Carbonic Anhydrase Activity in Early Developing Chick Embryos

Changes in tension of environmental CO₂ gas have been shown to affect cellular differentiation. In the presence of increased partial pressure of dissolved CO₂Loomis (1957) has demonstrated an increase in sexual differentiation of hydra and Flickinger (1958) has caused explanted frog ectoderm to differentiate into neural tissue. Loomis was able to show that the substance which stimulates sexual differentiation in crowded cultures of Hydra littoralis is dissolved CO₂ gas. Flickinger could produce a definite increase of neural tissue induction by bubbling CO₂ gas through the culture media for a 10–20-minute period. Trinkaus & Drake (1959) have reported an analysis of the stimulating effect of on development of the embryo of Fundulus heteroclitus. For these reasons it seemed important to study an enzyme which utilizes CO₂ as a substrate. The appearance of carbonic anhydrase activity in early developing embryos which are producing CO₂ might lead to a reduced CO₂ tension (H₂O+ CO₂⇆H₂CO₃). In turn this activity could control development by slowing cellular differentiation.

Previous measurements of carbonic anhydrase activity in chick embryos have dealt mainly with embryonic organs after the 4th day. Van Goor (1940) and Clark (1951) have shown a rise in retinal activity between the 4th and 6th days and both have failed to find activity in blood before the 11th day. The absence of enzyme activity in blood greatly simplified the measurement in tissue by obviating corrections for erythrocyte carbonic anhydrase.

This report deals with values for whole chick embryo carbonic anhydrase from days (stage 8) to 19 days (stage 45) and relates the activity to weight and content of protein and nitrogen. In addition, the intracellular particulate localization of protein, nitrogen, and carbonic anhydrase activity has been studied in embryos from days to days. Preliminary studies have also been made of the localization of activity in the organ systems of the early embryo.

Fertilized eggs from single comb White Leghorns were obtained from Heis-dorf and Nelson Hatcheries, Houghton, Washington, and incubated in a Brower forced-draft incubator (Model 50) at 38° C. and 60–65 per cent, humidity. The embryos were staged according to the method of Hamburger & Hamilton (1951). The embryo ages herein reported were obtained by first staging and then assigning the corresponding age as found in the data of Hamburger & Hamilton. In general these ages wereto day less than the actual incubation age.

Embryos under 2 days were obtained by excision and removal of the blastoderm in 0·25 M sucrose solution. The embryos were then trimmed and examined under a dissecting microscope. Embryos over 2 days were removed with forceps from the egg and the extraembryonic membranes and allantois were dissected free.

The embryos were weighed immediately on a Mettler analytic balance. The number of embryos weighed for each experiment was 10–15 for the day embryos and 5-10 for the older embryos.

The embryos were homogenized for 3 minutes in a Potter-Elvehjem glass homogenizer with a motor-driven Teflon pestle. The pestle was rotated at 830 rev./min. The suspending medium of 0·25 M sucrose was found to be satisfactory and similar results were obtained with 5 per cent, polyvinylpyroli-done (PVP) in 0·25 M sucrose except that a 6-minute period of homogenization was required for complete microscopic disruption of the tissue when the latter was used.

All steps of the cell fractionation were carried out at approximately 3° C. All homogenates were stained with methylene blue or cresyl echt violet and examined microscopically to assure complete disruption of the cell membranes.

The fractionation technique was based on the method of Schneider & Hogeboom (1950). The homogenate was carefully layered over 0·34 M sucrose and the nuclear fraction which was obtained by centrifuging the homogenate at 800 × g for 10 minutes was resuspended and washed twice. In spite of brief homogenization between washes there remained a small amount of non-nuclear debris in the final nuclear fraction. The mitochondrial fraction obtained by centrifuging (International PR-2) the combined supernatants from the nuclear fraction at 5,000×g for 10 minutes was also washed twice by resuspending in 0·25 M sucrose and centrifuging at 20,000 ×g. This fraction was labelled mitochondrial because under phase microscopy it was composed of small (1-2 μ) rod or spherically shaped particles which could be stained with Janus green. The microsomal fraction was obtained by centrifugation (Spinco Model L) of the supernatant from the mitochondrial fraction at 105,000 × g for one hour. This fraction, which revealed no structures under light microscopy, was washed once. The supernatant from the microsomal fraction was called the soluble fraction. It is apparent from the above definitions that the labelling of these fractions is arbitrary and done for the sake of simplicity in presentation of the material. The total number of embryos used for each fractionation were as follows: days, 120–50; days, 70–120; days, 20–35.

