Protein synthesis by chick (Gallus domesticus) extraembryonic membranes

Comp. Biochem. Physiol. Vol. 104B,No. 4, pp. 689-693, 1993

0305-0491/93 $6.00+ 0.00 © 1993 PergamonPress Ltd

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PROTEIN SYNTHESIS BY CHICK (GALLUS DOMESTICUS) EXTRAEMBRYONIC MEMBRANES LuIs S,~NCHEZPALAZON and ANTONIORODRiGUEZ-BURGOS Department of Microbiology, Faculty of Sciences, University of C6rdoba, C6rdoba, Spain (Tel. 957-2186-02; Fax 957-2186-06) (Received 28 September 1992; accepted 6 November 1992)

Abstract--1. The protein synthesis in chorioallantoic membrane (CAM), amniotic membrane (AM) and yolk sac (YS) were studied by in vitro culture with ~4C L-glutamic and immunoautoradiography. 2. The yolk sac synthesizes transferrin, alpha-fetoprotein (AFP) and serum albumin. These three proteins are also synthesized, but only in slight amounts, by AM. A light synthesis of AFP, transferrin and another unidentified protein is detected in the CAM. 3. At the ultrastructural level, AFP is localized in the rough endoplasmic reticulum of CAM mesodermal cells.

polyclonal polyspecific antiserum, that there is synthesis of other proteins besides AFP. On the whole, the results obtained in this paper together with the previous ones, give validity to these new patterns for the study of the biological function of AFP.

INTRODUCTION To date, alpha-fetoprotein is one of the most studied differentiation antigens. It is expressed in different stages of the embryo-fetal development during the ontogeny of mammals and other vertebrates, going with tissues derived from the three germ layers. This carrier globulin can be expressed in the adult stage associated with some types of tumors, mainly hepatocellular carcinoma and extraembryonic tumors as well as testicular and ovarian germinal cell tumors. Many years of research on this fetoprotein have provided a new tool for clinical diagnosis. AFP, among others, is used in the diagnosis of neural tube malformations and as a tumor marker (Ruoslahti and Sepp~il/i, 1979). Although copious data exist in the literature relating to the physical and chemical properties of A F P (Mizejewski, 1985), very little is known about the role that A F P plays in the different developmental stages. Perhaps this is due partly to the lack of patterns where specific A F P properties can be compared with specific stages of the development and of the cellular differentiation. In this context, and with the goal of finding suitable patterns, we are studying the ontogeny of A F P in the chorioallantoic membrane (CAM), where A F P had previously been detected (Rodriguez-Burgos, 1990), and now in the amniotic membrane (AM). Through the use of joint techniques such as in vitro cultures and immunoperoxidase, we found evidence of the synthesis of A F P in the aforesaid extraembryonic membranes (Sfinchez Palaz6n and RodriguezBurgos, 1991a,b). Nevertheless, there is the possibility that the A F P so detected would have been taken from an outward source and could have remained in the explant during its culture. To solve this problem, the membranes were cultured with ~4C L-glutamic and the synthesis was shown by immunoautoradiography. It has also been proved, using a

MATERIALS AND METHODS

Immunization

The antigenic solution was composed of serum, allantoic fluid and amniotic fluid of 14-day chick embryos. Each of the rabbits was first inoculated intramuscularly with 0.5 ml of the antigenic solution emulsified with 0.5 ml complete Freund's adjuvant (Serva, Heidelberg). A following three inoculations were done intramuscularly and/or subcutaneously each week, with 0.5ml of the antigenic solution emulsified with 0.5 ml of incomplete Freund's adjuvant that was prepared by mixing 85 parts of Bayol F with 1J parts of Arlacel A (v/v). One month later, the rabbits were inoculated in the same manner. Ten days after this last dose, the rabbits were bled and their serum was analyzed by immunoelectrophoresis. The antiserum so obtained was named anti-p, and was dispensed in aliquots and stored at -20°C. Organ cultures

CAM, AM, and YS were isolated under sterile conditions from 14-day embryos. Small pieces of these membranes were cultured in a medium with I4C t-glutamic. The culture medium was prepared with 2% of an amino acid stock solution minus glutamic acid (Van Furth, 1978) in Hanks' balanced salt solution plus 0.5% (w/v) ovalhumin, 0.5% (w/v) glucose, 1% (v/v) BME vitamin mixture (Sigma, St Louis, MO) and 25#g/ml gentamycin. For each culture, 1-1.5 ml of medium containing 1 #Ci/ml of 14C L-glutamic (136 mCi/mmol; ICN Biomedicals,

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Irvine, CA) was used. The cultures were incubated for 48 hr at 37°C under continuous rotation and then frozen at -20°C. After being thawed, the culture fluid was centrifuged at 16,000g for 25 min at 4°C. To remove the excess of radioactive amino acids and to decrease the ionic strength before lyophilization, the cell-free supernatants were dialyzed against 0.015M phosphate buffer, pH 7.2 (YS), and 0.0015 M (CAM, AM) for 72hr at 4°C.

