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.
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.


METHODS
Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 microg/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 microL 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-microg/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.
Esteves
Sandro C
SC
Androfert, Center for Male Infertility, Campinas, Sao Paulo, Brazil. s.esteves@androfert.com.br
Sharma
Rakesh K
RK
Thomas
Anthony J
AJ
Agarwal
Ashok
A
D020101:Acrosome Reaction / Q000502:physiology*; D000328:Adult; D018929:Cell Culture Techniques; D015925:Cryopreservation; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D009994:Osmolar Concentration; D019887:Peanut Agglutinin; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*
D005456:Fluorescent Dyes; D019887:Peanut Agglutinin