Tryptophan promotes sperm hyperactivation in hamsters via 5-hydroxytryptamine biosynthesis within sperm

Abstract

L-tryptophan (Trp), an essential amino acid, is a precursor of 5-hydroxytryptamine (5-HT; also known as serotonin) that promotes mammalian sperm hyperactivation. Since mammalian sperm contain Trp hydroxylase (TPH), they may contribute to 5‑HT biosynthesis. Therefore, this study aimed to examine the effect of Trp on hamster sperm hyperactivation and determine whether sperm are involved in 5-HT biosynthesis. Trp significantly enhanced sperm hyperactivation via the 5-HT₄ receptor and its associated signals. In contrast, D-tryptophan did not affect sperm hyperactivation. Furthermore, hamster sperm contained the 5-HT biosynthesis enzymes TPH and aromatic L-amino acid decarboxylase (AADC). Additionally, hamster sperm secreted 5-HT. Trp-enhanced hyperactivation and 5-HT secretion were significantly inhibited by TPH and AADC inhibitors. Overall, our findings suggest that Trp enhanced sperm hyperactivation through the biosynthesis of 5-HT within the sperm.

L-tryptophan (Trp) is one of the nine essential amino acids and one of the 20 standard amino acids that constitute proteins [1, 2]. Trp is a precursor of 5-hydroxytryptamine(5-HT; also known as serotonin), melatonin, and vitamin B₃. Both 5-HT and melatonin regulate mammalian reproductive functions, such as steroidogenesis and steroid secretion, spermatogenesis, oocyte maturation, sperm motility and capacitation, fertilization, and embryonic development [3,4,5]. Both 5-HT and melatonin enhance the acrosome reaction (AR) and hyperactivation [6,7,8,9,10,11,12]; these are key processes associated with the fertilization capacity of mammalian sperm [13, 14]. Only capacitated sperm can fertilize oocytes [13]. AR is an exocytotic process that involves the release of hydrolases from acrosomal vesicles [13]. Following the AR, sperm digest the oocyte envelope, which consists of the zona pellucida and cumulus cells, and then bind to the oocytes [13]. Hyperactivation is a specialized motility pattern exhibited by mammalian sperm during capacitation [13, 15]. Hyperactivated sperm swim through the viscous oviductal fluid and penetrate the oocyte envelope [13, 15]. Notably, hyperactivation capacity is correlated with in vitro fertilization (IVF) success [16,17,18,19]. In mice [10] and rats [12], 5-HT enhances sperm hyperactivation and increases IVF success.

Albumin, Ca²⁺, and HCO₃^- play important roles as key molecules during in vitro sperm capacitation [13, 15]. Albumin removes cholesterol from the cell membrane [20], stimulates CatSper Ca²⁺ channels, and induces Ca²⁺ influx [21]. In the absence of albumin, sperm do not undergo hyperactivation [7, 22]. Additionally, mice lacking the CatSper Ca²⁺ channel fail to exhibit hyperactivated motility in sperm [23]. Ca²⁺ and HCO₃^– activate soluble adenylate cyclase (sAC) and increase cAMP levels [14, 24,25,26]. Moreover, Ca²⁺ and cAMP regulate phosphorylation by activating protein kinases and phosphatases [13, 15, 27, 28]. Protein kinase A (PKA) induces the activation of CatSper Ca²⁺ channels in mouse sperm [29] but not in human sperm [30]. Hormones and neurotransmitters regulate sperm hyperactivation through specific membrane receptors [5]. CatSper Ca²⁺ channels, sAC, and PKA are activated by Ca²⁺ and cAMP following receptor stimulation. In hamsters and rats, progesterone binds to the membrane progesterone receptor, inducing phospholipase C (PLC) activation, inositol 1,4,5-trisphosphate receptor stimulation, as well as sAC and PKA activation [22, 31, 32]. In hamsters, 5-HT binds to 5-HT₂ and 5-HT₄ receptors in a dose-dependent manner [9]. 5-HT acts on the 5-HT₂ receptor and activates PLC, inositol 1,4,5-trisphosphate receptor, sAC, and PKA [33]. The 5-HT₂ and membrane progesterone receptors are found to trigger similar signals. On the other hand, stimulation of the 5-HT₄ receptor by 5-HT activates transmembrane adenylate cyclase (tmAC) [33], which further activates PKA by catalyzing the conversion of ATP to cAMP. Once activated, CatSper Ca²⁺ channels trigger the activation of sAC and PKA.

