1. Fluorescent analysis of boar sperm capacitation process in vitro
Eva Zatecka, Pavla Dostalova, Katerina Komrskova, Lukas Ded, Andrej Dorosh, Jana Peknicova Reprod Biol Endocrinol . 2019 Dec 19;17(1):109. doi: 10.1186/s12958-019-0554-z.
Background:Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry.Results:All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions).Conclusions:Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.
2. Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin
F Oosawa, E Prochniewicz-Nakayama, T Yanagida J Cell Biol . 1983 Dec;97(6):1663-7. doi: 10.1083/jcb.97.6.1663.
F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.
3. Electron microscopic localization of F-actin in acrosome reacted boar spermatozoa by means of a phalloidin-FITC complex
L Castellani-Ceresa, G Radaelli, M F Brivio J Submicrosc Cytol Pathol . 1991 Apr;23(2):347-9.
An immunocytochemical study at the ultrastructural level has been performed in boar spermatozoa in order to clarify the aggregation state of actin before and after the acrosome reaction. A new phalloidin probe has been used to detect F-actin: a phalloidin derivative conjugated with FITC, followed by incubation with an anti-FITC antibody. The protein A-gold technique was then applied for the localization of the antigenic sites. Gold particles were localized on the sperm surface only after the acrosome reaction which was induced by the ionophore A23187.
