Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.
The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.
Department of Obstetrics-Gynecology, University of Rochester Medical Center, New York 14642.
G M
GM
Centola
J H
JH
Mattox
S
S
Burde
J F
JF
Leary
D000177:Acrosome / Q000502:physiology; D000814:Aniline Compounds; D006690:Bisbenzimidazole; D002470:Cell Survival; D015925:Cryopreservation; D004801:Eosine Yellowish-(YS); D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D037102:Lectins; D008297:Male; D008856:Microscopy, Fluorescence; D037121:Plant Lectins; D011419:Propidium; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology*; D013194:Staining and Labeling; D013861:Thiocyanates
D000814:Aniline Compounds; D005452:Fluoresceins; D037102:Lectins; D037121:Plant Lectins; D013861:Thiocyanates; C036292:pea lectin; D011419:Propidium; C002712:nigrosin; D016650:Fluorescein-5-isothiocyanate; D006690:Bisbenzimidazole; D004801:Eosine Yellowish-(YS)