Serotonergic signals enhanced hamster sperm hyperactivation

Abstract

In the present study, we investigated the regulatory mechanisms underlying sperm hyperactivation enhanced by 5-hydroxytryptamine (5-HT) in hamsters. First, we examined the types of 5-HT receptors that regulate hyperactivation. Hyperactivation was significantly enhanced by 5-HT_2A and 5-HT₄ receptor agonists. Moreover, the results of the motility assay revealed that 5-HT_2A, 5-HT₃, and 5-HT₄ receptor agonists significantly decreased the velocity and/or amplitude of sperm. Under 5-HT₂ receptor stimulation, hyperactivation was associated with phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP₃) receptor, soluble adenylate cyclase (sAC), and protein kinase A (PKA). In contrast, under 5-HT₄ receptor stimulation, hyperactivation was associated with transmembrane adenylate cyclase (tmAC), sAC, PKA, and CatSper channels. Accordingly, under the condition that sperm are hyperactivated, 5-HT likely stimulates PLC/IP₃ receptor signals via the 5-HT_2A receptor and tmAC/PKA/CatSper channel signals via the 5-HT₄ receptor. After sAC and PKA are activated by these stimulations, sperm hyperactivation is enhanced.

Keywords: 5-HT2 receptor; 5-HT4 receptor; 5-Hydroxytryptamine (5-HT); Hyperactivation; Sperm.

Fig. 1.

Suppression of MS-enhanced hyperactivation by PLC, IP₃R, and PKC inhibitors. Percentages of hyperactivation were detected after sperm were cultured for 4 h with 100 fM MS and inhibitors, including 1 μM U73122 and 1 μM U73343 (A), 10 μM D609 (B), 15 μM ET-18-OCH3 (C), 65 μM neomycin (D), 1 mM spermine (E), 1 μM xestospongin C (F), and 10 nM Bis-1 (G). Data represent the mean ± standard deviation (SD). (A) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) ethanol as vehicle; (MS) medium with 100 fM MS and vehicle; (MS + U73122) medium with 100 fM MS, 1 μM U73122, and vehicle; (MS + U73343) medium with 100 fM MS, 1 μM U73343, and vehicle. (B) (Vehicle) same as above; (MS) medium with 100 fM MS and vehicle; (MS + D609) medium with 100 fM MS, 10 μM D609, and vehicle. (C) (Vehicle) same as above; (MS) medium with 100 fM MS and vehicle; (MS + ET-18-OCH3) medium with 100 fM MS, 15 μM ET-18-OCH3, and vehicle. (D) (Vehicle) same as above; (MS) medium with 100 fM MS and vehicle; (MS + Neomycin) medium with 100 fM MS, 65 μM neomycin, and vehicle. (E) (Vehicle) same as above; (MS) medium with 100 fM MS and vehicle; (MS + Spermine) medium with 100 fM MS, 1 mM spermine, and vehicle. (F) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MS) medium with 100 fM MS and vehicle; (MS + Xestospongin C) medium with 100 fM MS, 1 μM xestospongin C, and vehicle. (G) (Vehicle) same as above; (MS) medium with 100 fM MS and vehicle; (MS + Bis-1) medium with 100 fM MS, 10 nM Bis-1, and vehicle. * indicates significant differences compared with “Vehicle” (P < 0.05). ** indicates significant differences compared with “Vehicle,” “MS + U73122” (P < 0.05). # indicates significant differences compared with “Vehicle” and “MS + inhibitors” (P < 0.05). MS, α-methylserotonin maleate; Bis-1, bisindolylmaleimide 1; PLC, phospholipase C; PKC, protein kinase C; IP₃, inositol 1,4,5-trisphosphate.

Fig. 2.