5-HT is formed by the hydroxylation and decarboxylation of Trp [3] (Supplementary Fig. 1). Trp is hydroxylated by Trp hydroxylase (TPH) to synthesize 5-hydroxytryptophan (5-HTP). 5-HTP is an intermediate in 5-HT biosynthesis; it is decarboxylated by aromatic L-amino acid decarboxylase (AADC; also known as DOPA decarboxylase) to form 5-HT. Recently, TPH has been detected in human [34, 35] and stallion [36] sperm.

5-HT mediates its actions through 5-HT receptors [3, 37]. 5-HT receptors are classified into seven families: 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄, 5-HT₅, 5-HT₆, and 5-HT₇. 5-HT₁ and 5-HT₅ receptors suppress tmAC, whereas 5-HT₄, 5-HT₆, and 5-HT₇ receptors stimulate tmAC and activate cAMP signaling. Furthermore, the 5-HT₂ receptor stimulates PLC and activates Ca²⁺ signaling, whereas the 5-HT₃ receptor is a ligand-gated cation channel receptor that induces depolarization.

In the reproductive organs, 5-HT is produced in cumulus cells and secreted into follicles and oviductal fluids [3]. In humans, 5-HT levels in the preovulatory and cystically degenerated follicles are 14.3 ± 8.9 and 12.2 ± 6.2 μg/100 ml, respectively [38]. In rats, 5-HT levels in oviducts are 2.06–3.34 μg/g fresh tissue [39]. 5-HT increases human sperm velocity and motility [34, 35], with this increase in motility being associated with the 5-HT₂ receptor [35]. In hamster sperm, 5-HT enhances the AR and hyperactivation through the 5-HT₂ and 5-HT₄ receptors [6, 9, 11]. In mice, 5-HT promotes sperm hyperactivation through the 5-HT₂, 5-HT₃, 5-HT₄, and 5-HT₇ receptors, and increases IVF success through the 5-HT₄ receptor [10]. In rats, 5-HT boosts sperm hyperactivation and increases IVF success, mediated by the 5-HT₄ receptor alone [12].

Given that TPH has been detected in human and stallion sperm [34,35,36], we hypothesized that sperm produce and secrete 5-HT. Moreover, it is hypothesized that hyperactivation is enhanced by 5-HT secreted from sperm. To verify this, we examined whether Trp influences the motility and hyperactivation of hamster sperm via 5-HT biosynthesis within sperm.

Materials and Methods

Chemicals

Bovine serum albumin (BSA), cyproheptadine hydrochloride sesquihydrate (Cypro), 2ʹ,3ʹ-dideoxyadenosine (ddAdo), 2-hydroxyestradiol (2-CE), KH7, 2,4-dithenoyl-1,2,5-oxadiazone n2-oxide (HC-056456 [HC]), and GR113808 (GR) were purchased from Merck KGaA (Darmstadt, Germany). H-89, LP-533401 (LP), mibefradil (Mib), and NNC 55-0396 (NNC) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Serotonin ELISA kit (ADI-900-175) was purchased from ENZO Life Sciences Inc. (Farmingdale, NY, USA). The anti-TPH antibody (EP1311Y; ab52954) was purchased from Abcam Ltd. (Cambridge, UK). The anti-DOPA decarboxylase polyclonal antibody (10166-1-AP) was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Polyvinylidene difluoride membranes were purchased from Millipore (Bedford, MA, USA). The molecular weight marker set was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). EzWestLumi Plus was purchased from the ATTO Corporation (Tokyo, Japan). Benserazide (Ben) and other reagent-grade chemicals were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Animals

Syrian hamsters (Mesocricetus auratus) were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and bred at the Research Center for Laboratory Animals at the Dokkyo Medical University. The hamsters were housed in light- and temperature-controlled (12 h on/off and 25 ± 2°C, respectively) facilities with food and water provided ad libitum. All experiments were approved by the Animal Care and Use Committee of the university (approval numbers: 0107 and 1383) and were performed in accordance with the university guidelines for animal experimentation.