A F P serum was used diluted 1 : 750 with buffer phosphate saline (PBS, 0.01 M, pH 7.2) containing 0.5% bovine serum albumin (BSA; Sigma). Goat IgG anti-rabbit IgG labeled with 15nm gold particles (Bio-Cell, Cardiff, U.K. ) diluted 1:10 was used as secondary antiserum. The samples were examined under a Philips CM-10 transmission electron microscope.

Immunoelectrophoresis and autoradiography

Analysis of" a polyclonal serum against embryonic serum, amniotic fluid and allantoic ,fluid proteins

The lyophilized culture media were dissolved in 100 #1 twice-distilled water; therefore, they were concentrated 10-fold (YS) and 100-fold (CAM, AM). These solutions were analyzed by immunoelectrophoresis (Scheidegger, 1955). The electrophoresis was carried out in 1% agarose M ( M r = - - 1 3 ) in Tris-barbiturate buffer, pH 8.6, at 10°C and 10 V/cm for 50 min. After immunoprecipitation, the gels were washed in six changes of NaC1 0.15 M at 4~'C for 72hr, and then dried. Kodak X-Omat AR film (Sigma) was used for autoradiography. After 30 days of exposure at - 6 0 ° C , the films were developed in a Kodak X-Omat M35 processor.

Electron microscopic immunohistochemistry Small pieces of 14-day CAM were fixed at 4°C for 2-3 hr with the following fixatives dissolved in cacodylate buffer 0.1 M, pH 7.2: 0.5% glutaraldehyde; 2% paraformaldehyde-1% glutaraldehyde; 4% paraformaldehyde-picric acid containing 0.5% glutaraldehyde. After fixation, the samples were washed in cacodylate buffer and some of them were post-fixed with 1% osmium tetroxide in cacodylate buffer 0.1 M, pH 7.2, at 4°C for 1 hr. Then the samples were dehydrated through graded ethanol series and embedded in Agar 100 (Agar Scientific Ltd, U.K.). The A F P localization was studied in ultrathin sections collected on nickel grids, by the colloidal gold marker system (Roth, 1989). The best results were obtained with samples fixed in 4% paraformaldehyde-picric acid containing 0.5% glutaraldehyde, and post-fixed in osmium tetroxide. The ultrathin sections were treated with a saturated aqueous sodium metaperiodate solution for 60 min at room temperature before the immunocytochemical labeling, Then, the grids were washed with several changes of twice-distilled water. As primary antiserum, a rabbit anti-avian

RESULTS

The immunoelectrophoretic pattern of the antigenic mix composed of embryonic serum, amniotic fluid and allantoic fluid proteins of 14-day embryos, developed with anti-p serum, shows that this antiserum contains antibodies against at least seven different proteins: IgG, transferrin, AFP, two unidentified proteins, serum albumin and alpharacid glycoprotein (Fig. 1).

Protein synthesis by extraembryonic membranes The culture media of AM, CAM and YS of 14-day embryos that had been in vitro cultured with ~4C L-glutamic, as described in Materials and Methods, were analyzed by immunoelectrophoresis and autoradiography. The immunoelectrophoretic pattern of each extraembryonic membrane culture medium developed with anti-AFP serum is shown in Fig. 2(a). The autoradiography of this pattern shows the A F P synthesis in these membranes (Fig. 2b). The immunoelectrophoretic pattern of each extraembryonic membrane culture medium developed with anti-p serum that has been analyzed above is shown in Fig. 2(c). The autoradiography of this pattern shows the synthesis of transferrin, AFP and serum albumin in YS. These three proteins are also synthesized but in slight amounts by AM. Slight syntheses of AFP, transferrin and another unidentified protein are detected in the CAM (Fig. 2d). The negative control consisted of embryonic serum incubated with radioactive medium and no autoradiographic image was observed. When the cultures were treated with 25/~g/ml puromycin, a protein synthesis inhibitor, no autoradiographic image was observed. On the other hand, the scarce A F P localization by immunohistochemistry in the rough endoplasmic

Fig. 1. Immunoelectrophoretic pattern of an antigenic mix composed of embryonic serum, amniotic fluid and allantoic fluid of 14-day embryos, developed with anti-p serum. 1: IgG; 2: transferrin; 3: AFP; 4~5: unidentified proteins; 6: serum albumin; 7: alphal-acid glycoprotein.

