Chapter 9
Human Sperm Cells
ANITA RAUCH
Introduction
At least one third of human germ cells are carrying a chromosomal abnormality resulting from errors in meiosis. These chromosomal abnormalities often result in early abortions and sometimes in the birth of a disabled child. Since 1978 it has been possible to karyotype human sperm
cells by reactivating them with hamster ova (Rudak et al. 1978). However,
this technique is difficult, time-consuming, expensive, and yields only a
small number of karyotypes, whereas interphase FISH is a rapid and relatively easy alternative technique to study aneuploidy in human sperm
cells. As sperms are extremely condensed due to extensive intermolecular
disulfide cross-links, they must be decondensed prior to the FISH procedure to allow the DNA probes access to the chromatin (Fig. 1). As dithioerythritol quantitatively reduces disulfide groups by forming cyclic disulfide
(Cleland 1963), it was already used in 1977 to prepare mouse sperm for
protein analyses (Balhorn et al. 1977).
The described decondensation-solution was used by Otto to investigate Chinese hamster cell lines by flow karyotyping (Otto 1988). In order
to achieve a high hybridization efficiency we recommend pretreatment of
decondensed cells with RNase and pepsin. In addition, application of directly labeled probes reduces background fluorescence.
Anita Rauch, Institut rur Humangenetik, Schwabachanlage 10, Erlangen, 91054,
Germany (phone +49-9131-8522318; fax +49-9131-209297;
e-mail arauch@humgenet.uni-erlangen.de)
PROTOCOL
Human Sperm Cells
ANITA RAUCH
Introduction
At least one third of human germ cells are carrying a chromosomal abnormality resulting from errors in meiosis. These chromosomal abnormalities often result in early abortions and sometimes in the birth of a disabled child. Since 1978 it has been possible to karyotype human sperm
cells by reactivating them with hamster ova (Rudak et al. 1978). However,
this technique is difficult, time-consuming, expensive, and yields only a
small number of karyotypes, whereas interphase FISH is a rapid and relatively easy alternative technique to study aneuploidy in human sperm
cells. As sperms are extremely condensed due to extensive intermolecular
disulfide cross-links, they must be decondensed prior to the FISH procedure to allow the DNA probes access to the chromatin (Fig. 1). As dithioerythritol quantitatively reduces disulfide groups by forming cyclic disulfide
(Cleland 1963), it was already used in 1977 to prepare mouse sperm for
protein analyses (Balhorn et al. 1977).
The described decondensation-solution was used by Otto to investigate Chinese hamster cell lines by flow karyotyping (Otto 1988). In order
to achieve a high hybridization efficiency we recommend pretreatment of
decondensed cells with RNase and pepsin. In addition, application of directly labeled probes reduces background fluorescence.
Anita Rauch, Institut rur Humangenetik, Schwabachanlage 10, Erlangen, 91054,
Germany (phone +49-9131-8522318; fax +49-9131-209297;
e-mail arauch@humgenet.uni-erlangen.de)
PROTOCOL