1. The seminal plasma of Balanus balanus has been analysed for a number of organic substances including total nitrogen, protein, urea, protein-bound hexose, hexosamine, fucose, ‘sialic acid’, glycogen, and RNA. The material was separated into soluble and insoluble fractions by precipitation with ethanol.

  2. A large part of the ethanol-soluble material is inorganic salts and glycine; it contains a small quantity of protein-like material which on hydrolysis yields largely glycine and glutamic acid.

  3. The alcohol-precipitated material contains protein, protein-bound hexose, hexosamine, lipids, and RNA. Glycogen, fucose, and ‘sialic acid’ are absent.

  4. It is suggested that the plasma contains glycoproteins of a mucoid nature: the absence of both fucose and ‘sialic acid’ suggests that the mucoid may be of a relatively simple character.

  5. Attention is drawn to the similarity of this seminal plasma, which is produced in the absence of accessory male organs, to that of higher animals.

Some data have already been given concerning both the inorganic and organic constituents of the seminal plasma of the common boreo-arctic sublittoral cirripede, Balanus balanus (Barnes, 1962a, b). The seminal plasma contains some 50% of its total solids as organic material, part of which may be readily precipitated by 5 % trichloroacetic acid; it is well known, however, that many seminal proteins, peptones, and proteoses are not precipitated by this reagent (Mann, 1954). The function of the organic material in invertebrate seminal plasma is little understood; it is well known that in some invertebrates, at least, it is of little importance as a respiratory substrate, since the latter is derived from endogenous phospholipid material (Rothschild & Mann, 1950; Rothschild & Cleland, 1952; Mori, 1959a, b). The present analyses were made in order to determine in more detail the nature of the organic material present in the plasma, so that its contribution to the physical and biological properties might be more accurately assessed.

The plasma was obtained as previously described (Barnes, 1962a), although the samples were taken somewhat earlier (1−2 weeks) in the season; the spermatozoa within the seminal vesicles were, however, fully ripe. In an attempt partially to fractionate the organic substances present ethanol, rather than trichloroacetic acid, was used as a precipitant; ethanol at the required strength was added to the plasma and after stirring vigorously the mixture was centrifuged. The precipitate and supernatant were examined separately.

The supernatant alcoholic solutions

A sample (20 μl.) was spotted directly on Whatman No. 1 paper and chromatographed for amino acids. An aliquot of the supernatant was then used for the estimation of total nitrogen and the remainder was dialysed against distilled water for 24 hr. After dialysis the material was made up to a known volume, an aliquot taken for a second nitrogen estimation, and the remainder was hydrolysed (see below) and examined by paper chromatography for its amino-acid content. In one sample the total solids, organic and inorganic material (after ignition), nitrogen, phosphorus and protein were determined on the 80% ethanol-soluble material and after dialysis.

The precipitate insoluble in ethanol

The precipitate was washed first with absolute ethanol then with ether, and dried over calcium chloride; the loss on oven-drying (105°C.) was determined on a sample and although all the results are expressed on an oven-dry basis, the original material was normally used for analyses. Total nitrogen, total phosphorus, protein-bound hexose, hexosamine, fucose, ‘sialic acid’, and lipids were determined as described below on samples of the precipitates, and in one sample the glucosamine/galactosamine ratio and the RNA content were determined.

Hydrolytic and chromatographic methods

When amino acids were to be determined the material was hydrolysed by refluxing for 12−24 hr. under a long air condenser with a 1:1 mixture of 6 N-HCI and 90% formic acid; the presence of formic acid reduces the destruction of sulphur-containing amino acids (Block, Durrum & Zweig, 1955). The hydrolysate was evaporated to a small bulk under reduced pressure, water was added and the solution was again evaporated. This was repeated several times to remove most of the hydrochloric acid, after which the solution was taken to dryness and the residue was kept over solid KOH in a desiccator. The resultant solid was made up to a known volume with 10% isopropanol, centrifuged and an aliquot (usually 20 μl.) of the supernatant was applied to the paper (Whatman no. 1). Conventional descending two-dimensional chromatography was used, with butyl alcohol-acetic acid mixtures in the first direction and phenol-water in the presence of 3 % ammonia and KCN in the second direction. The spots were held over ammonium hydroxide before the first run and the papers were steamed before each run. Spots were identified by specific tests, Rf values, and a comparison of their positions with those of known amino acids. For estimation, the spots were first identified with dilute ninhydrin, cut out, and then fully developed. Six replicate papers were usually run. Calibration curves were set up from the mean of six replicate runs of the identified amino acids at each of three concentrations taken through the same hydrolysis procedure and the two-dimensional chromatography.

