Analysis of Formed Bodies in Urine Removed from the Crayfish Antennal Gland By Micropuncture

Numerous investigators have remarked upon the secretory appearance of the labyrinth and coelomosac of the crayfish antennal gland (e.g. Marchal, 1892; Peters, 1935 ; Maluf, 1939). It is readily observed in stained sections that vesicles are formed at the lumenad borders of the labyrinth cells. Furthermore, the cells of the coelomosac appear to be filled with vacuoles containing granular material. Parat & Feyel (1930) and Maluf (1941) observed the release of these vacuoles from the coelomosac cells. Kûmmel (1964) has made studies of the coelomosac using the electron microscope. In addition to noting the vacuoles seen by other investigators, he found that the regions of the bases of the cells exhibited considerable pinocytotic activity.

The urine samples obtained by micropuncture from parts of the antennal gland other than the labyrinth often appeared to have objects in them. These objects were first noticed by their diffraction of light. However, they could just be seen when the sample was properly illuminated and viewed at 40 ×. It was therefore decided to investigate the microscopic appearance of urine removed from all parts of the antennal gland. From this investigation it was ascertained that formed bodies are present in the urine from all parts of that organ, except the bladder. The investigation was continued to include chemical analyses of proteinaceous compounds associated with the formed bodies.

In the present report the microscopic appearance of the formed bodies will be discussed, and the results of the chemical analysis will be presented.

Specimens of Austropotamobius pallipes pallipes (Lereboullet) were used in this study. Micropuncture procedures were those described ear her (Riegel, 1963).

Urine samples were removed from each part of the antennal gland and deposited under a drop of liquid paraffin on a siliconed microscope slide. The paraffin was then covered with a coverslip. The slides were viewed under bright-field, phase-contrast and dark-field illumination at 130–1290 × using a Vickers Patholux microscope. Photomicrographs were taken under various illuminations at 320 × or 500 ×, using a Leitz Ortholux microscope fitted with a Leitz Orthomat automatic camera.

Three main analytical methods were used in this study. They will be presented separately in order to simplify the discussion.

The technique of gel filtration using a cross-linked dextran (Sephadex G 25) was found to be well suited to the separation of peptide and amino acid fractions of crayfish urine. Accordingly, columns were constructed and the type found to be best suited for the experiments reported here is illustrated in Text-fig. 1.

The column consisted of thin-walled capillary tubing (c. 0·5−1·0 mm. inside diameter) of variable length. It was inserted into a holder of the type illustrated in Text-fig. 1. The thick-walled capillary portion of the holder served to support the gel column. It also served as a means of attachment for thermometer clamps which held the column rigidly vertical. The holder had a stoppered opening A to permit the emptying of the bulb and the insertion of samples into the gel column. A second opening B was connected by flexible tubing to a reservoir containing the eluant. The flow in the flexible tubing was controlled by a pinchcock. The height of the reservoir could be adjusted to vary the pressure on the gel column. The total volumes of the gel columns used varied from 50 to 100μI. and the height:width ratios varied from 200:1 to 300:1. The tip of the column was drawn to a narrow bore and partially blocked with a minute fragment of glass. The volume of the drops delivered varied from 5 to 10μl.

The gel column was constructed and set up as follows. Pyrex tubing was drawn to a narrow bore taking care to keep the diameter as uniform as possible. The upper few mm. of the resulting capillary were widened into a funnel shape, while the lower end was drawn into a sharply tapered tip. The column was sealed into the capillary bore of the holder with sealing wax as illustrated in Text-Fig. 1.

The column was prepared for use by the following procedure. The delivery tip was inserted into a beaker of eluant and the bulb and column were filled. The pre-swollen gel was then introduced into the column with a pipette, and allowed to flow from the pipette until the column was filled. The bulb was then topped up and the opening A stoppered. The eluant was allowed to flow for 12–24 hr. During this packing process a small space appeared in the column above the gel. This served as a space in which to deposit samples.

