1. The O2 uptake of sea-urchin spermatozoa, Echinus esculentus, has been reexamined under varying conditions of sperm density, and in the presence of CuCl22H2O and sodium diethyldithiocarbamate (DDC).

2. The total O2 uptake of dilute sperm suspensions was previously thought to be higher than that of dense suspensions per unit quantity of spermatozoa (Dilution Effect I). This result is only obtained when the oxygen saturation of the dense suspension is inadequate, which may easily occur as the QOO2 of sea-urchin spermatozoa may reach 30 at 15.0° C. When oxygen saturation is satisfactory, the total O2 uptake of dense solutions is slightly greater than that of dilute ones. The experiment cannot be done in micro-respirometers of the normal Warburg type unless the density of spermatozoa per ml. suspension is less than about 109 corresponding to an initial semen dilution of 1:20 or 1:25. These figures apply to other manometric experiments on the O2 uptake of sea-urchin spermatozoa using normal amounts of material.

3. When movement ceases, there is a sharp increase in the O2 uptake of the suspension.

4. The addition of seminal plasma to dilute sperm suspensions does not inhibit the increased rate of O2 uptake, per unit quantity of spermatozoa, observed in these suspensions when compared with dense ones (Dilution Effect II). Dilution Effect II is therefore not caused by the dilution of an inhibitory substance in seminal plasma.

5. Sperm suspensions were prepared by diluting semen 1:50 with sea water and allowing them to respire for 45 mm. They were then centrifuged, the supernatant was discarded and the spermatozoa were re-suspended to different densities with sea water. This treatment has the following effects:

(a) Centrifugation irreversibly damages the spermatozoa and reduces their O2 uptake.

(b) Removal of the supernatant, which contains seminal plasma, and re-suspension in sea water also reduces O2 uptake.

(c) The treatment markedly reduces Dilution Effect II.

If the experiment is done in the same way but the suspensions are only allowed to respire for 10 min. before centrifugation, (a) and (b) are the same, but Dilution Effect II is normal. This shows that during metabolism, a regulatory substance is lost from dilute suspensions, as in mammalian spermatozoa; but this is not the cause of Dilution Effect II.

6. Dilution Effect II, considered as the reduced O2 uptake of dense suspensions, can be reversed by the addition of CuCl22H2O, 1 p.p.m., to the medium.

7. Dilution Effect II can be made to occur in sperm suspensions which do not normally exhibit it, by the addition of DDC, in concentrations as low as 3.64x10-5M (final concentration). The action of DDC is not greater when its concentration is increased to 10-3M, which suggests that in these conditions it acts as a chelating agent and not as a narcotic. For the same reasons its oxidation product, tetra ethyldithiocarbamyl disulphide, is unlikely to be responsible for DDC's inhibitory effect on sperm O2 uptake.

8. These results are consistent with the hypothesis that Dilution Effect II is due to the amounts of copper (or possibly zinc) in sea water being inadequate to satisfy the requirements of dense sea-urchin sperm suspensions. This situation is unlikely to arise during natural spawning as sperm densities are too low for the effect to occur in these conditions. Other interpretations of the stimulating action of copper and zinc are discussed.

9. The experiments remove several of the differences hitherto believed to exist between sea-urchin and mammalian spermatozoa.

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