Chemical analyses: methods

Total nitrogen and total phosphorus were determined on both the precipitated material and the dialysed supernatant by the methods already described (Barnes, 1962 a). Ammonia was determined by distillation and estimation as in total nitrogen procedure. Protein-bound hexose was determined by the method given by Winzler (1955) using a galactose-mannose (equal quantities) standard. Hexosamine was estimated, after hydrolysis with 3 N-HCI, by the Elson-Morgan technique as outlined by Winzler (1955) except that an amount of sodium chloride equivalent to that contained in the unknown after neutralization was added to the solutions used in setting up the calibration curves (see Ashwell, 1957). Fucose was estimated by the method of Dische & Shettles as given by Winzler (1955) with the following slight modification. It was found difficult to dissolve some of the material completely in 0·1 NaOH and the colour developed by sulphuric acid (before the addition of cysteine hydrochloride) was variable even with replicate samples. The sample was, therefore, split into two equal portions after the acid treatment, one half only receiving the Cysteine hydrochloride reagent and the other, after the addition of an equivalent amount of water, serving to estimate non-specific colour development. ‘Sialic acid’was estimated after hydrolysis of the sample with 5 % trichloracetic acid by the desoxypentose reaction with diphenylamine in an acetic-sulphuric acid solution (Winzler, 1955). The ratio of glucosamine to galactosamine was determined, in a single sample of the material precipitated by 80% ethanol, by Tracey’s method (see Gardell, 1958) which takes advantage of the differential depression of colour formation in the two hexosamines by borate in the Elson-Morgan reaction. Glycogen was estimated by the method of Kemp & van Kits Heijningen (1954). Urea was determined in untreated plasma by incubation with urease (B.D.H. preparation); after 3 hr. trichloroacetic acid was added, the precipitate was centrifuged off and the ammonia in the supernatant was distilled off and determined as in the total nitrogen procedure. A control portion of the untreated plasma, without the addition of urease, was taken through the whole procedure. Lipids were estimated as the material lost by repeated extraction of the solid with a hot 2:1 (by volume) chloroform-methanol mixture; such a simple extraction procedure, although subject to a number of errors (for a discussion see Sperry, 1955), is adequate for the present purpose. Nucleic acid (RNA) was determined in a lipid-free sample by the Schmidt-Thannhauser procedure, estimating the pentose by the Mejbaum orcinol reaction (see Volkin & Cohn, 1954). Protein in the material left after dialysis was determined by the biuret reaction with a casein standard (Layne, 1957).

The nitrogen content of the fractions

Table 1 gives the results obtained on two or three separate batches of pooled seminal plasma after precipitation with 80 % ethanol. It is evident that of the total nitrogenous material (3−4 mg. N per ml. plasma) about one-third is soluble in 80 % ethanol and of this only one-tenth remains after dialysis. Urea could not be detected and the concentration of ammonia was low. On treatment of the plasma with a series of graded concentrations of ethanol the amount of material precipitated is reduced as the concentration of alcohol is lowered (at 20 % ethanol only a turbidity is obtained) and at the same time the nitrogen content after dialysis increases (Table 2); as might be expected, more non-dialysable nitrogenous material remains in the supernatant as the concentration of the ethanol is reduced.

Table 1.

