Effects of 5-hydroxytryptamine on spermatozoal hyperactivation and in vitro fertilization in mice

Abstract

In this study, we examined the effects of 5-hydroxytryptamine (5-HT) on the motility and hyperactivation of mouse spermatozoa. In addition, we examined whether 5-HT increases the success of in vitro fertilization (IVF) in mice. Interestingly, 5-HT and agonists of the 5-HT₂, 5-HT₃, 5-HT₄, and 5-HT₇ receptors significantly increased the percentage of hyperactivated spermatozoa but did not affect the percentage of motile spermatozoa. Moreover, agonists of the 5-HT₂, 5-HT₃, and 5-HT₄ receptors significantly affected the velocities, linearity, straightness, wobbler coefficient, amplitude and/or frequency of spermatozoa. In particular, the improvement of hyperactivation by 5-HT was strongly inhibited by antagonists of the receptors 5-HT₄ and 5-HT₇ and was completely inhibited by a mixture of the four 5-HT-receptor antagonists. The increase in hyperactivation by the agonists was significantly inhibited by the corresponding 5-HT-receptor antagonist. Moreover, 5-HT significantly increased the percentage of two-cell embryos. The increase in the IVF success rate by 5-HT was significantly inhibited by a 5-HT₄-receptor antagonist. These results suggest that 5-HT increased hyperactivation through the 5-HT receptors and increased the success of IVF in mice.

Keywords: 5-Hydroxytryptamine (5-HT); Hyperactivation; In vitro fertilization (IVF); Spermatozoa.

Fig. 1.

Effects of 5-hydroxytryptamine (5-HT) and 5-HT-receptor agonists on percentages of motile and hyperactivated spermatozoa. The percentages of motile and hyperactivated spermatozoa are shown after spermatozoa were exposed to 100 fM, 100 pM, or 100 nM 5-HT (A), 17 nM or 100 nM sumatriptan succinate (Sumatriptan) (B), 100 fM α-methylserotonin maleate salt (MS) (C), 100 μM 1-(3-chlorophenyl)biguanide hydrochloride (mCPBG) (D), 10 pM 5-methoxytryptamine (MT) (E), 7.3 nM WAY-208466 dihydrochloride (WAY) (F), and 0.13 nM LP12 hydrochloride hydrate (LP12) (G). Data represent the mean ± SD. (A) (Vehicle) the medium with 0.1% (v/v) pure water as vehicle; (respective concentrations of 5-HT) the medium with indicated concentration of 5-HT and vehicle. (B) (Vehicle) same as above; (respective concentrations of Sumatriptan) the medium with indicated concentrations of Sumatriptan and vehicle. (C) (Vehicle) same as above; (MS) the medium with MS and vehicle. (D) (Vehicle) same as above; (mCPBG) the medium with mCPBG and vehicle. (E) (Vehicle) medium with 0.1% (v/v) ethanol as vehicle; (MT) medium with MT and vehicle. (F) (Vehicle) medium with 0.1% (v/v) pure water as vehicle; (WAY) medium with WAY and vehicle. (G) (Vehicle) same as above; (LP12) medium with LP12 and vehicle. * Significant difference compared with “Vehicle” (P < 0.05).

Fig. 2.