Preparation of hyperactivated sperm

Sperm were collected from the caudal epididymis of 10- to 20-week-old-male hamsters. Hyperactivated sperm were prepared as previously described [27]. Modified Tyrode’s albumin lactate pyruvate medium [40] was used as the capacitation medium. The caudal epididymis of each hamster was punctured with a 23-G (0.6 mm) needle (Terumo Corporation, Tokyo, Japan). A drop (approximately 5 μl) of sperm was collected and placed in a culture dish (35 mm in diameter; Iwaki Asahi Glass Co., Ltd., Tokyo, Japan), and 3 ml of medium was added to the dish. Sperm were incubated for 5 min at 37°C and allowed to swim up. The supernatant containing swimming sperm was placed in a new dish that contained the vehicle, inhibitors, or antagonists. Following incubation for 5 min, the supernatant was transferred to a new dish containing the vehicle, Trp, D-tryptophan (D-Trp), or 5-HTP. Sperm were incubated for 4 h at 37°C and 5% (v/v) CO₂ to induce hyperactivation. To prepare the stock solutions, Ben (5 mM), ddAdo (100 mM), Trp (10 mM), D-Trp (10 mM), and 5-HTP (100 mM) were dissolved in pure water. Cypro (1 mM) and 2-CE (20 mM) were dissolved in ethanol. GR (1 mM), H89 (100 mM), LP (1 mM), KH7 (10 mM), HC (30 mM), Mib (40 mM), and NNC (20 mM) were dissolved in dimethyl sulfoxide. In all experiments, the maximum concentration of the vehicle was 0.2% (v/v).

Measurement of motility and hyperactivation

Sperm motility and hyperactivation were measured as previously described [33]. Motile sperm in the dish were observed using a CCD camera (Progressive 3CCD; Sony Corp., Tokyo, Japan) attached to a phase-contrast microscope (IX70; Olympus Corp., Tokyo, Japan) and a small CO₂ incubator (MI-IBC; Olympus) at 37°C, and recorded for 1 min on a DVD recorder (RDR-HX50; Sony). Visual analyses of the movies included manual counts of total sperm, motile sperm, and hyperactivated sperm from eight different fields. Visual analyses were conducted in a blinded manner for all experiments. Motile sperm exhibiting asymmetric and whiplash-like flagellar movements were considered hyperactivated sperm [5, 33]. The percentages of motility and hyperactivation were calculated as the number of motile sperm/number of total sperm × 100 and number of hyperactivated sperm/number of total sperm × 100, respectively. Each experiment was repeated four times using four hamsters. If the proportion of motile sperm was ≤ 80%, the experiment was repeated.

Evaluation of motility kinematics

Motility kinematics were evaluated using the Sperm Motility Analysis System (SMAS) for Animals (v.3.18) with the loaded parameter file mouse_BM10×_ 640 nm _Bright59_150fps-shutter200.ini (Ditect Co., Ltd., Tokyo, Japan) as previously described [33]. The suspension containing motile sperm (20 μl) was transferred to an observation chamber (0.1 mm deep, 18 mm wide, and 18 mm long) constructed using two parallel strips of mending tape attached to the glass slide and covered with a cover glass. Sperm movement was recorded for 1 s on the hard disk drive of the SMAS using a high-speed digital camera (HAS-L2; Ditect) attached to a microscope (ECLIPSE E200; Nikon Corp., Tokyo, Japan) with phase-contrast illumination, a 650 nm band-pass filter, and a warm plate (MP10DM; Kitazato Corp., Shizuoka, Japan). The SMAS analyzed 150 consecutive images obtained from a single field at 10× magnification with a negative phase contrast. Velocity straight line (VSL) (μm/sec), velocity curved line (VCL) (μm/sec), velocity average path (VAP) (μm/sec), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH) (μm), and beat-cross frequency (BCF) (Hz) were automatically calculated by SMAS; wobbler coefficient (WOB; defined as VAP/VCL) was calculated manually [41]. The SMAS analysis was repeated four times using four hamsters. In each experiment, ≥ 300 sperm were detected. Only motile sperm from the detected sperm were analyzed.

Preparation of the sperm protein extract

Sperm proteins were extracted as previously described [27]. Sperm were homogenized at 10 mg sperm (wet weight)/ml in a urea solution containing 7 M urea and 10% (v/v) 2-mercaptoethanol, incubated on ice for 10 min, and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was used as the urea extract. The precipitate was re-homogenized in a urea–thiourea solution at the same volume as the urea solution. The urea–thiourea solution contained 5 M urea, 1 M thiourea, 10% (v/v) 2-mercaptoethanol, and 2% (v/v) NP-40. The homogenate was incubated on ice for 10 min and centrifuged at 15,000 × g for 10 min at 4°C. The resulting supernatant was used as the urea–thiourea extract.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was conducted as described by Laemmli [42] using a separating gel of 10% (w/v) polyacrylamide containing 0.1% (w/v) SDS. Following Coomassie brilliant blue staining, the gel was scanned using a densitometer (GS-800; Bio-Rad Laboratories).

