Analysis of the flow cytometer stain Hoechst 33342 on human spermatozoa

Abstract

Several procedures exist for processing sperm cells for sex preselection. Flow cytometric separation using the fluorochrome stain Hoechst 33342, chemically known as bisbenzimide, is the most promising. The objective of this study was to determine the effect of bisbenzimide on spermatozoa assessed by means of the sperm survival test and to analyse the beta-globin gene in sperm DNA after exposure to increasing concentrations of bisbenzimide. Donor (n = 16) sperm specimens were pooled and washed in a discontinuous Percoll gradient 95:47%, divided and incubated in tubes containing bisbenzimide at concentrations 0 (control), 0.9, 9, 90, 900 and 9000 microM at 25 degrees C and scanned on a computer-aided sperm motility analyser at 0, 1, 4 and 24 h. Spermatozoa were also incubated in a known mutagen, ethidium bromide, as positive control. After 24 h of incubation, the treated sperm cells were processed through DNA extraction and polymerase chain reaction (PCR) performed with primers targeting the beta-globin gene. The amplified DNA products were analysed for evidence of mutation in 5% polyacrylamide gel electrophoresis and 20:80 denaturing gradient gel electrophoresis (DGGE) and further confirmed in 30:40 DGGE. The results showed complete cessation of motility in sperm incubated in the presence of 900 microM or higher concentrations of bisbenzimide. The beat cross frequency sperm parameter was significantly different at the 90 microM or higher concentration of bisbenzimide compared with the control. At concentrations < 900 microM bisbenzimide, there were no differences in the remaining sperm kinematic parameters (percentage rapid progressive, percentage total progressive, sperm velocities, linearity, straightness, amplitude of lateral head displacement and percentage hyperactive motility). PCR and DGGE analyses of spermatozoa treated with bisbenzimide showed no evidence of mutation in the representative region of the beta-globin gene at concentrations < 900 microM. The data suggest an inhibitory effect of bisbenzimide on human sperm motility at 900 microM or higher concentrations of bisbenzimide. The decrease in sperm motility and rapid progression were not due to changes in pH. Point mutation in the representative region of the beta-globin gene in human spermatozoa was detected only at high concentrations (> or = 900 microM) of bisbenzimide. The data suggest that incubating sperm in low concentrations of bisbenzimide (< 90 microM) for up to 24 h does not significantly affect all the sperm kinematic parameters including the beat cross frequency parameter when compared with the control.

In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR

“…A previous study aimed at the evaluation of mutagenic effects of Hoechst 33342 conducted on human spermatozoa (Watkins et al 1996) revealed possible risks for mutagenesis at high concentrations of dye (O900 mM) after 24 h of exposition. In our study, the high concentrations of dye were used for no more than 15 min, after which sperms were diluted to a concentration that, in the same work, has been demonstrated to be non-mutagenic even after 24-h incubation.…”

Cinética de espermatozoides criopreservados de bovinos após sexagem por citometria de fluxo

“…Nessa técnica de sexagem, o espermatozoide é submetido a vários procedimentos que podem causar danos à célula, tais como exposição ao corante e ao laser, grande pressão na passagem pelo citômetro, queda em grande velocidade dentro do tubo de colheita e permanência durante algumas horas em temperatura ambiente antes de ser processado (Chastant-Maillard & Druat, 2005). Portanto, a queda na motilidade observada no sêmen sexado pode ter sido causada pela exposição, durante o processo de sexagem, ao corante Hoechst 33342, que é tóxico, como já foi demonstrado em espermatozoide humano (Watkins et al, 1996) e bovino (Hollinshead et al, 2004). Além disso, o efeito da exposição ao corante e, posteriormente, ao laser pode alterar a atividade mitocondrial, como foi demonstrado em trabalho realizado por Smith (1993) com ovócitos corados com Hoechst 33342 e submetidos à pequena irradiação de luz ultravioleta.…”

High-resolution melt analysis does not reveal mutagenic risk in sexed sperm andin vitro-derived bovine embryos

“…The evidence that bisbenzimide Hoechst 33342 at high concentrations (≥900 μ m ) is able to induce point mutations in human sperm was provided using PCR with primers targeting the ß‐globin gene, which was chosen as a representative gene (Watkins et al . ). In the same work, incubating the sperm at a concentration <90 μ m for up to 24 h did not significantly affect the sperm kinetics, nor did this treatment exert any mutagenic effect.…”