Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro.
Abstract: To facilitate the study of interactions between equine spermatozoa and homologous oviduct epithelial cells, we developed an assay to count labelled spermatozoa bound to oviduct epithelial cell (OEC) monolayers and used the assay to compare the binding ability of spermatozoa from different stallions. Washed spermatozoa from three stallions were incubated with the fluorochrome Hoechst 33342 (5 micrograms/ml) for 1 min. Spermatozoa were then layered over confluent monolayers of oviduct epithelial cells in 2 cm2 culture wells. Coculture treatments comprised five concentrations of spermatozoa (10(5), 5 x 10(5), 10(6), 2.5 x 10(6), and 5 x 10(6) per well). Cocultures were incubated for 30 min before unattached spermatozoa were aspirated in coculture supernatant. Fluorescent videoimages of attached spermatozoa were digitized, and attached spermatozoa were counted by image processing and analysis. Four wells (replicates) of each concentration were allocated within each ejaculate, and ejaculates were blocked by stallion for ANOVA. The total number of spermatozoa bound was not different between replicate wells (P > 0.1). Stallion, ejaculate, concentration, and all higher level interactions influenced total spermatozoa bound (P < 0.00001). Coefficients of variation between replicates were lowest for inseminate concentrations between 10(6) and 5 x 10(6) spermatozoa per well. Within the ejaculate, a log linear relationship exists between the number of bound spermatozoa and a spermatozoal concentration of the inseminate between 5 x 10(5) and 5 x 10(6) spermatozoa per well. This assay provides a reliable method of determining numbers of spermatozoa bound to somatic cells in vitro. Furthermore, differences exist in the ability of spermatozoa from different stallions to bind OEC monolayers.
Publication Date: 1996-01-01 PubMed ID: 8720113DOI: 10.1002/(SICI)1098-2795(199601)43:1<55::AID-MRD7>3.0.CO;2-TGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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Researchers have developed a cytofluorescent assay to measure the adherence of horse sperm to oviductal epithelial cells in the lab. The test potentially offers a means of assessing the fertility of different stallions by comparing the binding ability of their sperm.
Methodology
- The researchers first incubated the spermatozoa of three stallions with the fluorescent dye Hoechst 33342 for one minute to enable tracking.
- The marked spermatozoa were layered over a confluent monolayer of oviduct epithelial cells in 2 cm2 culture wells.
- The team conducted co-culture treatments using five sperm concentration levels, varying from 100,000 to 5 million sperm per well.
- After 30 minutes, unattached spermatozoa were aspirated from the co-culture supernatant.
- Attached spermatozoa were counted by digitizing fluorescent video images and analyzing the resulting image data.
- Each sperm concentration was allocated four replicate wells within each ejaculate. The ejaculates were grouped by stallion for comparison purposes using the ANOVA statistical method.
Results
- The total number of spermatozoa that bound to the oviduct epithelial cells was not significantly different between the replicate wells.
- All variables – the stallion, the ejaculation, the sperm concentration, and all higher level interactions – were found to significantly influence the number of bound spermatozoa.
- The disparity between replicates was deemed lowest for insemination concentrations ranging from 1 million to 5 million spermatozoa per well.
- A direct correlation was found between the number of bound spermatozoa and sperm concentration in the insemination sample – from 500,000 to 5 million spermatozoa per well.
- The assay test showed a difference in the ability of spermatozoa from different stallions to adhere to oviduct epithelial cell monolayers.
Implications
- The new cytofluorescent assay enables reliable in vitro measurement of the number of spermatozoa that bind to somatic cells.
- This research opens up potential avenues for fertility studies, offering a way to differentiate stallions based on the adhering capabilities of their spermatozoa.
Cite This Article
APA
Thomas PG, Ball BA.
(1996).
Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro.
Mol Reprod Dev, 43(1), 55-61.
https://doi.org/10.1002/(SICI)1098-2795(199601)43:1<55::AID-MRD7>3.0.CO;2-T Publication
Researcher Affiliations
- Department of Clinical Sciences, Cornell University, Ithaca, NY 14853, USA.
MeSH Terms
- Analysis of Variance
- Animals
- Benzimidazoles
- Cell Adhesion
- Cells, Cultured
- Coculture Techniques
- Epithelial Cells
- Epithelium / physiology
- Fallopian Tubes / cytology
- Fallopian Tubes / physiology
- Female
- Fluorescent Dyes
- Horses
- Male
- Microscopy, Fluorescence
- Regression Analysis
- Reproducibility of Results
- Spermatozoa / cytology
- Spermatozoa / physiology
Citations
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