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Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa
1Department of Molecular Embryology, AFRC Institute of Animal Physiology & Genetics Research, Babraham, Cambridge, UK.
Abstract:
Carboxyfluorescein diacetate and propidium iodide were used as fluorescent stains to assess membrane integrity in sperm populations from ram and boar. The living spermatozoa were immobilized with low concentrations of formaldehyde so that individual stained cells could be observed in a suspension with the aid of a fluorescence microscope. Intracellular esterases liberated impermeant-free carboxyfluorescein from the permeant carboxyfluorescein diacetate and caused the product to accumulate and fluoresce green within the acrosome and the mitochondria as well as within the cytoplasm. Most of the spermatozoa (the intact ones) accumulated carboxyfluorescein in all compartments; however, a few cells (those with damaged plasma membranes) accumulated the stain only in the acrosome and/or the mitochondria, while others (all of whose membranes were damaged) remained entirely unstained. The impermeant propidium iodide did not stain any of the (intact) spermatozoa that accumulated carboxyfluorescein throughout their length, but stained all the others (the heads fluoresced red). The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity (presence of normal apical ridge). The technique also demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress. Assessments of boar sperm samples during preparative incubation for in-vitro fertilization indicated a considerable increase in the percentage of cells with damaged plasma membranes as incubation proceeded, in advance of the increase in the percentage of cells with discharged acrosomes.
Insights
This study introduces a new fluorescent staining technique to accurately assess sperm membrane integrity in rams and boars. The method reliably identifies functional sperm cells, outperforming traditional assessments.
Area of Science:
- Reproductive Biology
- Cell Biology
- Biotechnology
Background:
- Assessing sperm membrane integrity is crucial for evaluating male fertility and optimizing assisted reproductive technologies.
- Traditional methods like motility and acrosomal integrity assessments have limitations in accurately reflecting functional sperm status.
Purpose of the Study:
- To develop and validate a novel fluorescent staining technique for precise assessment of sperm plasma membrane integrity in ram and boar spermatozoa.
- To compare the efficacy of this new method against conventional techniques for evaluating sperm viability and function.
Main Methods:
- Utilized carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) as fluorescent stains.
- Employed fluorescence microscopy to observe stained sperm populations after immobilization with formaldehyde.
- Analyzed intracellular esterase activity and membrane permeability to CFDA and PI to differentiate between intact and damaged sperm.
Main Results:
- The CFDA/PI staining method accurately distinguished between intact spermatozoa (staining green throughout) and those with damaged membranes (partial or no green staining, red staining with PI).
- This technique provided more reliable estimations of functional cells compared to motility and acrosomal integrity assessments.
- Demonstrated high sensitivity to cold shock, with rapid membrane permeability changes observed.
- Observed increased damaged boar sperm during in-vitro fertilization incubation prior to acrosome reaction.
Conclusions:
- The CFDA/PI fluorescent staining technique offers a reliable and sensitive method for assessing sperm membrane integrity.
- This method enhances the evaluation of sperm quality for fertility assessments and biotechnological applications.
- Highlights the susceptibility of sperm plasma membranes to stressors like cold shock and incubation conditions.