The effect of IBMX and prolactin on functional status of cryopreserved bull spermatozoa

Abstract

Purpose: investigate the effect of IBMX (activator of protein phosphorylation) and prolactin (PRL) on the functional state of cryopreserved bovine spermatozoa using inhibitory analysis.Materials and methods. Frozen-thawed semen samples from 60 black-and-white bulls was used in the experiments. For capacitation, cells were incubated in Sp-TALP medium supplemented with 6 mg/ml bovine serum albumin and various compounds: an inductor of capacitation (IBMX at concentrations of 1 μM, 10 μM, 50 μM, 100 μM), hormone (PRL at concentrations of 1 ng, 10 ng, 50 ng, 100 ng) and inhibitors of protein kinases C (Ro 31-8220 at a concentration of 10 ng/ml) and protein kinase A (H-89 at a concentration of 10 μM). The incubation was carried out at 38°C in an atmosphere of 5% CO2, 98% humidity for 4 hours. The functional status of the cells was determined by the chlortetracycline test.Results. It was shown that IBMX at all experimental concentrations did not affect the post-ejaculatory maturation (capacitation and acrosome reaction) of spermatozoa, while all concentrations of PRL (1-100 ng/ml) promoted the acrosome reaction in capacitated cells. In the presence of a protein kinase A inhibitor, there was a decrease in number of capacitated and an increase in number of acrosome-reactive spermatozoa under the action of IBMX at a concentration of 100 μM and no changes under the action of a protein kinase C inhibitor. Also, in case of protein kinase C inhibition the PRL-related stimulation of the acrosome reaction was canceled, while the usage of H-89 did not affect the functional status of spermatozoa, mediated by PRL. Thus, the influence of IBMX and PRL on the processes of post-ejaculatory maturation in thawed bovine spermatozoa was studied using the inhibitory analysis.Conclusion. At the capacital stage, all studied IBMX concentrations did not affect the ratio of deconved cells with various functional status. Prode also contributed to the passage of the acrosomous reaction in the rolled spermatozoa after defrosting. Inhibition of protein kinase A when incubating cells with IBMX has mediated the processes of acrosomal exocytosis in ripped cells and did not affect this process under the action of the PRR, while the protein kinase inhibitor C changed the ratio of cells with various functional status in the direction of increasing the percentage of cells at the rate of occasion I did not participate in intracellular action provided IBMX on deconved cells.