Protein synthesis by extraembryonic membranes

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Fig. 3. (a) Immunolocalization of AFP in rough endoplasmic reticulum of 14-day CAM mesodermal cells. (b) Gold particle label is not present in the control section that consisted of the absorption of anti-AFP serum with chick embryonic serum. Magnification:(a) x 52,000; (b) x 39,000. reticulum of CAM mesodermic cells (Fig. 3a), supports the slight synthesis of AFP detected by immunoautoradiography. A negative control of the AFP localization consisted by the absorption of primary antiserum with chick embryo serum (Fig. 3b). DISCUSSION

Fig. 2. (a) Immunoelectrophoretic pattern of amniotic membrane (AM) yolk sac (YS) and chorioallantoic membrane (CAM) culture medium developed with anti-AFP serum. (b) The autoradiography of this pattern shows the AFP synthesis in AM, YS and CAM. (c) Immunoelectrophoretic pattern of AM, CAM and YS culture medium developed with anti-p serum. (d) The autoradiography of this pattern shows the synthesis of I: transferrin; 2: unidentified protein; 3: AFP and 4: serum albumin.

Former studies point out that synthesis of serum albumin in the yolk sac stops from the 7th day of incubation, being replaced by the liver. That is why a type of humoral communication between these two organs has been suggested (Weller, 1976). Our results show a double disagreement with the former studies. On one hand, as far as time is concerned, we detected synthesis of serum albumin on the 14th day, and on the other hand as far as the place is concerned, the yolk sac as well as the amniotic membrane, synthesized serum albumin. Moreover, the synthesis of serum albumin by the yolk sac has also been shown, at 16-18 days, by other authors (Gitlin and Kitzes,

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1967). All this makes it improbable that the above possible humoral relationship between the liver and the yolk sac exists. The results obtained by immunoautoradiography confirm the synthesis suggested by means of the culture in vitro of CAM and AM and the later immunostaining with anti-AFP antibodies (S~inchez Palaz6n and Rodriguez-Burgos, 1991a,b). These facts show that the synthesis of AFP is not specific in the yolk sac endodermal cells and the hepatocytes, and comes from the extraembryonic and embryonic endoderm, respectively, seeing that extraembryonic mesodermal cells also synthesize AFP. The former results also show that the three extraembryonic membranes studied synthesize transferrin in major or minor amounts. This synthesis could be justified in vivo, at least partly, bearing in mind that on one hand, the transferrin has been identified as one of the essential factors in the culture medium of mammal cells (Sato, 1980; Ambesi-Impiombato et al., 1980) and, on the other hand, that the yolk sac as well as CAM and AM, are fetospecific organs. It is said they are not incorporated by the embryo after hatching and undergo active processes of growth and differentiation during a short time-period. The essentiality of the transferrin is due to the iron molecules which it carries. The iron internalization is made easy by transferrin receptors localized on the plasma membrane and by a later separation of the iron from the carrier protein at an intracellular level (May and Cuatrecasas, 1985). The iron-free transferrin is not degraded within the cell but is recycled by secretion into the extracellular medium (Dautry-Varsat et al., 1983). Transferrin aids in the cellular iron-carrying proliferation which is required for the enzymatic activity of the ribonucleotide reductase and, therefore, for the synthesis of DNA and cellular fission (Laskey et al., 1988). In this context we would like to emphasize that AFP of different species bind in a reversible form and with high-affinity fatty acids, especially a long polyunsaturated chain such as arachidonic acid (20:4, n-6) and docosahaexenoic acid (22:6, n-3) (Parmelee et al., 1978; Pifieiro et al., 1979; Benassayag et al., 1980). Other studies indicate that the biological role of AFP could be the transport and the internalization of these molecules through binding of the fatty acid-AFP complex to the AFP receptor localized on the plasma membrane (Geuskens et al., 1986; Uriel et al., 1987; Torres et al., 1989; Uriel, 1989). These fatty acids are essential molecules for the synthesis of the membrane phospholipids. It is known how some of them, such as 20:4 (n-6) have, by themselves and/or by their metabolites, regulatory effects over the function and proliferation of lymphocytes. They are also important regulators of inflammatory reactions (Bevan and Wood, 1987; Cathbert and Lipsky, 1986; Taill and Wirck, 1984). All this hints that both glycoproteins, transferrin and AFP aid the cellular proliferation in these three fetospecific

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