Uniform packing of the column was checked by running through it a small volume of egg albumen stained with Congo red or blue-dyed dextran 2000. If packing was complete, stained material could be seen to flow through the column as a discrete band.

Insertion of samples into the column was accomplished as follows. The flexible tubing was pinched off and the bulb was drained by suction through opening A. The eluant in the space above the gel was allowed to drain into the gel. The sample was diluted with 2–3 μl. of eluant, placed above the gel and allowed to drain into it. Then flow was begun as described above. The drops were collected on Parafilm for later chemical or chromatographic analysis.

When urine samples were chromatographed directly after collection they appeared to separate into two fractions which stained with ninhydrin (Pl. 2 A). It was found that the two fractions could be separated in the gel column (Pl. 2B). Therefore, in order to provide conclusive evidence as to the identity of the two fractions, the technique of acid hydrolysis was employed.

All of the micropuncture samples from a single antennal gland were pooled. This gave a total volume of c. 0·3-0·5 μl. The formed bodies in the urine were then disrupted, so far as possible, with distilled water (see Discussion). Sufficient distilled water was added to make up the volume to about ten times the original volume of the sample. The enlarged sample was then placed in the gel column. In order to avoid desalting procedures the eluant used was distilled water in which the two ninhydrin-positive substances appeared to be readily soluble. The drops containing the two fractions were collected on Parafilm and pooled. The pooled samples (c. 28–35 μl) were then dried by gentle warming with a desk lamp.

Hydrolysis vessels were made by drawing out Pyrex tubing of 2 mm. inside diameter. This resulted in vessels having a bowl of 5–10 μl. capacity with sharply tapered arms on either end. The vessels were then fixed with sealing wax into a larger piece of glass tubing. The latter was fitted with a mouthpiece to control the emptying and filling of the hydrolysis vessels.

The hydrolysis vessels were filled with 4N-HCI which was blown out on the dried material from the gel column. The resulting solution was mixed in an air jet and redrawn into the hydrolysis vessels. The tapered ends of the vessels were then sealed in the flame. The vessels were placed on sand in an oven at 105° C. for periods ranging from 30 min. to 30 h.

After hydrolysis, the contents of the vessels were blown out on siliconed watch glasses placed on a hot plate at 100° C. The hydrolysates were evaporated to dryness five times to remove excess HC1. Distilled water was added after each evaporation except the last. The hydrolysates were stored in a desiccator until they were to be analysed chromatographically.

The method of thin-layer chromatography was used for the identification of ninhydrin-positive substances. Silica gel G (Merck) layers, 250 μ thick, were used routinely. One-way chromatograms were run for distances of 10– 17 cm., using a solvent composed of n-butanol, acetic acid and water (60:20:20, w/w). Two-way chromatograms were run for distances of 10 cm. each way. The above-mentioned solvent was used on the first run; phenol:water (4:1, w/w) was used as the solvent on the second run. The unknown amino acids were identified by comparison with chromatograms of known amino acids run under identical conditions. The foregoing procedures are modified slightly from those described by Randerath (1963).

The formed bodies, which were observed in all parts of the antennal gland except the bladder, are illustrated in Pl. 1A-E. Those in the coelomosac are usually yellowish in colour when viewed under bright-field illumination. They take the form of spheroids. Generally, they are much smaller than 10 μ in diameter (see Pl. 1 A). In the labyrinth the formed bodies are of two types : first, there are large (c. 20 μ) irregularly shaped vesicles, and second, there are spheroids which appear to be identical with those in the coelomosac. The vesicles appear to be packed with granular material, and at times they contain spheroids which are similar to those in the coelomosac. The granular material of the vesicles appears yellowish to bright green when viewed under bright-field illumination. These colours correspond to the macroscopic appearance of the labyrinth. The formed bodies of the labyrinth are shown in Pl. 1B.

