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Acia Anat 1990;137:141-145
Immunohistochemical Detection of an Epithelial Membrane Protein in Rat Embryos at Different Stages of Development T. Tarmann, G. Dohr,
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Schlecht, S. Barth. M. Hartmann
Institut für Histologie und Embryologie. Universität Graz. Austria
Key Words. Rat embryos • Monoclonal antibodies • Membrane proteins Abstract. This study aimed to describe the distribution of a membrane protein called Gz-1 -Ag in embryonal rat tis sues using monoclonal antibodies. Three monoclonal antibodies recognizing different antigenic determinants of Gz-1Ag were tested on different stages of rat embryos-fertilized oocytes, two-cell stages, morulae, blastocysts and embryos up to 17.5 days old.The embryos were fixed by different methods; the Tokuyasu method was the most convenient. It yielded very good morphological conservation, good preservation of antigenicity and weak background fluorescence. Gz- I-Ag was detected in practically all early epithelial structures, but the intensity of fluorescence varied. Fluorescence was not associated with the germ layer from which the epithelium derived. The uninterrupted presence of G z-1-Ag from the fertilized oocyte to all subsequently arising epithelial structuressuggests a role of Gz-l-Ag in cell adhesion, secretory processes or the intercellular exchange of information or substances.
The present study describes the detection and distribu tion of a membrane protein called Gz I Antigen (Gz-lAg) in rat embryos at different stages of development. It continues recent studies which identified Gz-l-Ag with monoclonal antibodies (moAbs). The three moAbs were obtained using rat small intestine cells (Gz 1, Gz 2) or the isolated basolateral membrane (BLM) of these cells (Gz 20) as antigens [Schiechl et al., 1986; Schiechl and Dohr. 1987]. The moAbs are specific for Gz-l-Ag but recognize at least two different epitopes. Gz-l-Ag was first detected in the BLM or rat small intestine cells. Differentiated rat small intestine cells have an apical brush border mem brane that differs in composition and function from the BLM. The brush border membrane has been studied by various groups [Douglas et al.. 1972; Fujita et al., 1973; Ziomek et al.. 1980; Ouaroni. 1984]. A number of enzy matic complexes have been described, but no membrane
protein like Gz-l-Ag has been reported. Gz-l-Ag was found not only in the BLM of rat small intestine cells but also in many epithelial structures of organs of the adult rat [Schiechl and Dohr, 1987]. This prodded the current com plementary investigation of the appearance and distribu tion of Gz-l-Ag in embryonal tissue. We studied fertilized oozytes. two-cell stages, morulae, blastocysts and embryos of 8.5, 11.5, 14.5 and 17.5 days’ age.
Material and Methods Production o f the m oAbs Gz ¡, Gz 2 und Gz 20 Gz I was produced by fusion of P3-NS 1/Ag 4-1-myeloma cells with spleen cells of Balb-c mice [Köhler eta l.. !976:Goding. 1983] which had been immunized with rat small intestine cells (Weiser. 1973), as described by Schiechl etal. 11986|.Gz2 was produced in the same way in another fusion [Dohr et al.. 1987], For the production of Gz 20, instead of the isolated entire epithelial cells, the isolated BLM of the epithelial cells was used (Weiser. 1978|.