Nitrogen fractions (mg. N/ml. seminal plasma) fractionated with 80% ethanol

Nitrogen fractions (mg. N/ml. seminal plasma) fractionated with 80% ethanol
Nitrogen fractions (mg. N/ml. seminal plasma) fractionated with 80% ethanol
Table 2.

Weight of precipitate (mg. /ml. seminal plasma) and nitrogen content (mg. N/ml. seminal plasma) of fractions after precipitation with a graded series of ethanol

Weight of precipitate (mg. /ml. seminal plasma) and nitrogen content (mg. N/ml. seminal plasma) of fractions after precipitation with a graded series of ethanol
Weight of precipitate (mg. /ml. seminal plasma) and nitrogen content (mg. N/ml. seminal plasma) of fractions after precipitation with a graded series of ethanol

Composition of the ethanol-soluble material

The results of further analyses on one sample of the 80 % ethanol-soluble material are given in Table 3. On dialysis, salts and most of the organic matter are lost, the values confirming the nitrogen estimations before and after dialysis. Before dialysis the nitrogen content (8·0 %) of the organic matter is higher than that conventionally accepted for proteins; chromatography of the solution before dialysis shows that most of this material may be accounted for as glycine. The raw alcoholic extract also gave a spot near to the position of cystine, but this disappeared on hydrolysis; the previously reported presence of cystine in the plasma must, therefore, be regarded as erroneous (Barnes, 1962a). The material remaining after dialysis is completely accounted for by the protein analysis (casein standard) and chromatography after hydrolysis shows that glycine, accompanied by a smaller quantity of glutamic acid, are the chief components of this protein fraction; traces of alanine, valine, and probably leucine were also present as indicated by very weak spots in the appropriate positions after development with ninhydrin. It is of interest to note that in the original material (before dialysis) a spot was obtained in the position of ethanolamine; no tests were applied other than a measure of the Rf value. Ethanolamine may well have been derived from the breakdown of endogenous phospholipids utilized in respiration and their subsequent diffusion into the plasma.

Table 3.

Fractionation of material soluble in 80% ethanol

Fractionation of material soluble in 80% ethanol
Fractionation of material soluble in 80% ethanol

The composition of the ethanol-precipitated material (Table 4)

Both glycogen (on long standing) and glycoproteins (more readily) may be precipitated by ethanol. Glycogen, however, was not found in the precipitates, either free, or after hydrolysis (as glucose) with trichloroacetic acid in the presence of silver Sulphate. This suggests that the glycogen previously shown to be present in the plasma is either free or combined with the protein fraction soluble in 80 % ethanol; qualitatively the solution after dialysis, which contains little nitrogen (see above), was shown to react strongly positive to tests for glycogen. The total nitrogen in the precipitate, and also the hexose and hexosamine, in general increased as the concentration of ethanol was reduced, suggesting that, at least in part, all three may be associated in the plasma as mucoprotein. Any such material may, however, be accompanied by free proteins. The glucosamine/galactosamine ratio was 1:1. Fucose and ‘sialic acid’ were absent. (If the spectrophotometric readings were accepted they indicate 0·6 % ‘sialic acid’; such a small quantity determined by this method, particularly in view of the fact that the test solution was a blue colour as distinct from the purple given by ‘sialic acid’, may be considered as not significant.) Although the method used will tend to give high values for lipid, it is evident that a considerable quantity of material of this nature is precipitated by 80 % ethanol.

Table 4.

Composition (% oven-dry weight) of material precipitated by ethanol

Composition (% oven-dry weight) of material precipitated by ethanol
Composition (% oven-dry weight) of material precipitated by ethanol

The above analyses of the seminal plasma of Balanus balanus show that it contains considerable quantities of organic material, only a part of which is precipitated by 80 % ethanol. The results are summarized in Table 5. Apart from the inorganic salts the solid content of the plasma is largely made up of free glycine, a small quantity of non-dialysable proteinaceous material, mucoid substances, and lipids, while a considerable quantity of RNA is present.

Table 5.

