246 USE OF IONOPHORE A23187 ON BOVINE FROZEN SPERM INCREASES HYPERACTIVATED MOTILITY
| Autor: | A. A. Mutto, M. J. Franco, N. M. Ortega, T. Fanti, R. Garaguso |
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| Rok vydání: | 2015 |
| Předmět: |
endocrine system
Spermatozoon urogenital system Acrosome reaction Motility Reproductive technology Biology Sperm Andrology Endocrinology medicine.anatomical_structure Reproductive Medicine Capacitation Immunology Genetics medicine Animal Science and Zoology Molecular Biology Percoll Spermatogenesis reproductive and urinary physiology Developmental Biology Biotechnology |
| Zdroj: | Reproduction, Fertility and Development. 27:212 |
| ISSN: | 1031-3613 |
| DOI: | 10.1071/rdv27n1ab246 |
| Popis: | Sperm capacitation is critical for oocyte fertilization in mammals. The capacitated state is acquired by the spermatozoon during its passage through the female reproductive tract and can be induced in vitro. At a functional level, sperm capacitation is associated with changes in the motility pattern – the hyperactivated state (HA) – and terminates with the acrosome reaction (AR). These events are characteristically regulated by different Ca2+-signalling pathways. For this reason, Ca2+ ionophores, such as A23187, are commonly used in sperm capacitation studies. The induction of AR and IVF, as well as the assessment of protein phosporylation of tyrosine substrates are useful methods for the evaluation of the capacitation state of spermatozoa. Although the increase of protein tyrosine phosphorylation via PKA is associated with sperm capacitation, in the mouse, A23187 capacitates the spermatozoa, thus bypassing this pathway. The aim of the present work was to test the effect of the ionophore A23187 on acquisition of the capacitated state by evaluation of HA motility and protein phosphorylation pattern in frozen bovine spermatozoa. Motile bovine spermatozoa were isolated by gradient centrifugation (Percoll) from frozen/thawed samples and treated with different concentrations of A23187 (0.05, 0.1, 0.2, and 0.3 µM) in H-TYH medium without BSA. After 10 min of treatment, spermatozoa were washed in medium with BSA (11.2%) and incubated in H-TYH with BSA (6%) for 30 min. For control groups, sperm were incubated in H-TYH medium and DMSO. The motility pattern was visually identified and quantified using a computer-assisted sperm analysis system. The motile/immotile spermatozoa and the HA motility patterns of each group were statistically analysed by applying Fisher's tests. The protein tyrosine phosphorylation pattern was evaluated by a Western immunoblotting assay using heparin as a positive control of sperm capacitation. Our results showed that spermatozoa treated with A23187 had a significant increase in HA motility. The proportions of HA spermatozoa were 10.92, 31.27, and 75.4% for 0.1, 0.2, and 0.3 µM A23187, respectively (P |
| Databáze: | OpenAIRE |
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