylin, and dehydrated in a graded alcohol series and dodermal cells of both the periplacental yolk sac and the endodermal sinuses. No specific staining, except a n octoluene. casional island of yolk sac endoderm which was cut Tissue Preparation for Radial lrnrnunodiffusion Assay obliquely, was observed in other parts of the placenta Tissue samples of placenta and yolk sac were obtained including the chorioallantoic labyrinth. Figure 2B shows from 16-17-day pregnant mice under ether anesthesia. a control, using antiserum that was absorbed with The placenta was dissected into two regions: 1)the pla- highly purified CaBP. This region of intraplacental yolk cental intercept which consisted of a disk-shaped zone sac showed no immunoreactivity using the absorbed (36-78 mg wet weight) cut from the center of the placen- antiserum. M.E.H. BKUNS El' AL 5 16 n lntraplacental Bilaminar Omphalopleure (Yo1k Sac Periplacental Bilaminar Omphalopleure (Yolk Sac) (Vascular Splanchnopleure) Smooth Yolk Sac (Vascular Splanchnopleure) Juxtauterine Bilaminar Omphalopleure (Yolk Sac) Fig 1 A drawing depicting the relationships of the murine yolk sac membranes, placenta, uterus, and fetus in late gestation For details see the text The square outlines a region typically containing intraplacental yolk sac and is fox orientation in interpreting Figure 2 on p 532 of this issue Figure 3 demonstrates staining of 16-day intraplacen- mous endodermal cells on the opposite side of the sinus tal yolk sac and the relationship of several fetal and makes determination of their immunoreactivity for maternal vessels to it. This tissue was perfused through CaBP difficult at the light microscopic level; however, the maternal vessels; maternal blood spaces contain no immunolocalization was detected in these cells. In order to confirm the immunohistochemical localizamany fewer blood elements than do fetal vessels. The intensely stained columnar intraplacental endodermal tion of CaBP in endodermal cells of the intraplacental cells are often found in close proximity to the fetal ves- yolk sac, quantitative CaBP measurements were made sels. The attenuated cytoplasm of the cuboidal-to-squa- by a radial immunodiffusion assay. The 17-day placenta was dissected into two parts: the intraplacental yolk sac region was termed the placental intercept and the rest of the placenta was termed placenta minus intercept. Figure 4 indicates the CaBP concentration (pg CaBP/mg T protein) in these tissue and in extraplacental yolk sac. The placental intercept, which contained the intraplacental yolk sac but no villous yolk sac, showed by far .-c 6 the highest concentrations of immunoreactive CaBP. A al c greater amount of immunochemical CaBP in the fetal 2 side of the placenta has also been observed in rats (Dea 5lorme et al., 1979). The visual difference in the staining intensities of \ villous yolk sac and intraplacental yolk sac (Fig. 2) was 2c 4 matched by the quantitative CaBP determinations-2.6 al pg CaBP/mg protein versus 6.8 pg CaBP/mg protein for 5 the villous and intraplacental portions, respectively. The '3 3U placenta minus the intercept, although low in CaBP 0) concentration as compared to placenta plus intercept, had measureable amounts of CaBP--1.2 pg CaBP/mg protein. The placenta minus the intercept was stained using the immunohistochemical method; no specific placental staining was observed, except for intense staining in islands of yolk sac cells that happened to have been outside the intercept dissection. Thus the low concentraPlacenta Placental Splanchnopleuric tion of CaBP, which was quantitated in the placenta minus intercept Yolk Sac minus the intercept, may be contained in those cells of intercept the sinuses of Duval which deeply evaginate into the placental labyrinth. 'i F Fig. 4. Immunoreactive CaBP in placental intercept, placenta minus intercept, and splanchnopleuric yolk sac in the 17-daymouse placenta. The placenta intercept consisted of a disk-shaped zone (36-78 mg wet weight) cut from the center of the placenta's fetal surface surrounding the umbilical attachment. DISCUSSION The results described here indicate that the 10-kd CaBP is located not only within the extraplacental yolk CABPIN MOUSE PLACENTA AND YOLK SAC sac but is to be found as well within intraplacental yolk sac endoderm. This has been shown by immunohistochemical staining using a PAP technique and quantitated by radial immunodiffusion assay. The placental CaBP immunoreactivity is localized to columnar yolk sac cells lining the sinuses of Duval in close proximity to fetal vessels. Duval, in 1892, first described outpocketings of yolk sac that penetrate the fetal surface of the chorioallantoic placenta, intertwining among the branching roots of the allantoic blood vessels. These outpocketings of the yolk sac endoderm have been referred to as Duval’s entodermal sinuses (Amoroso, 19521, or endodermal sinuses (Padykula and Richardson, 19631, and have been postulated to play a role in the transport of nutrients and antibodies from fetus to mother (Amoroso, 1952; Brambell and Halliday, 1956; Padykula and Richardson, 1963). Although the precise gestational time for the initiation of these evaginations is not fully established, they have been observed to be present on the 15th day of gestation and to develop until term (Padykula and Richardson, 1963). Thus far, they have been observed only in genera which are members of the family Muridae (Mossman, personal communication). Mossman refers to endodermal sinuses as “placental pits” and describes them as “intraplacental diverticula of the periplacental yolk sac wall.” Endodermal sinuses are lined by two histologically distinct endodermal cell types, described by Wislocki and Padykula (1953) as visceral and parietal endodermal cells. In the present study, we refer to the different cells in the intraplacental yolk sac a s cuboidal to squamous yolk sac (parietal) and columnar yolk sac (visceral) cells. The availability of immunoreactive CaBP as a marker for intraplacental endoderm should facilitate anatomic studies of the three-dimensional structure of the sinuses. We anticipate temporal studies of the appearance of CaBP-containing cells in the placenta. By analogy to the intestine, the high concentration of CaBP in these cells suggests that active transcellular transport of calcium is a n important function of the intraplacental portion of the yolk sac in Muridea. This hypothesis proposes a specific function for the intraplacental yolk sac and carries important implications for future studies of the mechanisms of placental calcium transfer. ACKNOWLEDGMENTS This research was supported by United States Public Health Service grant HD 123345. The authors wish to thank Mrs. Grace Kohler and Mrs. Betty Roberts for their excellent secretarial assistance. Also we wish to thank Dr. William B. Rhoten for his valuable assistance in the immunoperoxidase methodology. The suggestions of Dr. Harland Mossman are greatly appreciated. 517 LITERATURE CITED Amoroso, E.C. (1952)Placentation. In: Marshall’s Physiology of Reproduction, 3rd Ed. AS. Parkes, ed. Longman, Green, New York, Vol. 2, pp. 127-311. Brambell, F.W.R., and R. 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