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Related Concept Videos

Feedback Regulation of Calcium Concentration01:27

Feedback Regulation of Calcium Concentration

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Calcium is an essential signaling molecule required for various cellular functions. Calcium pumps and ion channels on cell and organellar membranes, such as those on the endoplasmic reticulum (ER), regulate calcium concentrations inside the cell. They remain closed, keeping the cytosolic calcium levels low at a resting state.
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
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Updated: May 15, 2025

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
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Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

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Sperm Intracellular Calcium Evaluation.

Adriana Casao1, Sara Miguel-Jimenez2, Rosaura Pérez-Pe2

  • 1Grupo BIOFITER, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón, Universidad de Zaragoza, Zaragoza, Spain. adriana@unizar.es.

Methods in Molecular Biology (Clifton, N.J.)
|April 9, 2025
PubMed
Summary

This study details methods for evaluating intracellular calcium in sperm using chlortetracycline and calcium dyes. These techniques assess sperm capacitation and acrosome reaction by measuring calcium distribution and levels.

Keywords:
ChlortetracyclineEpifluorescence microscopyFlow cytometryFluo-4-AMPlate reader fluorometryRho-5-AM

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Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

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Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
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Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm
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Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

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Area of Science:

  • Reproductive Biology
  • Cellular Physiology
  • Biochemistry

Background:

  • Intracellular calcium (Ca2+) is crucial for sperm function, including capacitation and acrosome reaction.
  • Accurate measurement of Ca2+ is vital for understanding these processes.

Purpose of the Study:

  • To describe methods for evaluating Ca2+ distribution and levels in spermatozoa.
  • To correlate Ca2+ dynamics with sperm capacitation and acrosome reaction states.

Main Methods:

  • Chlortetracycline (CTC) staining for Ca2+ distribution relative to sperm capacitation.
  • Flow cytometry using low-affinity Rhod-5N-AM dye for Ca2+ levels.
  • Flow cytometry using high-affinity Fluo-4-AM dye for intracellular Ca2+ changes.
  • Plate reader fluorometry for measuring Ca2+ levels in Fluo-4-AM loaded spermatozoa.

Main Results:

  • CTC effectively visualizes Ca2+ distribution linked to sperm capacitation.
  • Rhod-5N-AM allows Ca2+ assessment in subcellular compartments.
  • Fluo-4-AM quantifies changes in intracellular Ca2+ levels.

Conclusions:

  • The described methods provide robust tools for studying Ca2+ in sperm.
  • These techniques are valuable for research on sperm capacitation and acrosome reaction.
  • Differential dye affinities enable targeted investigation of Ca2+ dynamics.