THE COMPOSITION OF THE SEMINAL PLASMA OF BALANUS BALANUS

Transcription

1 J. Exp. Biol. (1962), 39, Printed in Great Britain THE COMPOSITION OF THE SEMINAL PLASMA OF BALANUS BALANUS BY H. BARNES The Marine Station, Millport, Scotland {Received 19 March 1962) Studies of a small number of mammalian species, a few common fishes, and to a lesser extent some echinoderms, have provided much of the available data on the biochemistry of semen: the Crustacea have hardly been investigated. The present work is part of a study of the physiology and biochemistry of reproduction in the Cirripedia, particularly in relation to the ecology of this group. The swollen seminal vesicles of a sexually ripe balanomorph barnacle are very prominent, and in some species form a considerable part of the total body tissue. They lie near the surface of the body and may easily be dissected out; the semen, when expressed, is a sticky, white, viscous fluid, which on coming into contact with sea water, at first coagulates forming a stringy mass. Although the animals are hermaphrodites, cross-fertilization is usual and sometimes obligatory, the semen being introduced into the mantle cavity of the functional female by copulation. After copulation the coagulated semen is not, therefore, at first easily washed out of the mantle cavity of the functional female. After standing in sea water for some time, however, the semen may be more readily dispersed. The changes involved in coagulation and dispersal have not yet been elucidated, but under natural conditions this breakdown does not appear to take place so readily, since a white gelatinous mass may be exuded from the mantle cavity after copulation. MATERIAL AND METHODS Balanus balanus (L.) Da Costa has been used for the following reasons: first, it is one of the largest species found in British waters; secondly, it may easily be obtained locally in reasonably large quantities; thirdly, it has a single annual brood (Barnes & Barnes, 1954) so that not only is there a large production of spermatozoa at the breeding season but also the sexual products are in a similar state of ripeness throughout the population. Although when ripe the seminal vesicles are grossly distended with semen and may be removed easily and cleanly without adherent body tissue, the collection of a large quantity of semen is nevertheless laborious and presents certain difficulties; a moderate-sized animal contains only some cvoi-c-02 ml. of semen. The separation of the extremely viscous semen from the seminal vesicles in quantities sufficient for analysis would involve excessive evaporation. In this work the distended vesicles and contained semen were, therefore, transferred intact to a centrifuge tube and before separating off the seminal plasma by high-speed centrifugation the viscous mass was macerated with a glass rod. As a consequence, the residual sperm fraction was contaminated with vesicle tissue. From the dimensions of the seminal vesicles