Composition of seminal plasma: Balanus balanus

Composition of seminal plasma: Balanus balanus
Composition of seminal plasma: Balanus balanus

We have little information on the organic material of invertebrate seminal plasma, particularly as regards the nitrogenous fraction. Hayashi (1945) found 2·5 mg./ml. protein in the seminal plasma of Arbaciapunctulata—much less than that found in the present investigation (3−4 mg. N/ml.); A. punctulata has, however, a much higher plasma to spermatozoa ratio, and the quantities per ml. semen may be more comparable. In human seminal plasma there are 30-50 mg. of protein-like material per ml. of which 60% passes through membranes impermeable to blood serum proteins (Huggins, Scott & Heinen, 1942). If it were assumed that all the material precipitated by 80% ethanol would be retained if untreated plasma of Balanus balanus were dialysed, only 20−30 % of the nitrogenous material in the plasma is dialysable. If all the nitrogen is calculated as protein, using the conventional factor, the composition of the material precipitated by 80% ethanol is: protein 32·8%, hexose 10·5%, hexosamine 7·2 %, and lipids 20·2 %. The phosphorus may be in part extracted with the lipid fraction but would otherwise contribute to the deficit. There may, however, be substances present whose nitrogen content is somewhat less than that of proteins.

The presence of hexoses and hexosamines along with proteins clearly indicates the presence of glycoproteins and these are probably of a mucoid rather than a mucoprotein nature. The absence of both fucose and ‘sialic acid’suggests that the mucoid is of relatively ‘simpler’ nature. The mucoid is almost certainly the substance responsible for the relatively high viscosity of the plasma and may well coat the spermatozoa even in washed semen. It is well known that serum mucoids vary in composition and that several distinct entities are present in blood plasma. Oroso-mucoid, which up to the present is the best defined mucoid, contains about 40% total carbohydrate; the precipitated material obtained from the semen of B. balanus contains 35 % (on a lipid-nucleoprotein-free basis). Other mucoids are, however, known which contain only 4−6% carbohydrate. Further, some of the protein may be bound with lipid so that the proportion of carbohydrate in the mucoid material would be greater. A number of protein fractions of human seminal plasma have been recognized by electrophoretic methods (Gray & Huggins, 1942; Ross, Curzrok & Miller, 1941) as well as a mucoprotein which contains 9·3% nitrogen, 10·8% hexosamine and which after hydrolysis gives 26·8 % reducing substances; the nitrogen-hexosamine ratio (1:1) is similar to that found here. Gross analyses such as those given here for nitrogen and the carbohydrate moieties can give no information with regard to any specific entities, mucoids or protein, which may be present.

The results extend the number of substances known to be present in invertebrate seminal plasma to include proteins, mucoids, lipids, and RNA, and make it increasingly evident that in spite of the absence, as far as is known, of accessory male organs, the plasma has many resemblances to that of higher animals. A number of functions have been suggested for the free amino acids in seminal plasma (Tyler & Rothschild, 1951; Giese & Wells, 1952; Metz & Donovan, 1950), but the functions of most of the other components discussed here are largely unknown even in animals whose seminal plasma has received much more attention. The fact that they are present even in the absence of accessory organs suggests that, unless they are to be regarded as merely the by-products of the metabolism of the developing spermatozoa, they are of general importance to the functional activity of the whole semen. Lipids and RNA are known to be present in the spermatogenic cells of many species and more may remain in the semen when accessory glands are absent. The amino acids may result from the activity of proteolytic enzymes on the proteins during spermatogenesis. The presence of ethanolamine is of considerable interest since endogenous phospholipids are a major oxidizable substrate in some invertebrate spermatozoa; it may originate as residual material from the utilization of such substrates during development. When it is remembered that, although ergothioneine is absent, ascorbic acid is present in some quantity in B. balanus seminal plasma, the similarity with vertebrate semen becomes even more marked. Indeed the most marked difference is the absence of a glycolysable substrate, such as fructose which is produced by accessory glands.

The ‘sialic acid’ used as a standard in this investigation was kindly provided by a colleague Mr J. Doyle.

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