ABSTRACT
Capacitation is the final maturation step spermatozoa undergo prior to fertilisation. The efflux of cholesterol from the sperm membrane to the extracellular environment is a crucial step during capacitation but current methods to quantify this process are suboptimal. In this study, we validate the use of a BODIPY-cholesterol assay to quantify cholesterol efflux from spermatozoa during in vitro capacitation, using the boar as a model species. The novel flow cytometric BODIPY-cholesterol assay was validated with endogenous cholesterol loss as measured by mass spectrometry and compared to filipin labelling. Following exposure to a range of conditions, the BODIPY-cholesterol assay was able to detect and quantify cholesterol efflux akin to that measured with mass spectrometry. The ability to counterstain for viability is a unique feature of this assay that allowed us to highlight the importance of isolating viable cells only for a reliable measure of cholesterol efflux. Finally, the BODIPY-cholesterol assay proved to be the superior method to quantify cholesterol efflux relative to filipin labelling, though filipin remains useful for assessing cholesterol redistribution. Taken together, the BODIPY-cholesterol assay is a simple, inexpensive and reliable flow cytometric method for the measurement of cholesterol efflux from spermatozoa during in vitro capacitation.
Subject(s)
Boron Compounds/chemistry , Cholesterol/analysis , Spermatozoa/physiology , Animals , Cholesterol/chemistry , Filipin/chemistry , Flow Cytometry , Male , Mass Spectrometry , Sperm Capacitation , Staining and Labeling , SwineABSTRACT
In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.
Subject(s)
Cholesterol/metabolism , Fertilization in Vitro , Sperm Capacitation/drug effects , Sus scrofa/metabolism , beta-Cyclodextrins/pharmacology , Animals , Cell Membrane/drug effects , Cells, Cultured , Cholera Toxin , Dogs , Flow Cytometry , Fluorescence , Male , Phospholipids/metabolism , PyrimidinonesABSTRACT
In this study we used a new method to detect reactive oxygen species (ROS) induced damage at the level of the sperm plasma membrane in fresh and frozen-thawed stallion sperm. Lipid peroxidation (LPO) in sperm cells was assessed by a fluorescent assay involving the labeling of stallion sperm with the LPO reporter probe C11-BODIPY(581/591). The peroxidation dependent spectral emission shift of this membrane probe could be localized using inverted spectral confocal microscopy and quantified on living and deteriorated sperm cells using flow cytometry. Mass spectrometric analysis of the main endogenous lipid class, phosphatidylcholine (PC), was carried out to determine the formation of hydroxy- and hydroperoxyphosphatidylcholine in fresh sperm cells. Peroxidation as reported by the fluorescent probe corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of LPO. This allowed us to correlate endogenous LPO with localization of this process in the living sperm cells. In absence of peroxidation inducers, only relatively little peroxidation was noted in fresh sperm cells whereas some mid-piece specific probe oxidation was noted for frozen-thawed sperm cells. After induction of peroxidation in fresh and frozen-thawed sperm cells with the 0.1 mM of lipid soluble ROS tert-butylhydrogen peroxide (t-BUT) intense probe oxidation was produced in the mid-piece, whereas the probe remained intact in the sperm head, demonstrating antioxidant activity in the head of fresh sperm cells. At higher levels of t-BUT, probe peroxidation was also noted for the sperm head followed by a loss of membranes there. Frozen-thawed sperm were more vulnerable to t-BUT than fresh sperm. The potential importance of the new assays for sperm assessments is discussed.
