The chromosome constitution of human spermatozoa was determined after injecting individual spermatozoa into mouse oocytes. Of a total 279 eggs arrested at first cleavage metaphase, 200 (71.7%) were suitable for the analysis of sperm chromosomes. Incidences of spermatozoa with numerical and structural chromosome aberrations were 1.3 and 6.9% respectively in spermatozoa with normal head morphology, showing values comparable with those found in previous studies using the hamster oocyte-human sperm fusion system. The ratio of X- to Y-bearing spermatozoa did not differ significantly from the expected 1:1 ratio. The incidence of structural chromosome aberrations was about four times higher in spermatozoa with amorphous, round and elongated heads (26.1%) than in those with morphologically normal heads, whereas the incidence of aneuploidy was not significantly different between the two groups. No increase in chromosome aberrations was found in spermatozoa with large heads. The same was true for spermatozoa with small heads. Although the sample size used in this study is rather small, the results nevertheless indicate that some morphological abnormalities in the sperm heads are associated with their chromosome defects.

Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.

Two hundred and sixty-five unfertilized human metaphase II (M II) oocytes from an in vitro fertilization program were studied cytogenetically using our chromosomal technique, a gradual fixation-air drying method. Of the 265 oocytes, 185 (70%) were successfully karyotyped. There were 21 aneuploids (11.4%) consisting of 8 hyperhaploids (4.3%), 11 hypohaploids (5.9%) and 2 complex cases (1.1%). There were also 9 structural anomalies (4.9%) and 18 diploids (9.7%). In aneuploidy, the loss or gain of dyads (so-called nondisjunction) occurred more frequently than the loss or gain of monads (so-called predivision). The frequency of abnormally behaved chromosomes (segregation errors) due to nondisjunction, anaphase lag and predivision was studied among the seven chromosomal groups (A-G) and compared with the frequency expected from an equal probability of segregation errors in each of the 23 chromosomes. The observed frequency was somewhat higher than the expected frequency in groups E and G but the difference was not statistically significant in either group. These results were discussed in relation to previous studies on human M II oocyte chromosomes.

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acrosomes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.

We have developed an improved method for analyzing human sperm chromosome, using zona-free hamster ova. Our main improvements of methodology are as follows: (1) Fertilization rate of hamster oocytes by human spermatozoa was markedly raised by successive treatments of the spermatozoa with 5-15 piM ionophore A23 187 solutions and a capacitation medium (BWW medium) containing 3.5% HSA. The HSA most effective in inducing capacitation was selected from several kinds of HSA products commercially available. (2) Monospermic fertilization was ensured by inseminating oocytes with highly capacitated spermatozoa at a low concentration for a short time. (3) TC medium 199 was used for postinsemination culture of the eggs. (4) A medium containing podophyllotoxin and vinblastine (0.04 jig/ml each) was used to block karyogamy and first-cleavage spindle formation. (5) Chromosome slides were prepared with our gradual fixation-air-dry method instead of Tarkowski's method. Ninety-two to 177 spermatozoa corresponding in number to 43%-79% (mean: 62%) of the inseminated oocytes were successfully karyotyped in each experiment. In spite of above-mentioned quantitative improvements, quality of Q-banding was not necessarily satisfactory in our slides. Improvement of banding technique is an important problem to be solved in our method. Spontaneous incidence of chromosome aberrations was studied in a total of 1,091 spermatozoa obtained from nine semen samples from four donors. Incidences of aneuploidy and structural anomaly were 0.9% (hyperhaploidy, 0.45%; hypohaploidy, 0.45%) and 13.0%, respectively. Structural aberrations included breaks (45.1%), fragments