The proximal tubule contains only spheroids (Pl. 1C), which in general shape resemble those in the coelomosac. However, their average size appears to exceed that of the coelomosac spheroids. Urine removed from the proximal portion of the distal tubule contains masses of spheroids. Their general size appears to be somewhat smaller than that of the spheroids in the proximal tubule (see Pl. 1D). In the distal portion of the distal tubule the spheroids are again in evidence. However, the average size is here much larger than the average size of the spheroids in the preceding parts of the antennal gland (Pl. 1E).

The photographs in Pl. 1 illustrate a striking characteristic of the formed bodies: they vary considerably in density in relation to the fluid which surrounds them. This is indicated by two observations. First, the numbers of spheroids in any single plane of observation indicates the numbers of spheroids of equal density. Secondly, the depth of the plane of observation indicates the relative density of spheroids and medium. In the coelomosac (Pl. 1 A) the spheroids are either equally dense as or only slightly denser than the surrounding medium. It is difficult to determine the relative densities of medium and formed bodies in the labyrinth (Pl. 1B). Samples removed from the labyrinth are usually thickly packed with formed bodies. In the proximal portion of the tubule (Pl. 1 C) the formed bodies appear to be less dense than their medium. They rise to the surface of the sample. In the proximal portion of the distal tubule (Pl. 1D) the formed bodies appear to be more dense than their medium. The fact that they aggregate may cause them to settle, however. In the distal portion of the distal tubule (Pl. 1 E) the formed bodies are again less dense than the surrounding medium.

The reactions of the formed bodies to changes in the osmotic pressure of their medium is very strange. The vesicles of the labyrinth are extremely sensitive to the osmotic pressure of their medium. They swell and burst when the medium is diluted and shrink when it is allowed to dry out. The spheroids in the coelomosac and laby-rinth do not appear to be sensitive to dilution, even when diluted many times. The spheroids in the distal tubule rapidly swell and burst when diluted with distilled water.

Pl. 2 A, illustrates the result when samples of urine of c. 0· 09 μl. are plated directly on thin layers of silica gel and chromatogrammed. Generally, an intensely staining spot showed up on the origin, and several spots appeared above the origin. When the urine samples were put through the gel-filtration column prior to chromatography, a very characteristic pattern emerged (Pl. 2B). The material which had remained on the origin (Pl. 2 A) was separated by the gel filtration from the material which had moved in the solvent.

The gel column used to obtain the results shown in Pl. 2 had the following characteristics. Dyed egg albumen (mol. wt. = c. 45,000) or dextran 2000 (mol. wt. = 200,000), substances which are wholly excluded from the gel, passed out of the column in drops 3– 4. Sodium, a substance which travels wholly within the gel, passed out of the column in drops 10– 13. The material which ran in the solvent travelled wholly within the gel, indicating that it had a relatively low molecular weight. The material which remained on the origin (Pl. 2 B) appeared in drops 6-9. This indicated that neither is it wholly excluded from, nor does it travel wholly within, the gel. Sephadex G 25 has an exclusion limit of about 5000 molecular weight; the material in drops 6– 9 probably has a molecular weight of less than 5000. In order to avoid adsorptive effects, the eluant was varied from distilled water to ionic solvents (20 and 200 mM./l. NaCl, 180 mM./l. KC1). This had no affect on the pattern shown in Pl. 2B.

As far as could be ascertained no soluble material appeared in drops 1– 5 collected from the gel column. A possible explanation of this is that any large molecules (e.g. protein) would be in the formed bodies of the coelomosac and labyrinth (spheroids). As mentioned earlier, these formed bodies are extremely resistant to disruption by distilled water.

The material in drops 6– 9 was subjected to acid hydrolysis for periods ranging from 30 min. to 14 hr. After 30 min. the material would run in the chromatographic solvent, but several large indefinite spots were all that could be seen after treatment with ninhydrin. If subjected to hydrolysis for 6 hr. or longer, twelve amino acids were consistently discernible on two-way chromatograms. These were alanine, arginine, cystine, glutamic acid, glycine, leucine, lysine, phenylalanine, proline, tryptophane, tyrosine and valine. This is very strong evidence that the material in drops 6– 9 contained a peptide.

