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Sperm-macrophage interaction in the mouse: a quantitative assay in vitro using 111indium oxine-labeled sperm
D L Olive1, J B Weinberg, A F Haney
1Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
The role of reproductive tract macrophages in contraception and reproductive failure has become widely recognized. However, in vitro analysis of sperm phagocytosis by macrophages has relied upon a semi-quantitative method of sperm counting that is of limited accuracy and reproducibility. We have developed an assay using murine sperm labeled with 111indium oxine, and results indicate the labeling to be rapid and efficient. Incorporation of 111indium into sperm increased the dose and sperm concentration and reached 90% maximal uptake after 15 min incubation, with maximal uptake occurring at 30 min. No decrease in sperm motility was noted with levels of oxine in excess of those required for significant labeling. Maximal labeling efficiency occurred in phosphate-buffered saline (PBS), with Dulbecco's modified Eagle's medium (DMEM) + 10% adult bovine serum (ABS) producing significantly less uptake. Label dissociation was detectable in PBS at room temperature, but at 37 degrees C in DMEM + 10% ABS, loss of label occurred at a rate of 23.5%/h. Addition of labeled sperm to murine macrophage monolayers under optimal conditions resulted in uptake of 111indium by macrophages, while free label was unincorporated. Results indicated assay specificity for macrophage-limited uptake, with insignificant label uptake by nonphagocytic murine fibroblasts and better sensitivity than sperm counting. Macrophages from Bacillus Calmette-Guerin (BCG)-infected mice resulted in a decrease in sperm uptake. Female macrophages showed greater capacity for sperm uptake than those of the male mouse. These initial studies demonstrated the utility of this model system in enhancing the understanding of sperm-macrophage interaction in the female reproductive tract.
Insights
Researchers developed a new assay for studying sperm phagocytosis by macrophages using radiolabeled sperm. This method offers improved accuracy and sensitivity for understanding reproductive tract immune cell interactions.
Area of Science:
- Reproductive immunology
- Cell biology
- Assay development
Background:
- Reproductive tract macrophages play a crucial role in contraception and reproductive failure.
- Existing in vitro methods for analyzing sperm phagocytosis by macrophages lack accuracy and reproducibility.
- A more precise assay is needed to study sperm-macrophage interactions in the reproductive tract.
Purpose of the Study:
- To develop and validate a novel assay for quantifying sperm phagocytosis by macrophages.
- To utilize radiolabeled murine sperm (using 111indium oxine) for improved accuracy and sensitivity.
- To investigate factors influencing sperm labeling and uptake by macrophages.
Main Methods:
- Murine sperm were labeled with 111indium oxine, assessing labeling efficiency, sperm motility, and optimal labeling conditions (buffer, temperature).
- Label dissociation rates were evaluated under different conditions.
- Labeled sperm were incubated with murine macrophage monolayers, and 111indium uptake was measured to assess phagocytosis specificity and sensitivity compared to sperm counting and nonphagocytic cells.
Main Results:
- 111indium oxine labeling of sperm was rapid, efficient, and did not impair sperm motility.
- Optimal labeling occurred in phosphate-buffered saline (PBS), with maximal uptake at 30 minutes.
- The assay demonstrated specificity for macrophage uptake, higher sensitivity than sperm counting, and revealed decreased sperm uptake by macrophages from BCG-infected mice and greater uptake by female macrophages compared to male macrophages.
Conclusions:
- The developed 111indium-labeled sperm assay provides a sensitive and reproducible method for studying sperm-macrophage interactions.
- This assay enhances our understanding of the role of macrophages in reproductive processes and potential reproductive failure.
- The model system is valuable for investigating immune cell dynamics within the female reproductive tract.