Evaluation of in vitro capacitation of stallion spermatozoa.
Abstract: The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca(2+)-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO(2) on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca(2+) ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus approximately 3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10,000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca(2+) ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.
Publication Date: 2001-07-24 PubMed ID: 11466214DOI: 10.1095/biolreprod65.2.462Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study focuses on determining the capacitation status of stallion sperm using flow cytometry, comparing it with conventional staining methods. It also analyses the impact of bicarbonate on the process of capacitation, and found that a newer staining agent, merocyanine 540, detects changes related to capacitation earlier than the conventionally used chlortetracycline (CTC).
Research Methodology
- The spermatozoa from fresh semen were studied for their capacitation status, which is a vital functional transformation required for the sperm to attain the ability to fertilize an egg.
- The semen was washed and incubated in two types of mediums – with and without bicarbonate for 5 hours. Some samples were also incubated with or without a Ca(2+) ionophore, a substance which can induce the acrosome reaction (AR). The AR is a regulated exocytosis which allows spermatozoa to penetrate and fertilize an egg.
- The study used a technique called flow cytometry to examine the changes in the fluidity of the plasma membrane of the sperm. It compared this with the use of a conventional staining technique using CTC.
Findings of the Study
- The study revealed that merocyanine 540, a staining technique used in flow cytometry, detected capacitation-related changes earlier than the traditional staining technique CTC. This suggested that flow cytometry is a quicker and more accurate method for evaluating capacitation and AR in stallions’ sperm.
- It was also observed that the Ca(2+) ionophore could not induce the AR in samples without bicarbonate, indicating the crucial role bicarbonate plays in the induction of AR.
- The percentage of hyperactive sperm did not change with the time of incubation. Sperm hyperactivity is an enhanced movement mode which is commonly associated with capacitation.
Conclusion
- The study concluded that merocyanine 540 staining is a better method than CTC staining for evaluating early events of capacitation in stallion spermatozoa treated in vitro (in a lab).
- It underlined the key role of bicarbonate in activating sperm and inducing the AR in stallion spermatozoa.
Cite This Article
APA
Rathi R, Colenbrander B, Bevers MM, Gadella BM.
(2001).
Evaluation of in vitro capacitation of stallion spermatozoa.
Biol Reprod, 65(2), 462-470.
https://doi.org/10.1095/biolreprod65.2.462 Publication
Researcher Affiliations
- Department of Equine Sciences, Graduate School of Animal Health, Utrecht University, 3584 CL Utrecht, The Netherlands.
MeSH Terms
- Acrosome Reaction / drug effects
- Animals
- Bicarbonates / pharmacology
- Calcimycin / pharmacology
- Chlortetracycline
- Coloring Agents
- Flow Cytometry
- Fluorescein-5-isothiocyanate
- Fluorescent Dyes
- Horses
- Ionophores / pharmacology
- Male
- Microscopy, Fluorescence
- Peanut Agglutinin
- Pyrimidinones
- Sperm Capacitation / drug effects
- Sperm Motility / drug effects
- Spermatozoa / drug effects
- Spermatozoa / physiology
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