Western blotting

Western blotting was performed as previously described [33]. After SDS-PAGE, the proteins were transferred onto polyvinylidene fluoride membranes. After washing with Tris-buffered saline (TBS) containing 0.15 M NaCl and 20 mM Tris-HCl (pH 7.5), the membranes were incubated with BSA-TBS containing 5% (w/v) BSA and TBS for 1 h at 25°C. Following three washes with TBS, the membranes were incubated with primary antibodies for 1 h at 25°C (anti-TPH antibody diluted 500-fold in BSA-TBS and anti-DOPA decarboxylase polyclonal antibody diluted 1000-fold in BSA-TBS). After washing thrice with TBS, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000 dilution with BSA-TBS). The membrane was washed three times with 0.05% (w/v) Tween-20 containing TBS. Chemiluminescence was detected using EzWestLumi plus, and antibody reactivity was analyzed using Ez-Capture MG (ATTO).

Measurement of 5-HT levels

The levels of 5-HT secreted by hamster sperm were measured using a serotonin ELISA kit. Hamster sperm (171 × 10⁶ sperm/ml) were suspended in modified Tyrode’s albumin lactate pyruvate medium containing the vehicle, Trp, 5-HTP, LP, and/or Ben and incubated for 4 h at 37°C and 5% CO₂. The suspension containing motile sperm was centrifuged at 15,000 × g for 10 min at 4°C. Finally, the supernatant was collected and the 5-HT concentration was measured according to the manufacturer’s instructions.

Statistical analyses

Data were obtained from experiments that were repeated four times using four hamsters. Data are presented as mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance and post hoc test (Student–Newman–Keuls test) using Microsoft Excel with the ystat2018 add-on (Igakutosho Shuppan, Saitama, Japan). When data were found to be significant by one-way analysis of variance, they were further analyzed using post hoc tests to determine detailed significance. Statistical significance was set at P < 0.05.

Results

Effects of Trp on sperm motility, hyperactivation, and motility kinematics

As shown in Figs. 1A and B, Trp significantly enhanced sperm hyperactivation after incubation for 1.5 h and 2 h, but it did not affect sperm motility. Following 1.5 h of incubation (Fig. 1C), 1 nM (10^-9 M) and 10 nM (10^-8 M) Trp significantly increased sperm hyperactivation compared with the vehicle and 1 fM to 100 pM (10^-15 to 10^-10 M) Trp. Moreover, 100 nM to 10 μM (10^-7 to 10^-5 M) Trp significantly increased sperm hyperactivation compared with the vehicle and 1 fM to 10 nM (10^-15 to 10^-8 M) Trp. As shown in Fig. 1D, following 2 h of incubation, 100 nM (10^-7 M) and 1 μM (10^-6 M) Trp significantly increased sperm hyperactivation compared with the vehicle and 1 fM to 100 pM (10^-15 to 10^-10 M) Trp. In addition, 10 μM (10^-5 M) Trp significantly increased sperm hyperactivation compared with the vehicle and 1 fM to 1 nM (10^-15 to 10^-9 M) Trp. Notably, D-Trp, an optical isomer, did not affect sperm motility or hyperactivation (Figs. 1E and F). Since Trp enhanced sperm hyperactivation (Figs. 1B, 1C, and 1D), we examined whether Trp affected motility kinematics and enhanced hyperactivation. As presented in Supplementary Tables 1 and 2, 100 nM and 1 μM Trp exhibited no effect on sperm motility kinematics following incubation for 1.5 h and 2 h (Supplementary Tables 1, 2).

Fig. 1.

Effects of L-tryptophan (Trp) on sperm motility and hyperactivation. Percentages of sperm motility (A) and hyperactivation (B) after incubation for 1.5 h (C) and 2 h (D) post incubation for 4 h in the presence and absence of Trp. Percentages of sperm motility (E) and hyperactivation (F) after incubation for 4 h in the presence and absence of D-Trp (100 nM and 1 μM). Experiments were repeated four times using four male hamsters. Data are represented as the mean ± standard deviation. (A–D): Vehicle, medium with 0.1% (v/v) pure water as vehicle; 1 fM Trp or –15, medium with 1 fM Trp and vehicle; –14, medium with 10 fM Trp and vehicle; –13, medium with 100 fM Trp and vehicle; 1 pM Trp or –12, medium with 1 pM Trp and vehicle; –11, medium with 10 pM Trp and vehicle; –10, medium with 100 pM Trp and vehicle; 1 nM Trp or –9, medium with 1 nM Trp and vehicle; –8, medium with 10 nM Trp and vehicle; –7, medium with 100 nM Trp and vehicle; 1 μM Trp or –6, medium with 1 μM Trp and vehicle; –5, medium with 10 μM Trp and vehicle. (E and F): Vehicle, medium with 0.1% (v/v) pure water as vehicle; 100 nM D-Trp, medium with 100 nM D-Trp and vehicle; 1 μM D-Trp, medium with 1 μM D-Trp and vehicle. * Significant differences compared with the Vehicle, –15, –14, –13, –12, –11, and –10 groups (P < 0.05). ** Significant differences compared with the Vehicle, –15, –14, –13, –12, –11, –10, –9, and –8 groups (P < 0.05). ^# Significant differences compared with the Vehicle, –15, –14, –13, –12, –11, and –9 groups (P < 0.05).

