An Improved, Efficient Method for Analyzing Human Sperm Chromosomes Using Zona-Free Hamster Ova

Abstract

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

Freeze-Dried Sperm Fertilization Leads to Full-Term Development in Rabbits1

“…Approximately 5 h after ICSI of mouse oocytes, the oocytes with two distinct pronuclei and second polar bodies were placed in CZB medium [17] containing 6 ng/ml of vinblastin to arrest them at metaphase of the first cleavage. The eggs were then freed from the zona pellucida with 0.5% pronase and processed for the preparation of chromosome spreads onto clean glass slides [18]. Haploid sets of mouse and rabbit chromosomes were easily recognizable from each other because of species-specific differences in the structure and number of chromosomes.…”

Germ Cell Research: A Personal Perspective

“…Later, we used the same method to examine human sperm chromosomes [37]. This karyological technique was greatly improved by Kamiguchi and Mikamo [38]. Although fluorescence in situ hybridization (FISH) is a faster and easier alternative to detect aneuploidy in human spermatozoa [39], the hamster method was the only way to directly examine individual sperm chromosomes until we later developed another method (i.e., microsurgical sperm injection into eggs [e.g., mouse eggs]) [40,41].…”

Decondensation of the mouse sperm nucleus within the interphase nucleus

“…Some sperm-injected pronuclear eggs were fixed for 1 h in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and processed for electron microscopy. Others were incubated in CZB for 4-5 h, then in CZB containing 0.006 ug/ml vinblastin for 5-7 h. They were fixed and then airdried on slides for examination of metaphase chromosomes in the first cleavage (Kamiguchi & Mikamo, 1986).…”