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Introduction
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Embryo Collection We used Sprague-Dawlcy rats from Himberg. Austria. Regular oestrous cycles were verified by vaginal smears during a 2 week period. Approximately 4-month-old female rats weighing 220-230 g were eaged overnight when in the oestrous stage with male rats at a ratio of 1 male to I female. A sperm-positive vaginal smear on the following morning was considered proof of pregnancy and counted as day I post conceptionem (p.c.). To confirm pregnancy, vaginal smears were also taken during the following days; if they did not show cyclic alterations, pregnancy was ascertained. Pregnant females were sacrificed by cervical dislocation or with chloroform at 2.5.3.5.5 .5 .8 .5 . 11.5. 14.5 and 17.5 days p.c. Fertilized oocytes and two-cell stages were obtained by rinsing the tubes. Morulae and blastocysts were obtained by rinsing the uterine horns on day 5.5 p.c. The older embryos were collected from the uterine horns, and those of 8.5 and 11.5 days were treated in toto. Older embryos were tran sected at the level of the diaphragm. Fixation and Tissue Preparation Three different methods of fixation were used to find out which would render the best results for indirect immunofluorescence testing. According to Tokuyasu [1986], the embryos were fixed immediately after collection for 1 h at 20 C in 2% formaldehyde + 0.1% glutaraldehyde dissolved in 0.1 M Na phosphate (pH 7.3) and consequently washed in PBS. To block unspecific binding sites due to fixation, the embryos were put into 50 pi glycine in PBS for I It at 20 C. After a short washing in PBS, the embryos were brought into a solution of 1 M sucrose dissolved in 0 .1 M Na phosphate at a pi I of 7.0 for 6-8 It at 0 C and were subsequently frozen in liquid propane. The frozen embryos were seri ally sectioned on a crvocut at a thickness of 5 urn. According to Wick et al. 11976], the material was fixed in 96% ethanol or acetone and then embedded in paraffin. Serial sections of 5 (tin thickness were done on a conventional microtome. Some embryos were frozen in liquid propane without previous fixation. Sections of 5 pm thickness were done on a cryocut. Inwutnocytocliemical Methods The immunohistochemical localization of Gz-l-Ag was performed on frozen and paraffin-embedded sections. Thesections were incubated at 20 °C for 30 min with undiluted Gz I. G z2 or G z2 0 supernatant (cen trifuged for 10 min at 12.000 g). washed 3 times with PBS (20 min) and
then incubated for 30 min at 20 C in a mixture of 50 pi fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and 20 pi inacti vated rat serum in 1.000 pi PBS. After further rinses in PBS and FLO. the sections were dehydrated in 100% ethanol, sealed in Moviol and examined unler a Zeiss fluorescence microscope (Axiophot). Control slides in which the first incubation had been omitted were used to recog nize unspecifically labelled sites. Suspension Marking Oocytes, two-cell stages, morulae and blastocysts were transferred with a Pasteur pipette into a buffered washing solution (PBS. pH 7.4. with 1% bovine serum albumin and 0.1% sodium azide). Then evert specimen was incubated with 200 pi undiluted moAh supernatant for 3(1 mi n. washed 3 times and incubated for 60 min in 200 pi FITC-conjugated goat anti-mouse IgG diluted 1:1 with washing solution, preabsorbed with rat serum, at room temperature. A fter3 more washing cycles, the specimens were examined under the Axiophot.
Results Comparison o f the Three Methods o f Preparation The best preservation of antigenicity combined with the weakest background fluorescence, but the worst mor phological conservation, was obtained by conventional freezing without previous fixation. The worst preserva tion of antigenicity combined with strong background fluorescence but good morphological conservation was obtained by the method of Wick et al. f 1987]. The best morphology combined with good antigenicity and weak background fluorescence was obtained by the modified Tokuyasu method [ 1986]. Consequently, the following results stem from serial sections of material treated according to Tokuyasu - with the exception of the 14.5-day-old embryos, which were conventionally frozen. Immunocytochemical Results The fertilized oozytes. two-cell stages and morulae showed bright but rapidly fading fluorescence of their plasmalemmata. but in the morula stage it seemed that not all cells were positive (fig. 1—1). In the blastocyst stage the trophoblast cells as well as the inner-cell mass (embryoblast) reacted strongly. The zona pellucida. though very well visible in the light microscope and phase contrast, did not give any fluorescence. In the 8.5-day embryo (fig. 5), at the stage of the threegerm layer, the ectoderm and entoderm fluoresced strongly, while the mesoderm remained dark in the indi rect immunofluorescence test (IIFT). In the 11.5-day embryo (fig. 6), all epithelial structures regardless of their origin and including those of mesoder mal origin such as the Wolffian duct and the coelomic epi-
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Antigen Detection To identify the antigen specificity of the moAbs. the proteins of the isolated BLM of rat small intestine epithelium were separated by SDS polyacrylamide gel electrophoresis. The protein transfer from gel to nitrocellulose paper was performed as described by Towbin et al. 11979). Then followed incubation with the undiluted ntoAb superna tant as the primary antibody and with conjugated goat anti-mouse IgG as the secondary antibody. All specifically labelled protein bands were then visualized by HRP detection with 11.0- and o-dianisidin. To elim inate unspecific reactions, the incubation with the specific antibodies was omitted in blind probes. The specificity of the three moAbs for proteins of the BLM is as follows: Gz 1 is specific for a 42-kD protein, designated Gz-l-Ag. Gz 2 is specific for the same band but also stains an additional 35-kD broad band. Gz 20 shows the same staining pattern as Gz 2. Since all three ntoAbs stain band 1. they are specific for Gz-lAg. As Gz 2 and Gz 20 also stain band 2. they appear to be directed against other antigenic determinants of the same protein (Schicchl et al.. 1986).
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