5-HT receptors are associated with Trp-enhanced sperm hyperactivation

As TPH has been detected in sperm [34,35,36], 5-HT may be synthesized from Trp within the sperm. In hamster sperm, 5-HT stimulates the 5-HT₂ and 5-HT₄ receptors to enhance hyperactivation [9, 33]. We next tested which 5-HT receptors mediated Trp-enhanced hyperactivation (Fig. 2). Following 1.5 h of incubation, sperm hyperactivation enhanced by 1 μM Trp was slightly inhibited by Cypro (a 5-HT₂ receptor antagonist), albeit not completely (Fig. 2B). Following 2 h of incubation, Cypro did not significantly affect Trp-induced hyperactivation (Fig. 2B). GR, a 5-HT₄ receptor antagonist, significantly inhibited sperm hyperactivation enhanced by 1 μM Trp 1.5 h of incubation (Fig. 2D). Additionally, GR significantly inhibited sperm hyperactivation enhanced by 100 nM and 1 μM Trp following 2 h of incubation (Fig. 2D). Neither Cypro nor GR affected cell motility (Figs. 2A and C).

Fig. 2.

5-hydroxytryptamine (5-HT) receptors are associated with Trp-enhanced sperm hyperactivation. Percentages of sperm motility (A) and hyperactivation (B) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the 5-HT₂ receptor antagonist, cyproheptadine (Cypro). Percentages of sperm motility (C) and hyperactivation (D) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the 5-HT₄ receptor antagonist GR113808 (GR). Experiments were repeated four times using four male hamsters. Data are represented as the mean ± standard deviation. (A and B): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) ethanol as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; Cypro, medium with 1 μM Cypro and vehicle; 100 nM Trp + Cypro, medium with 100 nM Trp, 1 μM Cypro and vehicle; 1 μM Trp + Cypro, medium with 1 μM Trp, 1 μM Cypro, and vehicle. (C and D): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; GR, medium with 1 μM GR and vehicle; 100 nM Trp + GR, medium with 100 nM Trp, 1 μM GR, and vehicle; 1 μM Trp + GR, medium with 1 μM Trp, 1 μM GR, and vehicle. * Significant differences compared with the Vehicle, Antagonist, and respective concentrations of Trp + Antagonist groups (P < 0.05). ** Significant differences compared with the Vehicle, respective concentrations of Trp, and Antagonist groups (P < 0.05). ^# Significant differences compared with the Vehicle and Antagonist groups (P < 0.05).

5-HT biosynthesis by sperm

5-HT is synthesized from Trp via two reactions (Supplementary Fig. 1). During the first reaction, Trp is converted into 5-HTP by TPH. As shown in Figs. 3A and B, LP, a TPH inhibitor, significantly inhibited sperm hyperactivation that was enhanced by 100 nM and 1 μM Trp after incubation for 1.5 and 2 h; however, it exerted no effect on sperm motility. The second reaction involves the conversion of 5-HTP to 5-HT by AADC (Supplementary Fig. 1). As presented in Fig. 3D, Ben, an AADC inhibitor, significantly inhibited hyperactivation enhanced by 100 nM and 1 μM Trp following 1.5 h of incubation, but did not inhibit sperm hyperactivation following 2 h of incubation. Moreover, Ben had no effect on sperm motility (Fig. 3C). TPH was detected in the urea extract (Fig. 3E, lanes c and d), whereas AADC was detected in both urea and urea–thiourea extracts of hamster sperm (Fig. 3E, lanes e and f). As shown in Fig. 3F, 5-HT was detected in the Trp-only medium, but not in the medium containing Trp, LP, or Ben, in which hamster sperm were cultured.

Fig. 3.