Suppression of MS-enhanced hyperactivation by adenylate cyclase inhibitors. Percentages of motility (A, C, and E) and hyperactivation (B, D, and F) were detected after sperm were cultured for 4 h with 100 fM MS and inhibitors, including 50 and 100 μM ddAdo (A and B), 20 and 50 μM 2-CE (C and D), and 10 and 25 μM KH7 (E and F). Data represent the mean ± standard deviation (SD). (A and B) (Vehicle) medium with 0.2% (v/v) pure water as vehicle; (MS) medium with 100 fM MS and vehicle; (50 μM ddAdo) medium with 50 μM ddAdo and vehicle; (MS + 50 μM ddAdo) medium with 100 fM MS, 50 μM ddAdo, and vehicle; (100 μM ddAdo) medium with 100 μM ddAdo and vehicle; (MS + 100 μM ddAdo) medium with 100 fM MS, 100 μM ddAdo, and vehicle. (C and D) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) ethanol as vehicle; (MS) medium with 100 fM MS and vehicle; (20 μM 2-CE) medium with 20 μM 2-CE and vehicle; (MS + 20 μM 2-CE) medium with 100 fM MS, 20 μM 2-CE, and vehicle; (50 μM 2-CE) medium with 50 μM 2-CE and vehicle; (MS + 50 μM 2-CE) medium with 100 fM MS, 250 μM 2-CE, and vehicle. (E and F) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MS) medium with 100 fM MS and vehicle; (10 μM KH7) medium with 10 μM KH7 and vehicle; (MS + 10 μM KH7) medium with 100 fM MS, 10 μM KH7, and vehicle; (25 μM KH7) medium with 25 μM KH7 and vehicle; (MS + 25 μM KH7) medium with 100 fM MS, 25 μM KH7, and vehicle. * indicates significant differences compared with “Vehicle” and “Inhibitors” (P < 0.05). ** indicates significant differences compared with “Vehicle,” “MS,” “Low concentration of inhibitor,” and “MS + inhibitors” (P < 0.05). # indicates significant differences compared with “Vehicle” and “MS” (P < 0.05). ## indicates significant differences compared with “Vehicle,” “MS,” “Low concentration of inhibitor,” and “MS + low concentration of inhibitor” (P < 0.05). ¢ indicates significant differences compared with “Vehicle,” “MS,” “High concentration of inhibitor,” and “MS + High concentration of inhibitor” (P < 0.05). ¢¢ indicates significant differences compared with “Vehicle,” “Inhibitors,” and “MS + Inhibitor” (P < 0.05). $ indicates significant differences compared with “Vehicle,” “Inhibitors,” and “MS + High concentration of inhibitor” (P < 0.05). $$ indicates significant differences compared with “Vehicle,” “High concentration of inhibitor,” and “MS + High concentration of inhibitor” (P < 0.05). MS, α-methylserotonin maleate; 2-CE, 2-hydroxyestradiol; ddAdo, 2’,3’-dideoxyadenosine.

Fig. 3.

Suppression of motility and hyperactivation by H-89 (PKA inhibitor). Percentages of motility (A and C) and hyperactivation (B and D) were determined after sperm were cultured with various concentrations of H-89 for 4 h in the absence and the presence of 100 fM MS. Data represent the mean ± standard deviation (SD). (A and B) (Vehicle) medium with 0.1% (v/v) dimethyl sulfoxide as vehicle; (1 µM H-89) medium with 1 µM H-89 and vehicle; (10 µM H-89) medium with 10 µM H-89 and vehicle; (100 µM H-89) medium with 100 µM H-89 and vehicle. (C and D) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MS) medium with 100 fM MS and vehicle; (MS + 1 µM H-89) medium with 100 fM MS, 1 µM H-89, and vehicle; (MS + 10 µM H-89) medium with 100 fM MS, 10 µM H-89, and vehicle; (MS + 100 µM H-89) medium with 100 fM MS, 100 µM H-89, and vehicle. * indicates significant differences compared with “Vehicle,” “1 µM H-89,” and “10 µM H-89” (P < 0.05). ** indicates significant differences compared with “Vehicle” and “1 µM H-89” (P < 0.05). # indicates significant differences compared with “Vehicle” (P < 0.05). ## indicates significant differences compared with “Vehicle,” “MS,” “MS + 1 µM H-89,” and “MS + 10 µM H-89” (P < 0.05). $ indicates significant differences compared with “Vehicle” (P < 0.05). $$ indicates significant differences compared with “Vehicle” and “MS + 10 µM H-89” (P < 0.05). ⁂ indicates significant differences compared with “Vehicle,” “MS + 1 µM H-89,” and “MS + 10 µM H-89” (P < 0.05). MS, α-methylserotonin maleate.

Fig. 4.

Suppression of MT-enhanced hyperactivation by adenylate cyclase inhibitors. Percentages of motility (A, C, and E) and hyperactivation (B, D, and F) were determined after sperm were cultured for 4 h with 10 pM MT and inhibitors, including 50 and 100 μM ddAdo (A and B), 20 and 50 μM 2-CE (C and D), and 10 and 25 μM KH7 (E and F). Data represent the mean ± standard deviation (SD). (A and B) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) ethanol as vehicle; (MT) medium with 10 pM MT and vehicle; (50 μM ddAdo) medium with 50 μM ddAdo and vehicle; (MT + 50 μM ddAdo) medium with 10 pM MT, 50 μM ddAdo, and vehicle; (100 μM ddAdo) medium with 100 μM ddAdo and vehicle; (MT + 100 μM ddAdo) medium with 10 pM MT, 100 μM ddAdo, and vehicle. (C and D) (Vehicle) medium with 0.2% (v/v) ethanol as vehicle; (MT) medium with 10 pM MT and vehicle; (20 μM 2-CE) medium with 20 μM 2-CE and vehicle; (MT + 20 μM 2-CE) medium with 10 pM MT, 20 μM 2-CE, and vehicle; (50 μM 2-CE) medium with 50 μM 2-CE and vehicle; (MT + 50 μM 2-CE) medium with 10 pM MT, 50 μM 2-CE, and vehicle. (E and F) (Vehicle) medium with 0.1% (v/v) ethanol and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MT) medium with 10 pM MT and vehicle; (10 μM KH7) medium with 10 μM KH7 and vehicle; (MT + 10 μM KH7) medium with 10 pM MT, 10 μM KH7, and vehicle; (25 μM KH7) medium with 25 μM KH7 and vehicle; (MT + 25 μM KH7) medium with 10 pM MT, 25 μM KH7, and vehicle. * indicates significant differences compared with “Vehicle,” “Inhibitors,” and “MT + Inhibitors” (P < 0.05). # indicates significant differences compared with “Vehicle” and “MT” (P < 0.05). ## indicates significant differences compared with “Vehicle,” “Inhibitors,” and “MT + High concentration of inhibitor” (P < 0.05). $ indicates significant differences compared with “Vehicle,” “MT,” “Low concentration of inhibitor,” and “MT + Inhibitors” (P < 0.05). $$ indicates significant differences compared with “Vehicle,” “MT,” and “High concentration of inhibitor” (P < 0.05). ¢ indicates significant differences compared with “Vehicle,” “MT,” “Low concentration of inhibitor,” and “MT + Low concentration of inhibitor” (P < 0.05). ¢¢ indicates significant differences compared with “Vehicle,” “MT,” “High concentration of inhibitor,” and “MT + High concentration of inhibitor” (P < 0.05). £ indicates significant differences compared with “Vehicle” and “Inhibitors” (P < 0.05). MT, 5-methoxytryptamine; 2-CE, 2-hydroxyestradiol; ddAdo, 2’,3’-dideoxyadenosine.