The embryos of 9 days of age and over were homogenized for 3 minutes in a Waring blendor or by a specially constructed small volume (1·5 ml.) steel bladed homogenizer.

The biuret determinations were carried out using the method of Gornall, Bardawill, & David (1949) and read at 550 m/u. in a Beckman Model DU spectrophotometer. Bovine serum albumin was used as a standard for each set of determinations. If cloudiness of the solution was seen a small amount of desoxycholic acid was added.

Nitrogen determinations were carried out by the method of Conway (1958).

As a measure of this activity the catalysed rate of hydration of the substrate (CO₂) was determined. The reaction vessel described by Maren (1960) was immersed in an Aminco constant temperature circulating water-bath and the temperature of the reaction mixture (0·8±0·02° C.) was standardized and measured by a constantin-copper thermocouple attached to a galvanometer. The CO₂ flow was monitored by a Fischer-Porter flowrater (Type 1700). The reaction vessel contained phenol red (0·4 ml.), Tris (hydroxymethyl) amino methane (Tris) buffer (0·2 ml.), and enzyme solution with water to make a final volume of 1·0 ml. Preparation of the phenol red and Tris solution was as detailed by Datta & Shepard (1959a). The timed reaction was started by diverting CO₂ into the temperature equilibrated reaction vessel and the colour end point determined visually. By equilibrating the solution at this relatively high pH (9·0) the precipitation of protein, including carbonic anhydrase, encountered at pH 4·5 during CO₂ equilibration was prevented. Reaction times under 20 seconds were limited by availability of substrate, but a direct relationship between the amount of added enzyme and derived units was obtained when the catalysed reaction was adjusted to 30–50 seconds. A unit of enzyme activity was defined as the difference between the non-enzymatic time (To) and the enzymatic test time (Te) divided by the enzymatic test time:= units.

All reported determinations were done in duplicate and agreed within less than 5 per cent, of one another.

The increase in weight of the embryos is illustrated in Text-figs. 1 and 2 and the average weights and the standard deviation of the average weight for each experiment are given in Table 1.

The total protein as measured by the biuret reaction did not increase appreciably between and days (t = 1·73 D.F. = 16 p = < 0·2, > 0·1; Table 1 and Text-fig. 1). Careful cleaning of the embryos did not alter this finding appreciably. After days there was a steady increase in the protein content. The nitrogen accumulation was proportional to the weight gained from day until day (Text-fig. 2; Table 2).

A sharp increase in carbonic anhydrase activity occurred between and 5 days (t = 3·33, D.F. = 14, p = < 0·01) and was maintained until some time after days (Table 1; Text-fig. 3). A possible increase occurs after the 14th day. In four experiments between and days a number of embryos were divided at a point just anterior to the heart and the separate parts were pooled and analysed for carbonic anhydrase activity. The anterior portion invariably had more activity although its specific activity was generally less than twice that of the posterior portion. The increase in activity at the 4th to 5th day was observed in both portions of the embryo. A sharp increase in retinal activity at around 5 days was confirmed and no activity was detected in the blood until after 11 days. There were 8·6 units per mg. of protein in the -day retina. Allantoic membrane was found to have a very low specific activity (0·28 units/mg. protein). Yolk and embryonic membranes had specific activities of less than 0·01 units/mg. of protein.

The proportion of protein and nitrogen in the different particulate fractions is shown in Text-fig. 4. Only fractionations which yielded protein recoveries within 10 per cent, of 100 per cent, were plotted. Although all fractions showed some carbonic anhydrase activity during the first 4 days there was no consistent localization. In fractionations determined following the increase in specific activity the soluble portion was significantly higher than the others. Representative findings are illustrated in Table 3.

No enzyme inhibiting substance was found in any of the early chick embryo fractions.

Although previous weights for embryos of the Heisdorf and Nelson strain are not available the wet weights reported here are similar to other reported figures for chick embryos (Romanoff & Romanoff, 1933, White Leghorn; Palmer & Levy, 1940, Rhode Island Red; Needham, 1931, several strains). The use of a direct reading analytical balance allowed for more rapid measurement which decreased the loss of weight due to evaporation. In the case of embryos below 10 mg. the difficulty in complete removal of fluid from the surface of the embryo led to rather gross errors in weight determination. Dry weights would be more accurate in this low range.

