Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test
Int. braz. j. urol
;
33(3): 364-376, May-June 2007. ilus, graf
Article
in English
| LILACS
| ID: lil-459858
ABSTRACT
OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 µg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 µL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-µg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.
Full text:
Available
Index:
LILACS (Americas)
Main subject:
Semen Preservation
/
Sperm Motility
/
Spermatozoa
/
Cryopreservation
/
Acrosome Reaction
Type of study:
Evaluation studies
Limits:
Adult
/
Humans
/
Male
Language:
English
Journal:
Int. braz. j. urol
Journal subject:
Urology
Year:
2007
Type:
Article
/
Project document
Affiliation country:
Brazil
Institution/Affiliation country:
Androfert/BR
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