Fig. 5.

Suppression of MT-enhanced hyperactivation by PKA inhibitors. Percentages of motility (A) and hyperactivation (B) were determined after sperm were cultured for 4 h with 10 pM MT and various concentrations of H-89. Data represent the mean ± standard deviation (SD). (A and B) (Vehicle) medium with 0.1% (v/v) ethanol and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MT) medium with 10 pM MT and vehicle; (MT + 1 µM H-89) medium with 10 pM MT, 1 µM H-89, and vehicle; (MT + 10 µM H-89) medium with 10 pM MT, 10 µM H-89, and vehicle; (MT + 100 µM H-89) medium with 10 pM MT, 100 µM H-89, and vehicle. * indicates significant differences compared with “Vehicle,” “Inhibitors,” and “MT + Inhibitors” (P < 0.05). ** indicates significant differences compared with “Vehicle,” “MT,” “MT + 1 µM H-89,” and “MT + 10 µM H-89” (P < 0.05). $ indicates significant differences compared with “Vehicle,” “MT + 1 µM H-89,” and “MT + 10 µM H-89” (P < 0.05). MT, 5-methoxytryptamine; PKA, protein kinase A.

Fig. 6.

Suppression of MT-enhanced hyperactivation by CatSper inhibitors. Percentages of motility (A, C, E, and G) and hyperactivation (B, D, F, and H) were determined after sperm were cultured for 4 h with 10 pM MT and inhibitors such as 3 μM HC (A and B), 10 μM HC (C and D), 30 and 40 μM Mib (E and F), and 10 and 20 μM NNC (G and H). Data represent the mean ± standard deviation (SD). (A and B) (Vehicle) medium with 0.1% (v/v) ethanol and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MT) medium with 10 pM MT and vehicle; (3 µM HC) medium with 3 µM HC and vehicle; (MT + 3 µM HC) medium with 10 pM MT, 3 µM HC, and vehicle. (C and D) (Vehicle) same as above; (MT) medium with 10 pM MT and vehicle; (10 µM HC) medium with 10 µM HC and vehicle; (MT + 10 µM HC) medium with 10 pM MT, 10 µM HC, and vehicle. (E and F) (Vehicle) same as above; (MT) medium with 10 pM MT and vehicle; (30 µM Mib) medium with 30 µM Mib and vehicle; (MT + 30 µM Mib) medium with 10 pM MT, 30 µM Mib, and vehicle; (40 µM Mib) medium with 40 µM Mib and vehicle; (MT + 40 µM Mib) medium with 10 pM MT, 40 µM Mib, and vehicle. (G and H) (Vehicle) same as above; (MT) medium with 10 pM MT and vehicle; (10 µM NNC) medium with 10 µM NNC and vehicle; (MT + 10 µM NNC) medium with 10 pM MT, 10 µM NNC, and vehicle; (20 µM NNC) medium with 20 µM NNC and vehicle; (MT + 20 µM NNC) medium with 10 pM MT, 20 µM NNC, and vehicle. * indicates significant differences compared with “Vehicle,” “inhibitors,” and “MT + inhibitors” (P < 0.05). ** indicates significant differences compared with “Vehicle,” “MT,” and “MT + inhibitors” (P < 0.05). # indicates significant differences compared with “Vehicle” and “inhibitors” (P < 0.05). ## indicates significant differences compared with “inhibitors” and “MT + inhibitors” (P < 0.05). $ indicates significant differences compared with “Vehicle” and “MT” (P < 0.05). MT, 5-methoxytryptamine; HC, 2,4-dithenoyl-1,2,5-oxadiazone n2-oxide; Mib, mibefradil.