Neither the expected nor a significant accumulation of biuret protein was observed between and days of development. In spite of more than a four-fold increment in wet weight between the and the -day embryos there was only a 39 per cent, increase in the amount of protein. A similar but less marked finding was seen on comparison of the - and -day embryo protein. This discrepancy in biuret accumulation appears in fig. 5 of the paper by Mahler, Wittenberger, & Brand (1958), but these authors do not give actual figures and make no comment about the finding. During the first 4 days of embryonic life their total biuret protein is less than the values here reported. The amount of nitrogen calculated from the biuret (conversion factor 0·16) was over twice the actual amount of nitrogen found until about the 5th day (Table 2). This suggests the presence of some substance which enhances the biuret reaction during the first 5 days of development. Meticulous care in cleaning any yolk particles from the embryo did not change the findings.

The observation that the activity of carbonic anhydrase increases between and days has not been previously reported except for the retina (Van Goor, 1940; Clark, 1951). The finding that the specific activity of both the anterior and the posterior portion of the young embryo shows a rise seems to make unlikely the possibility that the overall increase is related to a localized retinal build-up. As the anterior portion of the embryo had more activity the possibility of the increment being due to enzyme in the functionally developing mesonephros is unlikely. The allantoic membrane was also found to be low in activity. The moderate rise in. activity after 15 days is probably a reflection of enzyme appearance in the stomach and kidneys as reported by Clark (1951). Whether the increase in activity at days is of any developmental significance in relation to the metabolic and structural changes occurring remains an open question. The exact intracellular role of carbonic anhydrase has never been completely explained. By its hydration of CO₂ it is possible that secondary pH changes occur which in turn alter enzyme reactions or solubilize stored protein. In regard to the earlier-mentioned hypothesis that carbonic anhydrase might decelerate cellular differentiation little can be said except that the time of increasing enzyme activity does coincide with the end of the embryonic period when differentiation is slowing down. There is also at this period a rather marked drop in the daily percentage of weight increment. It should be possible to explore the above further by study of the embryos of other species and by observing the effect of carbonic anhydrase inhibitors on embryonic development. The carbonic anhydrase inhibitor Diamox (acetylaminothiadiazole sulphonamide) has been administered to pregnant rats and rabbits but no deleterious effects on the embryos have been noted (Lutwak-Mann, 1955; Hay et al., 1960). In preliminary studies with the same inhibitor we have been unable to produce malformations in developing chick embryos.

The intracellular distribution of the enzyme has been reported to be largely in the soluble fraction in rat tissues (Datta & Shepard, 1959; Karler & Woodbury, 1960). After days of embryonic life a similar distribution was found in the chick embryo. It will be noted in Table 3 that the -day cell fractionation shows an increase in mitochondrial activity. Other experiments occasionally showed an increase in the microsomal fraction. Because this particulate localization was not consistent, and because of the small number of units present, the author does not consider the carbonic anhydrase localization under days to be reliable. Karler & Woodbury emphasize that the small amounts of activity found in rat mitochondrial preparations cannot be eluted. These presently reported findings do not give any clue as to the site of synthesis of the enzyme.

The percent distribution of protein and nitrogen in the fractions at different ages did not seem to vary. This agrees with the work of Mahler et al. (1958) who found that the protein distribution was unchanged in chick embryos between 2 and 7 days. Because the technique of homogenization and fractionation used are crude and subject to considerable error, it is certainly not possible to be sure that the percentage of total protein or nitrogen of particles does not vary during the embryonic period under discussion. Microscopic techniques such as those used by Shaver (1957) in the study of mitochondrial populations in developing sea urchins may lead to more knowledge in the chick embryo.

1. Le poids frais d’embryons de Poulet Heisdorf et Nelson est comparable à celui d’autres types d’embryons de Poulet.

2. On a observé, de manière inattendue, de grandes quantités de protéines dans des homogénats d’embryons de Poulet, jusqu’au 5^e jour du développement.

3. L’activité spécifique (unités par mg. d’azote ou de protéines) de l’anhydrase carbonique d’embryons entiers a augmenté brusquement entre le 4^e et le 5^e jour. Cet accroissement a eu lieu à la fois dans la partie antérieure et dans la partie postérieure de l’embryon, et n’était pas seulement due à une accumulation dans la rétine.

4. Après 3 j de développement, la localisation intracellulaire de l’anhydrase carbonique est apparue dans la fraction soluble.

5. D’après les techniques utilisées, il n’y a pas eu de modification, entre le 1^er et le 5^e jour, dans la répartition en pourcentage des protéines ou de l’azote dans les fractions centrifugées.

The author gratefully acknowledges the generous technical consultations by Dr. Richard Blandau of the Department of Anatomy, University of Washington, and Dr. Cecil F. McClary of Heisdorf and Nelson Hatcheries, Houghton, Washington. The work was supported by United States Public Health Service Grant RG-6329 (Cl) and Playtex Park Grant No. 74TR.