Suppressive effects of 5-hydroxytryptamine (5-HT)-receptor antagonists on the increase of hyperactivation by 5-HT. The percentages of motile and hyperactivated spermatozoa are shown after spermatozoa were exposed to 100 fM, 100 pM, or 100 nM 5-HT after exposure to 5-HT-receptor antagonists (A–D) or a mixture of antagonists (E) for 5 min. The mixture contained 1 μM cyproheptadine hydrochloride sesquihydrate (Cypro), 3.8 nM dolasetron mesylate hydrate (DS), 1 μM GR113808 (GR), and 10 μM SB-258719 (SB). The data represent the mean ± SD. (A) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) ethanol as vehicle; (respective concentrations of 5-HT) medium with respective concentration of 5-HT and vehicle; (Cypro) the medium with 1 μM Cypro and vehicle; (respective concentrations of 5-HT + Cypro) medium with respective concentration of 5-HT, 1 μM Cypro, and vehicle. (B) (Vehicle) medium with 0.2% (v/v) pure water as vehicle; (respective concentrations of 5-HT) medium with respective concentration of 5-HT and vehicle; (DS) medium with 3.8 nM DS and vehicle; (respective concentrations of 5-HT + DS) medium with respective concentration of 5-HT, 3.8 nM DS, and vehicle. (C) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide (DMSO) as vehicle; (respective concentrations of 5-HT) medium with respective concentration of 5-HT and vehicle; (GR) medium with 1 μM GR and vehicle; (respective concentrations of 5-HT + GR) medium with respective concentration of 5-HT, 1 μM GR, and vehicle. (D) (Vehicle) same as above; (all concentrations of 5-HT) medium with respective concentration of 5-HT and vehicle; (SB25) medium with 10 μM SB-258719 (SB25) and vehicle; (respective concentrations of 5-HT + SB25) medium with respective concentration of 5-HT, 10 μM SB25, and vehicle. (E) (Vehicle) medium with 0.2% (v/v) pure water, 0.1% (v/v) ethanol, and 0.2% (v/v) DMSO as vehicle; (respective concentrations of 5-HT) medium with the respective concentrations of 5-HT and vehicle; (Mix) medium with the mixture of four antagonists and vehicle; (respective concentrations of 5-HT + Mix) medium with all concentrations of 5-HT, the mixture of four antagonists, and vehicle. * Significant difference compared with “Vehicle” and “antagonist” (P < 0.05). ** Significant difference compared with “Vehicle”, “antagonist”, and “respective concentrations of 5-HT + antagonist” (P < 0.05). ^# Significant difference compared with “Vehicle”, “Mix”, and “respective concentrations of 5-HT + Mix” (P < 0.05).

Fig. 3.

Inhibition of the effect of 5-hydroxytryptamine (5-HT)-receptor agonists by 5-HT-receptor antagonists. The percentages of motile and hyperactivated spermatozoa are shown when spermatozoa were exposed to 5-HT-receptor agonists after exposure to 5-HT-receptor antagonists for 5 min. The data represent the mean ± SD. (A) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) ethanol as vehicle; (MS) medium with 100 fM α-methylserotonin maleate salt (MS) and vehicle; (Cypro) medium with 1 μM cyproheptadine hydrochloride sesquihydrate (Cypro) and vehicle; (MS + Cypro) the medium with 100 fM MS, 1 μM Cypro, and vehicle. (B) (Vehicle) medium with 0.2% (v/v) pure water as vehicle; (mCPBG) medium with 100 μM 1-(3-chlorophenyl)biguanide hydrochloride (mCPBG) and vehicle; (DS) medium with 3.8 nM dolasetron mesylate hydrate (DS) and vehicle; (mCPBG + DS) medium with 100 μM mCPBG, 3.8 nM DS, and vehicle. (C) (Vehicle) medium with 0.1% (v/v) ethanol and 0.1% (v/v) dimethyl sulfoxide (DMSO) as vehicle; (MT) medium with 10 pM 5-methoxytryptamine (MT) and vehicle; (GR) medium with 1 μM GR113808 (GR) and vehicle; (MT + GR) medium with 10 pM MT, 1 μM GR, and vehicle. (D) (Vehicle) medium with 0.1% (v/v) pure water and 0.1% (v/v) DMSO as vehicle; (LP12) medium with 0.13 nM LP12 hydrochloride hydrate (LP12) and vehicle; (SB25) medium with 10 μM SB-258719 (SB25) and vehicle; (LP12 + SB25) medium with 0.13 nM LP12, 10 μM SB25, and vehicle. * Significant difference compared with “Vehicle”, “antagonist”, and “agonist + antagonist” for the same incubation time (P < 0.05).