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

Radiation- and chemical-induced structural chromosome aberrations in human spermatozoa

“…Since the development of an interspecific in vitro fertilization system between human spermatozoa and zona-free golden hamster oocytes [1,2,3,4], a number of cytogenetic studies with human spermatozoa have been carried out during the last two decades [5,6,7,8]. This method has been applied to chromosomal studies of spermatozoa exposed in vitro to various mutagens.…”

Live rats resulting from injection of oocytes with spermatozoa freeze‐dried and stored for one year

“…Chromosomal integrity of rat spermatozoa was analyzed in rat oocytes according to the method reported in mouse oocytes (Kamiguchi and Mikamo, 1986). All the sperm samples after rehydration for ICSI experiments were re-frozen and stored in LN 2 for a few months until the analysis.…”

Detection of aneuploidy in human spermatozoa using fluorescencein situ hybridization (FISH)

“…In 1978, Rudak et al reported a new cytogenetic 382 H. HU el aL technique using human sperm/hamster egg interactions which first permitted a direct analysis of the chromosomal constitution in individual human sperm (Rudak et al, 1978). In this way, human sperm chromosomes have been studied for both numerical and structural aberrations (Brandriff et al, 1984;Martin et al, 1982;Kamiguchi and Mikamo, 1986). However, this method is labor intensive and time consuming, and may reflect natural meiotic selection, since only fertilized eggs can be studied.…”