Toluidine blue, applied to frog sperm under appropriate conditions, inactivates specifically the sperm nucleus, leaving the extranuclear parts of the cell undamaged. Thus, the dye-treated spermatozoa stimulate eggs to cleave normally, but contribute no chromosomes to the resulting embryos, which develop as typical gynogenetic haploids.
The concentration of dye required to produce this inactivation varies with pH. Measurements made over the entire pH range which can be tolerated by sperm cells showed that in the lower part of the range (5 to 7) the effective dye concentration was about 5 x 10–6 M; in the intermediate range (7 to 8.5) it was 1 x –6 to 1 x –7 M; and for the higher pH values (8.5 to 10.0) it was about 5 x –8 M. Using sperm suspensions containing 1500 cells per c. mm. these concentrations of dye produced specific inactivation of the sperm nuclei within 7 to 60 minutes at 18°C.
Tests of the reversibility of the inactivation were made by transferring the sperm from the dye to a dilute Ringer's solution after a known degree of inactivation had been produced. Following removal of the dye the sperm cells were tested on eggs over a period of 2 hours. During this time there was no indication of a reversal of the inactivation.
Microscopic observations of sperm treated with –5 M or 5 X –5 M dye show that the dye is taken up by the sperm nucleus, which is faintly but definitely stained. The dye appears to be uniformly distributed in the nucleus, while extranuclear structures remain unstained.
Measurements of the amount of dye bound per sperm nucleus indicate that the minimal quantity required for complete inactivation is about 6.7 x –18 mole, while the maximal amount which can be bound without injury to extranuclear structures is about 1.5 x –16 mole. The value obtained for the minimal requirement (6.7 X 16 mole = 4 X 6 molecules) suggests that there are roughly 4 million binding sites in the nucleus which, when blocked by dye molecules, somehow prevent the sperm chromosomes from participating in the development of the egg.