2 346 H. BARNES it is estimated that their tissue made up about 4% by volume of the whole. The seminal plasma, after separation by centrifugation, was poured off and, unless analysed immediately, was stored at 20 0 C. Density was determined by weighing a known volume, ph using B.D.H. multirange papers, total solids after drying to constant weight at C, and ignition residue after heating at 6oo C. in an electric muffle. Inorganic ions were determined by the methods of Robertson & Webb (1939) with the minor modifications indicated by Rothschild & Barnes (1953, 1954). Proteins were precipitated with trichloracetic acid (TCA, equal volume of 20%) and the nitrogen in both precipitate and supernatant was estimated by a Kjeldahl procedure; the material was wet-ashed, using a copper sulphate-selenium catalyst, and after neutralization the ammonia was distilled off into o-i N-HC1 and estimated in an aliquot by the method of Riley (1953), the colour being developed with a sodium hypochlorite-phenol reagent freshly prepared each day (Barnes, 1959). The transmittancies were determined in a 1 cm. cell at 625 m/x. Phosphorus was determined by the method of Fiske & Subbarow (1925) after wetashing with concentrated sulphuric acid and clearing with a drop of 100 vol. hydrogen peroxide (M.A. Reagent, B.D.H.). When this method, which is so satisfactory for many kinds of organic materials, is used it is advisable to add a small portion of the sulphite reagent before the ammonium molybdate reagent and the rest after it in the usual manner. In this way any traces of peroxide which still remain even after reheating and evaporating several times, are reduced and no yellow colour is produced on addition of the molybdate reagent. The glucose content of deproteinized seminal plasma was determined by the method of Folin & Malmros (see Umbreit, Burris & Stauffer, 1945), and glycogen by the same method after first separating it by precipitation and then hydrolysing according to the procedure of Dales (1957). Fructose was estimated according to Roe (see Umbreit, Burris & Stauffer, 1945; Mann, 1946). Lactic acid was determined by the Barker & Summer8on method (1941) with the modifications suggested by Umbreit et al. (1945). Calibration curves were prepared using standards of analytical reagent quality. Phosphatase activity was examined qualitatively by incubating at room temperature 2 mg. of i-phosphoglucose with 0-2 ml. seminal plasma diluted with 2 ml. sea water for 4 hr.; 5-nucleotidase by a similar procedure with adenosine 5-phosphoric acid, and ATP-ase by incubation with ATP. Phosphate was estimated after incubation. The appropriate controls and blanks were run. The reducing action towards 2,6-dichlorophenolindophenol was measured (Roe, 1954). Plasma was treated with an equal volume of 5% metaphosphoric acid, the precipitate centrifuged off and an aliquot of the supernatant titrated with a solution of the indophenol from a micro-burette. The reagent was standardized against freshly prepared ascorbic acid. Ascorbic acid was determined by 2,4-dinitrophenylhydrazine (Roe, 1954). After treating the plasma with metaphosphoric-acetic acid solution and removing the precipitate, active charcoal was added and the mixture vigorously shaken. The charcoal was removed and an aliquot of the solution treated with the 2,4-dinitrophenylhydrazine-thiourea reagent. After maintaining at 37 C. for 3 hr. 85 % sulphuric acid was added slowly, and the optical density measured after 30 min.

3 The composition of the seminal plasma of Balanus balanus 347 at 540 m^.. Ascorbic acid taken through the procedure gave the appropriate calibration curve. Chromatography was carried out by standard procedures using the descending method and a variety of solvents. The estimation of the number of spermatozoa per unit volume of semen was made by diluting a known amount of semen with sea water and counting the spermatozoa in replicate samples under high power in a haemocytometer cell. RESULTS In view of the various modes of expression of the composition of semen used by different workers the relevant data for a number of conversions are given in Table 1. The analytical results are shown in Table 2. The seminal plasma is a slightly yellow, somewhat viscous fluid. It has a density of 1*044 an(^ ph of TZ~1'\- This is distinctly more acid than sea water, and although more acid than the value of y-6-^'g recorded by Hayashi (1945) for the seminal fluid of Arbacia punctulata, is similar to that given by Rothschild (1951) for the seminal fluid of Echinus esculentus. Table 1. Conversion factors for Balanus balanus semen 100 ml. semen 23-I ml. plasma (d 1-04) 76-9 ml. spermatozoa (d = t-i8) 24-1 g. plasma 90-8 g. spermatozoa 33-7 g. vacuum dried (37-1 %) 30-9 g. oven dried (34-0 %) ml. semen ml. spermatozoa 1 14 x io 10 spermatozoa 1-48 x io 10 spermatozoa g. wet spermatozoa 1-25 x io 10 spermatozoa g. vacuum-dried spermatozoa g. oven-dried spermatozoa 3'37* IC " 10 spermatozoa 3'7 x ' 10 spermatozoa Balance of the constituents The inorganic ions show a cation deficit of 178 m-equiv./l. The amount of TCAsoluble phosphorus is small, less than mg./ml., and even if all this were assumed to be inorganic phosphorus (1-5 charges being assigned to each phosphorus atom) it could only contribute 4-8 cationic m-equiv./l. It is almost certain that the cation deficit is largely made up by free amino acids present in the plasma and estimated in the non-protein fraction. If the 1-84 mg./ml. non-protein nitrogen corresponded to a monobasic acid it would account for 131 m-equiv./ml.; dibasic acids may well be present and indeed only a small quantity of the latter would be required to establish the ionic balance. Chromatography showed considerable quantities of cystine in the plasma. If chloride were assigned to sodium, and the remainder of this element together with potassium and calcium were calculated as carbonates, the ash on ignition should be 31-3 mg./ml.; this is in good agreement with 33 mg./ml. found experimentally. The total solids determined directly amounted to 64*3 mg./ml. and

