Sperm Nuclei Isolation
Sperm Nuclei Isolation - Related Videos
05:46
Isolation and In vitro Activation of Caenorhabditis elegans Sperm
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Cited by 26 •
2011
A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.
10:12
Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue
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Cited by 61 •
2012
Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.
07:40
Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm
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Cited by 21 •
2018
This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.
10:58
Neuronal Nuclei Isolation from Human Postmortem Brain Tissue
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Cited by 136 •
2008
The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.
03:55
Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples
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2023
This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.
FAQs
Protamines compact sperm chromatin, while disulfide bonds help stabilize that highly condensed structure. These features make sperm nuclei more resistant to chemical disruption than many other cellular components. A successful isolation strategy must therefore remove surrounding membranes and cytoplasmic material without excessively compromising nuclear structure, so the recovered fraction remains suitable for evaluating chromatin and genomic DNA.
Detergents primarily disrupt nonnuclear structures, including surrounding membranes, by altering their chemical organization. Reducing agents address the disulfide bonds that stabilize the compact sperm nucleus. Using these chemical functions together helps separate cellular material while accommodating the unusual structural properties of sperm chromatin. Their controlled application supports recovery of a nuclear fraction for downstream reproductive biology studies.
Centrifugation separates the disrupted cellular mixture into fractions according to the behavior of its components during sedimentation. After chemical lysis, it helps collect the sperm nuclei while leaving nonnuclear material in other portions of the preparation. This separation step is essential because the analytical value of the sample depends on enriching the nuclear material rather than examining an unfractionated cell mixture.
Chemical treatment must be strong enough to remove membranes and cytoplasmic components but sufficiently controlled to preserve the nuclear fraction. Excessive disruption could reduce the integrity of the material being analyzed, whereas insufficient treatment could leave unwanted cellular components associated with it. Balancing these effects improves the relevance of measurements involving DNA integrity, chromatin organization, and epigenetic features.
A typical workflow begins by exposing sperm cells to controlled chemical lysis, using detergents to disrupt nonnuclear structures and reducing agents to address stabilizing disulfide bonds. The resulting mixture then undergoes centrifugation to separate the nuclear fraction from other cellular material. The recovered nuclei can subsequently support focused analysis of sperm chromatin and genomic DNA.
The nuclear fraction enables researchers to examine genomic DNA integrity, chromatin organization, and epigenetic features without the same emphasis on surrounding cellular material. These measurements help investigate the nuclear contribution of sperm to fertilization and early development. They also provide a basis for assessing sperm nuclear quality in studies of reproductive health and experimental conditions.
This technique is useful when researchers need to determine whether an environmental or experimental condition affects the nuclear quality of sperm. By focusing analysis on isolated nuclei, investigators can examine changes in DNA integrity, chromatin organization, or epigenetic features. The resulting information helps connect altered sperm nuclear properties with broader questions about reproductive health and paternal contributions to early development.