5-HT biosynthesis by sperm. Percentages of sperm motility (A) and hyperactivation (B) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the Trp hydroxylase (TPH) inhibitor, LP-533401 (LP). Percentages of sperm motility (C) and hyperactivation (D) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the aromatic L-amino acid decarboxylase (AADC) inhibitor, benserazide (Ben). Detection of TPH and AADC (E) extracted from sperm. Detection of 5-HT (F) secreted by sperm. Experiments were repeated four times using four male hamsters. Data are represented as the mean ± standard deviation. (A and B): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; LP, medium with 1 μM LP and vehicle; 100 nM Trp + LP, medium with 100 nM Trp, 1 μM LP, and vehicle; 1 μM Trp + LP, medium with 1 μM Trp, 1 μM LP, and vehicle. (C and D): Vehicle, medium with 0.2% (v/v) pure water as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; Ben, medium with 5 μM Ben and vehicle; 100 nM Trp + Ben, medium with 100 nM Trp, 5 μM Ben, and vehicle; 1 μM Trp + Ben, medium with 1 μM Trp, 5 μM Ben, and vehicle. (E): Lanes a and b show the Coomassie brilliant blue-stained gel. Lanes c and d show the western blotting results using the anti-TPH antibody. Lanes e and f show the western blotting results using the anti-AADC antibody. Lanes a, c, and e show the urea-extract. Lanes b, d, and f show the urea–thiourea extract. Numbers on the left side of lane a represent the molecular weight markers. The volume of the extracts loaded in each lane was 15 μl. Arrow indicates the antibody reaction. (F): Vehicle, medium with 0.2% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; 10 μM Trp, medium with 10 μM Trp and vehicle; 10 μM Trp + LP, medium with 10 μM Trp, 1 μM LP, and vehicle; 10 μM Trp + Ben, medium with 10 μM Trp, 5 μM Ben, and vehicle. * Significant differences compared with the Vehicle, Inhibitor, and respective concentrations of Trp + Inhibitor groups (P < 0.05). ** Significant differences compared with the Vehicle, respective concentrations of Trp, and Inhibitor groups (P < 0.05). ^# Significant differences compared with the Vehicle and respective concentrations of Trp groups (P < 0.05). ^## Significant differences compared with the Vehicle and Inhibitor groups (P < 0.05).

Effect of 5-HTP on sperm hyperactivation

Thereafter, we examined the effects of 5-HTP, a metabolic intermediate in 5-HT biosynthesis, on sperm hyperactivation. As illustrated in Supplementary Figs. 2A and B, 5-HTP significantly enhanced sperm hyperactivation after incubation for 1, 1.5, and 2 h, but did not affect motility. Following 1 h of incubation (Supplementary Fig. 2C), 100 nM (10^-7 M) and 1 μM (10^-6 M) 5-HTP significantly increased sperm hyperactivation compared with the vehicle. As shown in Supplementary Fig. 2D, following 1.5 h of incubation, 100 nM to 10 μM (10^-7 to 10^-5 M) 5-HTP significantly increased sperm hyperactivation compared with 1 pM to 100 pM (10^-12 to 10^-10 M) 5-HTP. Moreover, 100 μM (10^-5 M) 5-HTP significantly increased sperm hyperactivation compared with the vehicle. As presented in Supplementary Fig. 2E, following 2 h of incubation, 100 pM (10^-10 M) 5-HTP significantly increased sperm hyperactivation compared with the vehicle. Furthermore, 1 nM to 1 μM (10^-9 to 10^-6 M) and 100 μM (10^-4 M) 5-HTP significantly increased sperm hyperactivation compared with the vehicle, 1 pM (10^-12 M) 5-HTP, and 10 pM (10^-11 M) 5-HTP. Additionally, 10 μM (10^-5 M) 5-HTP significantly increased sperm hyperactivation compared with the vehicle and 1 pM to 100 pM (10^-12 to 10^-10 M) 5-HTP.

In the presence of 5-HTP, Cypro did not affect sperm motility or hyperactivation (Supplementary Figs. 3A and B). GR inhibited sperm hyperactivation that was enhanced by 5-HTP, but did not affect sperm motility (Supplementary Figs. 3C and D). Following 1 h of incubation (Supplementary Fig. 3E), sperm hyperactivation, which was increased by 100 nM 5-HTP, was significantly inhibited by GR. As shown in Supplementary Fig. 3F, following 1.5 h of incubation, sperm hyperactivation, which was increased by 100 nM to 100 μM 5-HTP, was significantly inhibited by GR. However, following 2 h of incubation, GR did not inhibit sperm hyperactivation enhanced by 5-HTP treatment (Supplementary Fig. 3G).

Ben inhibited sperm hyperactivation enhanced by 5-HTP, but had no effect on cell motility (Supplementary Figs. 4A and B). Following 0.5 h of incubation, Ben significantly inhibited sperm hyperactivation that was increased by 100 nM 5-HTP (Supplementary Fig. 4C). Following 1 h of incubation, Ben significantly inhibited sperm hyperactivation that was increased by 100 nM and 1 μM 5-HTP (Supplementary Fig. 4D). As shown in Supplementary Fig. 4E, following 1.5 h of incubation, Ben significantly inhibited sperm hyperactivation that was increased by 10 nM to 100 μM 5-HTP. Notably, following 2 h of incubation, Ben did not exhibit any effect on sperm hyperactivation (Supplementary Fig. 4F). As indicated in Supplementary Fig. 4G, 5-HT was detected in the medium in which hamster sperm were cultured with 5-HTP, but not in the medium with 5-HTP and Ben.