4 348 H. BARNES this is reasonably well accounted for by the analyses: inorganic ions account for 25-8 mg./ml. leaving 33-5 mg./ml. of organic material. Conversion of the nitrogen and phosphorus by factors of 7 and 15 and addition of the small amounts of glucose, glycogen, and lactic acid, gives 25-3 mg./ml. of organic material leaving only 8-2 mg./ ml. not accounted for by the above analyses. Table 2. Composition of seminal plasma, Balanus balanus Density ph 7-3-7'4 Total solids (oven dry, 105 C.) mg./ml. Residue on ignition 33-0 mg./ml. Inorganic constituents Sodium mg./ml. 470 m-equiv./l. Potassium 1-26 mg./ml. 3a m-equiv./l. Calcium 0-77 mg./ml. 38 m-equiv./l. Chloride mg./ml. 362 m-equiv./l. Organic solids, by difference mg./ml. Nitrogen fractions TCA-soluble 1-84 mg./ml. TCA-inaoluble 1-3 mg./ml. Phosphorus fractions Total 0*14 mg./ml. TCA soluble "O95 mg./ml. Lipid fractions, insoluble mg./ml. Non-lipid fractions 0-04 mg./ml. Glycogen O-88 mg./ml. Glucose (reducing sugar) 0-17 mg./ml. Lactic acid 0-14 mg./ml. Fructose Not detected < 5 pg./ml. Phosphatase Negative 5-nucleotidase Positive ATP-ase Positive The ionic composition With the exception of potassium the cation concentrations differ little from those of sea water (Barnes, 1954; Na 485, K 10, Ca 22 m-equiv./l. at Cl = 19*37 %), although the total cationic value is somewhat greater, namely 540 as compared with 517 m-equiv./l.; the difference is largely the result of the higher potassium and somewhat higher calcium content, the former being more than twice that of sea water. The only other analyses of the inorganic ions in invertebrate seminal plasma appear to be those given by Rothschild (1948) for the plasma of Echinus esculentus. The mean of several estimations of potassium gave 1*55 mg./ml., that is, 40 m-equiv./l. This high value for potassium is common, therefore, to both animals, although less marked in B. balanus than in the sea-urchin. Neither value reaches those found for the higher mammals, where secretions from the accessory glands make an important contribution to the high potassium content (Mann, 1954). The organic material The inorganic ions account for 40% of the total solids, and of the remainder 35-1 % is protein precipitable by TCA. According to Mann (1954) some seminal proteins are often not precipitated by TCA so that they may well make up more than the 35 % indicated above. On the other hand, most of the non-precipitated nitrogen may well be in the form of free amino-acids (much of which in the present instance is cystine)