The material of drops 10-13 probably contained only amino acids, although the presence of dipeptides could not be ruled out. No new spots showed up on the chromatograms after even 30 hr. of hydrolysis.

As shown previously (Riegel, 1966), the spheroids of the coelomosac contain an acid protease and the vesicles of the labyrinth contain an alkaline protease. This makes clear much of the data presented here, and it also clarifies a number of observations made previously. The site of these enzymes within the formed bodies cannot be ascertained from the present experiments. However, as mentioned earlier, the formed bodies have granules within them which in colour and size resemble the granules seen in the cells of the coelomosac and labyrinth. The granules in the cells of the coelomosac and labyrinth appear to occupy clear spaces (Peters, 1935 ; personal observation). It is possible that the digestive enzymes are connected with the granules.

As shown in Pl. 2 A, the products of protein digestion, peptides and amino acids, are most concentrated in the distal tubule. A large amount of peptide is also present in the labyrinth. The latter observation possibly indicates that the alkaline-active protease in the labyrinth has only peptidase properties.

The concentration of peptides and amino acids in the distal tubule can be accounted for partially by water reabsorption there (Riegel, 1965). However, probably most of the concentration is due to the fact that digestion occurs there.

As mentioned in the results section, the spheroids in the coelomosac and labyrinth are extremely resistant to dilution. The spheroids in the distal tubule are not resistant to dilution. This suggested that the osmotic behaviour of the spheroids is connected with the distribution of osmotically active particles within them. It was inferred, correctly, as it turned out, that there is a change of pH between the proximal portion of the antennal gland and the distal portion. This suggested experiments of the kind outlined below.

Samples of urine (c. 0·1 μl.) were removed from the coelomosac and placed under liquid paraffin in siliconed watch glasses. Then, whilst being observed at 80 × in a stereo-microscope, 0·1 μl. of 200 mM./I. NaCl (made acid, pH = 6, with HC1) was added. The spheroids were seen to swell for periods up to 5 min. Then c. 0·2 μl. of neutral distilled water was added. The spheroids were seen to swell greatly and the larger of them burst.

Experiments of the kind outlined above provided a useful method for the study of enzymes in the spheroids of the crayfish and other animals (Riegel, 1966). However, the results are also relevant to the present discussion. It is probable that large, un-digested molecules are present in the spheroids in the proximal portions of the antennal gland. When the spheroids reach the distal portion of the gland, probably acids conditions are met, digestion occurs, and the products are osmotically active.

The puzzling feature of this is that it would be expected that large molecules would be less active osmotically. However, it would not be expected that formed bodies containing such particles would be completely impervious to distilled water. This suggests that the large undigested molecules in the spheroids are bound in some way into their structure. This would certainly explain why no soluble material of molecular weight greater than 5000 was separated in the gel-filtration column. As will be discussed later, Kümmel (1964) has presented evidence that the vacuoles in the apical regions of the coelomosac cells are formed by the fusion of tiny vesicles which appear to be produced by the Golgi apparatus. It is likely that the apical vacuoles are the same as the spheroids which are collected from the lumen of the coelomosac. Therefore, it is possible that the spheroids are structured elements rather than bags of accumulated loose materials.

As was observed by Maluf (1941) and by the writer, the protein-binding dye, Congo red, selectively stains the coelomosac. Furthermore, the writer has observed that the dye is localized in the spheroids in urine removed from the coelomosac some hours after injection. During the pre-moult (D) stage of the moult cycle the blood of crayfishes often contains an orange pigment (in the species here studied), the so-called ‘lipochrome’. The writer has observed that the coelomosac of such crayfishes is often deeply stained the same colour as the blood. Furthermore, spheroids in urine samples removed from the coelomosac of D-stage crayfishes often have the same colour as the blood. This strongly suggests that the blood lipochrome is accumulated and eliminated by the coelomosac cells. Protein labelled with fluorescent dye is also accumulated by the coelomosac cells (Kirschner & Wagner, 1965).