Regulatory signals of sperm hyperactivation are enhanced by Trp

In hamster sperm, the 5-HT₄ receptor stimulates adenylate cyclase, CatSper Ca²⁺ channels, and PKA [33]. Therefore, we examined the relationship between Trp and adenylate cyclase (Fig. 4). As shown in Figs. 4A and B, ddAdo, a tmAC inhibitor, significantly inhibited sperm hyperactivation, which was enhanced by 100 nM and 1 μM Trp after incubation for 1.5 and 2 h, but did not affect sperm motility. As presented in Fig. 4C, 2-CE, a sAC inhibitor, significantly inhibited sperm motility after incubation for 2.5, 3, and 4 h in the presence and absence of 100 nM and 1 μM Trp. Moreover, 2-CE significantly inhibited sperm hyperactivation that was increased by 100 nM and 1 μM Trp after incubation for 0.5, 1, and 1.5 h (Fig. 4D). After incubation for 2, 2.5, 3, and 4 h, 2-CE strongly inhibited sperm hyperactivation in the presence and absence of 100 nM and 1 μM Trp. In contrast, KH7, another sAC inhibitor, did not affect sperm motility and hyperactivation in the presence and absence of 100 nM and 1 μM Trp (Figs. 4E and F). Adenylate cyclase catalyzes the conversion of ATP into cAMP. As shown in Fig. 5B, H89, a PKA inhibitor, significantly inhibited sperm hyperactivation that was enhanced by 1 μM Trp following 1.5 h of incubation. Furthermore, H89 significantly inhibited sperm hyperactivation that was enhanced by 100 nM and 1 μM Trp following 2 h of incubation. However, H89 did not affect sperm motility in the presence and absence of 100 nM and 1 μM Trp (Fig. 5A).

Fig. 4.

Adenylate cyclase is associated with Trp-enhanced sperm hyperactivation. Percentages of sperm motility (A) and hyperactivation (B) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the transmembrane adenylate cyclase (tmAC) inhibitor, 2’,3’-dideoxyadenosine (ddAdo). Percentages of sperm motility (C) and hyperactivation (D) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the soluble adenylate cyclase (sAC) inhibitor, 2-hydroxyestradiol (2-CE). Percentages of sperm motility (E) and hyperactivation (F) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and another sAC inhibitor, KH7. Experiments were repeated four times using four male hamsters. Data are represented as the mean ± standard deviation. (A and B): Vehicle, medium with 0.2% (v/v) pure water as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; ddAdo, medium with 100 μM ddAdo and vehicle; 100 nM Trp + ddAdo, medium with 100 nM Trp, 100 μM ddAdo, and vehicle; 1 μM Trp + ddAdo, medium with 1 μM Trp, 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; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; 2-CE, medium with 20 μM 2-CE and vehicle; 100 nM Trp + 2-CE, medium with 100 nM Trp, 20 μM 2-CE, and vehicle; 1 μM Trp + 2-CE, medium with 1 μM Trp, 20 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; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; KH7, medium with 25 μM KH7 and vehicle; 100 nM Trp + KH7, medium with 100 nM Trp, 25 μM KH7, and vehicle; 1 μM Trp + KH7, medium with 1 μM Trp, 25 μM KH7, and vehicle. * Significant differences compared with the Vehicle, Inhibitor, and respective concentrations of Trp + Inhibitor groups (P < 0.05). ** Significant differences compared with the Vehicle and respective concentrations of Trp groups (P < 0.05). ^# Significant differences compared with the Inhibitor and respective concentrations of Trp + Inhibitor groups (P < 0.05). ^## Significant differences compared with the Vehicle and Inhibitor groups (P < 0.05). ^§ Significant differences compared with the Vehicle and 100 nM Trp groups (P < 0.05).

Fig. 5.

Protein kinase A (PKA) is associated with Trp-enhanced sperm hyperactivation. Percentages of sperm motility (A) and hyperactivation (B) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the PKA inhibitor, H89. Experiments were repeated four times using four male hamsters. Data are represented as the mean ± standard deviation. (A and B): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; 100 nM Trp + H89, medium with 100 nM Trp, 1 μM H89, and vehicle; 1 μM Trp + H89, medium with 1 μM Trp, 1 μM H89, and vehicle. * Significant differences compared with the Vehicle, 100 nM Trp, and 100 nM Trp + H89 groups (P < 0.05). ** Significant differences compared with the Vehicle and respective concentrations of Trp + H89 groups (P < 0.05).