5 The composition of the seminal plasma of Balanus balanus 349 of which some quantity has been seen to be required to make up for the inorganic cation deficit. It appears that free amino acids are also required for other functions; they may be important for their metal-binding capacity (Tyler & Rothschild, 1951) or for other beneficial effects (Giese & Wells, 1952; Metz & Donovan, 1950). The only other record of the protein in invertebrate seminal plasma is that of Hayashi (1945), 2-5 mg./ml. for Arbacia punctulata, and this is much less than the value found in the present investigation. This apparent discrepancy could be in part compensated for by the relatively much larger volume of seminal fluid in the semen of A. punctulata so that the protein concentration per ml. of semen may be more similar in the two species. According to Hayashi (1945) the reducing-sugar content of A. punctulata is less than 2 /ig./ml. The value found for the seminal fluid of B. balanus is much higher, 0-17 mg./ml., and this together with a glycogen value of 0-88 mg./ml. is a significant part of the organic content. The body tissues of the animal contain glycogen reserves in the early part of the year and these are utilized in gonadal development (Barnes, unpublished; Barnes & Finlayson, in preparation). The presence of glycogen, glucose and a small quantity of lactic acid suggests that some glycolysis takes place in natural semen. The absence of fructose is in accordance with the fact that this substance appears to be particularly associated with some mammals, where it is produced by accessory organs of reproduction. Anaerobic fructolysis would not be possible, and one would expect that as with sea-urchins the spermatozoa could not survive in the absence of oxygen. During storage in the vesiculae seminales, however, the respiration is low but it is possible that glycogen and glucose are being metabolized. With the extreme viscosity of the semen it does indeed seem that there may well be problems concerning the access of oxygen to the spermatozoa in the living animal and that there may be a change in endogenous substrate when the spermatozoa are released from the storage organs. According to Rothschild & Cleland (1952) the seminal plasma of Echinus esculentus contains little free reducing sugar and no glycogen, although there is 2 mg./ml. of an anthrone-reactive carbohydrate possibly a mucopolysaccharide. Glycogen reserves are much more common in crustaceans than in echinoderms. The amount of phosphorus is small, although three fractions which correspond to phosphorylated esters (0-095 in S-l n^-)^ lipids (0-005 m g-/ nr J-)> an(^ residual including DNA and phosphoproteins (0-04 mg./ml.), are present. It is not certain how far any of these represent functional substances in the metabolism of the spermatozoa or seminal plasma breakdown of glycogen to give respirable glucose, for example or how far they are merely residual or released from the spermatozoa or from their precursors. This will be dealt with subsequently when an account of the composition and properties of the spermatozoa is given. Phosphatases are well known in mammalian semen but their functional significance has not been elucidated. Phosphomonesterases were not detected but 5-nucleotidase and adenosine-triphosphatase (ATP-ase) were present which is interesting in view of the fact that Mazia (1949) has shown the presence of a nuclease in sea-urchin semen. Their function in invertebrates is, as yet, unknown. The seminal plasma shows a strong reducing action towards a number of noncarbohydrate reactive reagents including 2,6-dichlorophenol-indophenol. Ergothioneine was shown to be absent, but the reaction characteristic of ascorbic acid was

6 350 H. BARNES obtained with 2,5-dinitrophenylhydrazine (Barnes & Finlayson, 1962). The plasma contains 21 fig./ml. ascorbic acid. The presence of ascorbic acid in invertebrate semen had not previously been reported, although reducing substances are well known to occur in mammalian semen (Mann, 1954). This is particularly interesting since the seminal vesicles of B. balanus are usually considered to serve only as storage organs and not to perform any secretory function. The presence of ascorbic acid along with considerable quantities of cystine may be of considerable significance; the former may take part indirectly in a system which serves to protect -SH enzymes against heavy metals and other thiol-reactive groups (Brachet, 1944). On the other hand, the stimulation of mass copulation in Balanus sp. on the addition to the water of traces of ascorbic acid (i4^xg/l.), reported by Collier, Ray & Wilson (1956), suggests a further function for this substance in the seminal plasma. Release of semen will give ascorbic acid in the water and, stimulating copulation by other individuals, will result in multiple copulation which may well be more efficient in causing release of the ova into the mantle cavity. SUMMARY 1. The results of some chemical analyses for inorganic and organic constituents of the seminal plasma of Balanus balanus are presented. 2. The inorganic ions show a cation deficit of 178 m-equiv./l., which is probably made up by free amino-acids. 3. Cystine is a prominent amino acid present. 4. Potassium and calcium are present in excess of their quantities in sea water. 5. Reducing sugars, compared with the amount found in sea-urchin spermatozoa, are found in moderate quantities, 1 mg./ml. 6. Phosphorus of all kinds is present in only small quantities (total of o-14 mg./ml.). 7. Some phosphatases are present. 8. There are 21 ^.g./ml. of ascorbic acid; the function of this is discussed in relation to its possible contribution to the protective action against the poisoning of-sh groups by thiol-reactive agents. REFERENCES BARKER, S. B. & SUMMERSON, W. H. (1941). The colorimetric determination of lactic acid in biological material. J. biol. Chem. 138, BARNES, H. (1954). Some tables for the ionic composition of sea water. J. Exp. Biol. 31, BARNES, H. (1959). Apparatus and Methods of Oceanography. I. Chemical, 341 pp. London: George Allen and Unwin Ltd. BARNES, H. & BARNES, M. (1954). The general biology of Balanus balanus (L.) Da Costa. Oikos, 5, BARNES, H. & FINLAYSON, D. M. (1962). The presence of ascorbic acid in cirripede semen. Limnol. Oceanogr. 7, 98 only. BRACHET, J. (1944). Embryologie chimique. Paris-Liege: Masson-Desoer. COLLIER, A., RAY, S. & WILSON, W. B. (1956). Some effects of specific organic compounds on marine organisms. Science, 124, 220 only. DALES, R. P. (1957). Preliminary observations on the role of coelomic cells in food storage and transport. in certain polychaetes. J. Mar. Biol. Ass. U.K. 36, FISKE, C. H. & SUBBAROW, Y. (1925). The colorimetric determination of phosphorus. J. biol. Chem. 66, GIESE, A. C. & WELLS, P. H. (1952). Protective effect of glycine on sperm exposed to light. Science, " HAYASHI, T. (1945). Dilution medium and survival of the spermatozoa of Arbada ptmctulata. I. The effect of the medium on fertilizing power. BioL BulL, Woods Hole, 89,

