ABSTRACT
The copper content of semen, seminal plasma and coelomic fluid of Echinus esculentus has been determined. The average figures for sea-urchins examined immediately after collection were : semen, 1·33μg/ml.; seminal plasma, 0·50μg/ml.; coelomic fluid, 0·07μg./ml. The accuracy of the method of estimation was ± 0·08μg. Cu/ml. original sample, which means that the amount of Cu in coelomic fluid is not significant. This figure for coelomic fluid is considerably lower than that obtained by Webb (1937) in the only previous examination.
The copper content of sea water (surface, off Keppel Pier, Millport) was 0·0064μg./ml.
Introduction
The addition of copper salts to dense suspensions of unwashed sea-urchin spermatozoa in sea water markedly stimulates the O2 uptake of the suspension (Rothschild & Tuft, 1950). An investigation of the Cu content of sea-urchin semen and seminal plasma was therefore of interest and is the subject of this note. At the same time the coelomic fluid of Echinus esculentus and sea water were analysed for the same metal.
The only recent investigation of the Cu content of sea-urchins was carried out by Webb (1937). He found that the coelomic fluid of E. esculentus contained 1·04 μg./ml. Cu, eighty times as much as the sea water (Loch Ine) from which the sea-urchins were taken. The Cu content of this sea water was 0·013 μg./ml., assuming a chlorinity of 19%. According to Sverdrup, Johnson and Fleming (1942), the Cu content of sea water varies from 0·001 to 0·01 μg./ml.
Material
Semen, seminal plasma (obtained by centrifugation) and filtered or centrifuged coelomic fluid of E. esculentus; unfiltered surface sea water collected immediately before analysis in a Pyrex flask off Keppel Pier, Millport.
Before examination, some of the sea-urchins had been kept in the station aquarium. Other sea-urchins were examined immediately after collection.
Method
Collection of semen
Semen was obtained as described by Rothschild & Tuft (1950).
Sperm counts
Fuchs-Rosenthal haemocytometer.
Cu analysis
Samples were wet-ashed: dry-ashing in a muffle at a temperature just below red heat resulted in considerable losses of Cu, even though ignition was carried out in the presence of H2SO4. The sample was transferred to a 5 ml. flask and heated with several portions of H2SO4 (total 1 ml.) to fuming, and then under reflux in the later stages. After thorough digestion, the sample was cleared with 1 ml. of 100 vol. H2O2 M.A.R. (Barnes, 1946). After boiling to remove excess peroxide, the clear digest was cooled, transferred to a 10 ml. standard flask and made up to the mark. 2 ml. of 20% (w/v) citric acid was added to prevent interference by iron, followed by 3 ml. ammonia s.g. 880 (ultimate pH 9·2), the solution again being cooled. After transferring to a small separating funnel, 0·5 ml. of 0·5% (w/v) sodium diethyldithiocarbamate was slowly added, and, after standing 5 min., 1 ml. chloroform ; the yellow Cu complex was extracted by shaking for 2 min. After allowing the chloroform layer to settle, it was run off into a 1 cm. micro-Spekker cell and its absorption measured, using violet filters (Ilford 601). A calibration curve was constructed using known amounts of Cu; blanks were run in all experiments.
2 μg Cu/ml. original sample corresponded to a drum-reading difference of about 0·300 divisions. The accuracy of the method is approx. ± 0·08 μg. Cu/ml. original sample.
Results
The results of the sea-urchin analyses are shown in Table 1.
Three analyses of sea water were carried out. The results, in μg./ml., were 0·0064, 0·0063 and 0·0066, or an average of 0·0064 μg./ml-Table 1 and the sea-water analyses show that Cu is concentrated from sea water to the extent that there is about 200 times as much Cu in semen as in sea water. This, however, applies to semen, less than half of which consists of spermatozoa. Comparison of semen and seminal plasma shows that the spermatozoa concentrate Cu to a greater extent than the figures at first suggest.
The volume of a spermatozoon is about 15μ3 (Rothschild, 1950). In the case of sea-urchin no. 6, which can be examined as an example, 1 ml. semen, in which there were 2·76 × 1010 spermatozoa, contained 1·01μg. Cu, while 1 ml. seminal plasma contained 0·42μg. Cu. This means that 2·76 × 1010 spermatozoa contained 0·76μg. Cu. The concentration of Cu per ml. spermatozoa was therefore about 1·84μg., some 300 times as much as in sea water, or about 3 × 105 atoms of Cu per spermatozoon. In a dense suspension of spermatozoa, containing 109 sperm/ml. of sea water, there will be some 6 × 104 atoms of Cu available per spermatozoon, per ml. sea water apart from the Cu they contain and the small amount derived from the seminal plasma.