Cyclic AMP efflux through MRP4 regulates actin dynamics signalling pathway and sperm motility in bovines

Abstract

Previously we demonstrated that multidrug resistance-associated protein 4 transporter (MRP4) mediates cAMP efflux in bovine spermatozoa and that extracellular cAMP (ecAMP) triggers events associated to capacitation. Here, we deepen the study of the role of MRP4 in bovine sperm function by using MK571, an MRP4 inhibitor. The incubation of spermatozoa with MK571 during 45 min inhibited capacitation-associated events. MRP4 was localized in post-acrosomal region and mid-piece at 15 min capacitation, while at 45 min it was mainly located in the acrosome. After 15 min, MK571 decreased total sperm motility (TM), progressive motility (PM) and several kinematic parameters. The addition of ecAMP rescued MK571 effect and ecAMP alone increased the percentage of motile sperm and kinematics parameters. Since actin cytoskeleton plays essential roles in the regulation of sperm motility, we investigated if MRP4 activity might affect actin polymerization. After 15 min capacitation, an increase in F-actin was observed, which was inhibited by MK571. This effect was reverted by the addition of ecAMP. Furthermore, ecAMP alone increased F-actin levels while no F-actin was detected with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the regulation of actin polymerization and motility.

Figure 1

Effect of MK571, an MRP4 inhibitor, on sperm capacitation. Bovine spermatozoa (SPZs) were incubated in non-capacitating (NC) or capacitating (CAP) conditions in the presence or absence of MK571 (50 μM). After 45 min, samples were incubated or not for 15 min with 5 μM l-α-lysophosphatidylcholine (LPC) to induce acrosomal reaction. Cell viability was assessed with Hoechst 33258 (2 μg/ml, 5 min incubation). Cells were fixed, permeabilized and stained with PSA-FITC in order to evaluate acrosomal reaction. At least 200 spermatozoa per condition were examined with a fluorescence microscope (magnification 1,000 ×). (a) Live spermatozoa were identified as those without a bright and homogeneous signal in its head. n = 3, p > 0.05. Each n corresponds to a different straw from a different bull. (b) Percentage of capacitation was estimated as the difference between the number of live and reacted sperm in the presence of LPC and the number of alive cells spontaneously reacted. n = 3, p < 0.05. Each n corresponds to a different straw from a different bull. (c) In a similar way, after 45 min spermatozoa were incubated with 500 μM chlortetracycline (CTC) and examined with a fluorescence microscope. The percentage of cells with a capacitated pattern (B pattern) was quantified. n = 3, p < 0.05. Each n corresponds to a different straw from a different bull (d) Spermatozoa were co-incubated with a monolayer of bovine oviductal epithelial cells (BOECs). After 60 min, cells were washed and incubated or not with MK571 (50 μM). Spermatozoa release from the monolayer was induced with NC or CAP medium, supplemented or not with MK571 (50 μM) and cAMP (10 nM). After fixation, photographs were taken (upper panel, magnification 200 ×) and the number of bound sperm was quantified in at least 20 fields of 0.11 mm² (lower pannel). n = 6, p < 0.001 (left panel) and p < 0.005 (right panel). Each n corresponds to a different straw from a different bull. Different letters indicate statistically significant differences.

Figure 2

Evaluation of MRP4 localization through capacitation. Cryopreserved bovine sperm were thawed and selected by wool glass columns (T0) and capacitated for 15 or 45 min (CAP15 and CAP45, respectively). An indirect immunofluorescence assay was performed in order to detect MRP4. (a) Slides were examined with a fluorescence microscope (magnification 1,000 ×). Control condition represents cells incubated with secondary antibody without primary antibody. Fluorescence and bright field images are displayed for this condition. (b) MRP4 presence was quantified as fluorescence intensity relative to the area of each cellular compartment. Fluorescence intensity in each region of the spermatozoa was normalized to the T0 condition. At least 200 spermatozoa were examined for each condition. n = 4, p < 0.05 (acrosome), p < 0.05 (equatorial segment), p < 0.05 (postacrosomal region), p < 0.005 (mid-piece). Each n corresponds to a different straw from a different bull. Different letters indicate statistically significant differences.

Figure 3

Effect of MK571 and non-permeable cAMP on sperm motility. Cryopreserved bovine sperm were thawed and incubated for 15 or 45 min in CAP conditions with or without MK571 (50 μM) or non-permeable cAMP (10 nM). Spermatozoa were examined with a Computer-assisted Sperm Analysis (CASA) system set for bovine sperm. Total and progressive motility were assessed in 5 videos for each condition. n = 7, p < 0.05 in all cases. Each n corresponds to a different straw from a different bull. Different letters indicate statistically significant differences.

Figure 4

F-actin modulation during capacitation of cryopreserved bovine spermatozoa. Cryopreserved bovine sperm were thawed and incubated in NC and CAP conditions. After 15 (a) or 45 min (b) incubations, cells were subjected to Alexa Fluor 488-phalloidin staining (6.6 μM, 1 h) and examined with a fluorescence microscope (upper panels, magnification 1,000 ×). The fluorescent signal of F-actin was quantified with the Fiji software for the entire cell, its head or its tail (lower panels). F-actin levels were quantified as fluorescence intensity relative to the area of each cellular compartment and normalized to the NC condition. At least 200 spermatozoa were examined for each condition. n = 5, *p < 0.05 significantly different from NC spermatozoa. Each n corresponds to a different straw from a different bull. (c) Alternatively, after 15 min, cells were fixed, permeabilized and incubated with Alexa Fluor 488-phalloidin (6.6 μM, 60 min) and the examined with a flow cytometer. FL1 medians were obtained and normalized to the NC condition. n = 3, *p < 0.05 significantly different from NC spermatozoa. Each n corresponds to a different straw from a different bull.

Figure 5

Effect of MK571 and extracellular cAMP on actin polymerization. (a) Cryopreserved bovine sperm were thawed and incubated for 15 min in CAP conditions in the presence or absence of MK571 (50 μM) and cAMP (10 nM). (b) Alternatively, spermatozoa were incubated in NC medium for 15 min with or without cAMP (10 nM). Cells were then fixed, permeabilized and incubated with Alexa Fluor 488-phalloidin (6.6 μM, 60 min). Cells were examined with a fluorescence microscope (upper panel, magnification 1,000 ×) and the fluorescent signal was quantified with the Fiji software for the entire cell, its head or its tail (lower panel). F-actin levels were quantified as fluorescence intensity relative to the area of each cellular compartment and normalized to the CAP (a) or NC (b) condition. At least 200 spermatozoa were examined for each condition. n = 6, *p < 0.05 significantly different from sperm incubated in the absence of cAMP. Each n corresponds to a different straw from a different bull. Different letters indicate statistically significant differences.

Figure 6

Role of PKA in actin polymerization induced by non-permeable cAMP. Cryopreserved bovine sperm were thawed and incubated for 15 min in NC conditions in the presence or absence of 50 µM H89 (a); 100 nM KT5720 (b) or 1 mM Rp cAMPS (c). The F-actin staining technique was performed with Alexa Fluor 488-phalloidin (6.6 μM, 60 min) and cells were examined with a fluorescence microscope (upper panels, magnification 1,000 ×). The fluorescent signal was quantified with the Fiji software for the entire cell, its head or its tail (lower panel). F-actin levels were quantified as fluorescence intensity relative to the area of each cellular compartment and normalized to the NC condition. At least 200 spermatozoa were examined for each condition. n = 3, *p < 0.05 in all cases. Each n corresponds to a different straw from a different bull. Different letters indicate statistically significant differences.