7 The composition of the seminal plasma of Balanus balanus 351 MANN, T. (1946). Studies on the metabolism of semen. 3. Fructose as a normal constituent of seminal plasma. S'te of formation and function of fructose in semen. Biochem. J. 40, MANN, T. (1954). The Biochemistry of Semen, 244 pp. Methuen. London. MAZIA, D. (1949). The distribution of desoxyribonuclease in the developing embryo (Arbaciapunctulata). J. Cell. Comp. PhysioL 34, 17. METZ, C. B. & DONOVAN, J. E. (1950). Adjuvant action of amino acids and peptides in fertilizin agglutination of starfish sperm. Science, 11a, RILEY, J. P. (1953). The spectrophotometric determination of ammonia in natural waters with particular reference to sea water. Arudyt. cmm. Acta, 9, ROBERTSON, J. D. & WEBB, D. A. (1939). The micro-estimation of sodium, potassium, calcium, magnesium, chloride, and sulphate in sea water and body fluids of marine animals. J. Exp. Biol. 16, ROE, H. R. (1954). Chemical determination of ascorbic dehydroascorbic and diketogluconic acids. In Methods of Biochemical Analysis (ed. D. Glick), pp New York: Interscience Publishers Inc. ROTHSCHILD, LORD (1948). The physiology of sea-urchin spermatozoa. Lack of movement in semen. J. Exp. Biol. 35, ROTHSCHILD, LORD (1951). Sea-urchin spermatozoa. Biol. Rev. 36, ROTHSCHILD, LORD & BARNES, H. (1953). The inorganic constituents of the sea-urchin egg. J. Exp. Biol. 30, ROTHSCHILD, LORD & BARNES, H. (1954). Constituents of bull seminal plasma. J. Exp. Biol. 31, ROTHSCHILD, LORD & CLELAND, K. W. (195a). The physiology of sea-urchin spermatozoa. The nature and location of the endogenous substrate. J. Exp. Biol. 39, TYLER, A. & ROTHSCHILD, LORD (1951). Metabolism of sea-urchin spermatozoa and induced anaerobic motility in solutions of amino-acids. Proc. Soc. Exp. Biol., N. Y., 76, 52. UMBREIT, W. W., BLTRRIS, R. H. & STAUFFER, J. F. (1945). Manometric Techniques and Related Methods for the Study of Tissue Metabolism, 203 pp. Minnesota, U.S.A. Burgess Publ. Co. 23 Exp. BioL 39, 3

8