Fluorescent analysis of boar sperm capacitation process in vitro

Abstract

BackgroundCapacitation 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.ResultsAll 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).ConclusionsOur 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.

Perfluorooctane Sulfonate (PFOS) and Perfluorohexane Sulfonate (PFHxS) Alters Protein Phosphorylation, Increase ROS Levels and DNA Fragmentation during In Vitro Capacitation of Boar Spermatozoa

“…The in vitro boar sperm capacitation methodology gave optimal results, obtaining levels of capacitation, similar to others previously reported [ 27 , 36 ]. The CTC technique is useful for differentiating among non-capacitated, capacitated, and AR spermatozoa in normal in vitro sperm, based on the CTC fluorescence patterns when they are in contact with intracellular Ca 2+ bound to membrane proteins [ 37 ].…”

“…On the day of analyses, the medium was supplemented with 1 mM of sodium pyruvate and 6 mg/mL of bovine serum albumin fraction V, and then adjusted to pH 7.4. To induce capacitation, an aliquot of 5× 10 6 washed spermatozoa was transferred to 1 mL of TALP-HEPES and incubated for 4 h at 39 °C in a semi-humid atmosphere, with or without PFCs (concentrations indicated below) [ 25 , 26 , 27 ].…”

Effect of insulin-like growth factor-1 complex of Simmental bull seminal plasma on post-thawed Kacang buck semen fertility

“…The sperm capacitation and acrosome reaction statuses were evaluated using chlortetracycline (CTC) fluorescence staining [29]. A 135-µL measure of the sample was added to 15-µL H33258 solution (10-µg H33258/mL PBS) and then incubated for 10 min at room temperature.…”

“…Meanwhile, capacitated sperms had green fluorescence over the acrosomal region and a dark post-acrosomal region. Acrosomereacted sperms showed a mottled green fluorescence Available at www.veterinaryworld.org/Vol.14/August-2021/12.pdf over the head, green fluorescence only in the post-acrosomal region, or no fluorescence over the head [29].…”

In Memoriam: Lynn R Fraser

“…The importance of cAMP, 8 potassium and sodium ions, 9,10 decapacitation factors, 11 calcium, 12 a glycolyzable substrate, 13 protein phosphorylation 14 and the stimulatory role of adenosine 15 were all established at this time. This research was highly influential and internationally recognized, many papers generating over 200 citations and involving the description of techniques, such as the use of chlorotetracycline to monitor sperm capacitation, that are still proving their value today 16 . In the late 1990s, Lynn used her knowledge of mouse sperm biology to investigate their human counterparts, providing the field with new insights into the mechanisms underpinning capacitation and acrosomal exocytosis in these cells and introducing the world to FPP (the fertilization promoting peptide), one of a series of important physiological regulators of adenylate cyclase in these cells 17,18 .…”