The possible mechanism by which materials are accumulated by the coelomosac cells has been clarified by Kümmel (1964). He has shown that pinocytotic vesicles are formed at the bases of the coelomosac cells. Furthermore, he has presented evidence that the large vacuoles which occupy the apical regions of the coelomosac cells are produced by the fusion of small vesicles which appear to be produced by the Golgi apparatus. Probably the apical vacuoles are the same structures as the spheroids which are found free in the lumen of the coelomosac.

It is not clear what difference in function, if any, exists between the coelomosac and labyrinth (in an overall sense). The labyrinth vesicles are found only in the labyrinth. They probably become disrupted distally. However, they often appear to be filled with spheroids which appear to be identical with the spheroids found in the other parts of the antennal gland. It is probable that only the outer membrane of the vesicles is disrupted, freeing the spheroids and other materials.

Material accumulated in the coelomosac presumably must first pass through a filtering mechanism (Kümmel, 1964; Kirschner & Wagner, 1965; however, see, discussion by the writer, 1966). Kirschner & Wagner (1965) have shown that the antennal gland of the crayfish is freely permeable to dextrans of 20,000 to 40,000 molecular weight; that is, the urine:blood ratios for those dextrans are identical with the urine:blood ratios for inulin. It is possible that the labyrinth cells accumulate materials which are either too large or are otherwise unsuitable for entry into the urine via the coelomosac cells. The dyes phenol red and cyanol, though of relatively small molecular weight, are accumulated by the labyrinth cells (Maluf, 1941). Further, cyanol can be seen in the labyrinth vesicles (personal observation). It is possible that relatively neutral molecules (such as lipids, sugars and conjugated proteins) are eliminated by the coelomosac, whilst charged molecules (dyes, unconjugated proteins, etc.) are eliminated by the labyrinth.

In the foregoing portions of this paper and in the preceding paper (Riegel, 1966) evidence has been presented that the antennal gland of the crayfish performs a digestive function. Presumably the materials which are normally accumulated and released by the cells of the coelomosac and labyrinth are surplus to the requirements of the animal’s metabolism. Direct supporting evidence exists only for the digestion of proteinaceous compounds, but it is likely that other kinds of materials are involved

The digestive mechanism in the antennal gland of the crayfish represents an elegantly balanced arrangement. Digestion products which are of further use to the animal can be reabsorbed distally. In conclusion, it must be said that evidence is accumulating that it is the bladder of the antennal gland which is mainly responsible for reducing the concentration of the urine. It is hoped that more information on the role of the bladder will be forthcoming from studies of that tissues, in isolation, which are being pursued currently in this laboratory.

I am indebted to the Medical Research Council for their financial support of the research.

Photomicrographs of urine samples removed from various parts of the crayfish antennal gland. All photomicrographs were taken at a magnification of 320 × using phase-contrast illumination on a Leitz Ortholux microscope fitted with a Leitz Orthomat automatic camera. A, Coelomosac ; B, labyrinth ; C, proximal tubule; D, proximal portion of the distal tubule; E, distal portion of the distal tubule.

A. Reproduction of a typical plate resulting from the chromatography of 0·09 μl. samples of urine. Abbreviations: C, coelomosac; L, labyrinth; PT, proximal tubule; PDT, proximal portion of the distal tubule; DDT, distal portion of the distal tubule; BLAD bladder. B. Reproduction of a typical plate resulting from the chromatography of a large sample of urine after running it through a gel-filtration column. The numbers under the origin points refer to the sequence of drops as they fell from the column orifice. US refers to a sample which was plated directly on the thin-layer plate.