As shown in Fig. 6B, HC, a potent CatSper Ca²⁺ channel inhibitor, significantly inhibited sperm hyperactivation that was enhanced by 100 nM and 1 μM Trp after incubation for 1.5 and 2 h. After incubation for 2.5, 3, and 4 h, HC significantly inhibited sperm hyperactivation but had no effect on sperm motility in the presence and absence of 100 nM and 1 μM Trp (Fig. 6A). Mib and NNC are typical T-type voltage-activated Ca²⁺ channel inhibitors used as potent CatSper Ca²⁺ channel inhibitors in sperm studies [43,44,45]. As indicated in Figs. 6C–F, Mib and NNC significantly inhibited sperm motility and hyperactivation in the presence and absence of 100 nM and 1 μM Trp.

Fig. 6.

CatSper Ca²⁺ channel is associated with Trp-enhanced sperm hyperactivation. Percentages of sperm motility (A) and hyperactivation (B) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the CatSper Ca²⁺ channel inhibitor, HC-056456 (HC). Percentages of sperm motility (C) and hyperactivation (D) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and the CatSper Ca²⁺ channel inhibitor, mibefradil (Mib). Percentages of sperm motility (E) and hyperactivation (F) following 4 h of incubation in the presence and absence of Trp (100 nM and 1 μM) and another CatSper Ca²⁺ channel inhibitor, NNC 55-0396 (NNC). Experiments were repeated four times using four male hamsters. Data are represented as the mean ± standard deviation. (A and B): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide (DMSO) as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; HC, medium with 10 μM HC and vehicle; 100 nM Trp + HC, medium with 100 nM Trp, 10 μM HC, and vehicle; 1 μM Trp + HC, medium with 1 μM Trp, 10 μM HC, and vehicle. (C and D): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; Mib, medium with 40 μM Mib and vehicle; 100 nM Trp + Mib, medium with 100 nM Trp, 40 μM Mib, and vehicle; 1 μM Trp + Mib, medium with 1 μM Trp, 40 μM Mib, and vehicle. (E and F): Vehicle, medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as vehicle; 100 nM Trp, medium with 100 nM Trp and vehicle; 1 μM Trp, medium with 1 μM Trp and vehicle; NNC, medium with 20 μM NNC and vehicle; 100 nM Trp + NNC, medium with 100 nM Trp, 20 μM NNC, and vehicle; 1 μM Trp + NNC, medium with 1 μM Trp, 20 μM NNC, and vehicle. * Significant differences compared with the Vehicle, Inhibitor, and respective concentrations of Trp + Inhibitor groups (P < 0.05). ** Significant differences compared with the Vehicle, respective concentrations of Trp, and respective concentrations of Trp + Inhibitor groups (P < 0.05). ^# Significant differences compared with the Vehicle and respective concentrations of Trp groups (P < 0.05). ^## Significant differences compared with the Vehicle, both concentrations of Trp, and 1 μM Trp + HC groups (P < 0.05). ^§ Significant differences compared with the Vehicle and both concentrations of Trp groups (P < 0.05). ^§§ Significant differences compared with the Inhibitor and respective concentrations of Trp + Inhibitor groups (P < 0.05).

Discussion

Previous human and stallion studies [34,35,36] have suggested the presence of TPH and 5-HT receptors in sperm. Given that TPH is an enzyme involved in 5-HT biosynthesis, 5-HT may be synthesized and secreted within sperm. Moreover, in humans, hamsters, mice, and rats, 5-HT is associated with the regulation of sperm motility, velocity, hyperactivation, and AR [6, 9,10,11,12, 34, 35]. It boosts IVF success by enhancing sperm hyperactivation in mice and rats [10, 12]. In this study, we investigated whether 5-HT was synthesized and released in hamster sperm, and whether it plays a regulatory role in sperm hyperactivation. Both Trp and 5-HTP enhanced hamster sperm hyperactivation (Fig. 1B; Supplementary Fig. 2B). However, D-Trp exhibited no such effect on hamster sperm hyperactivation (Fig. 1F). In addition, the 5-HT₄ receptor antagonist suppressed these enhancements by Trp and 5-HTP (Fig. 2; Supplementary Fig. 3). Moreover, hamster sperm contained two 5-HT synthesis enzymes, TPH and AADC, and secreted 5-HT (Fig. 3; Supplementary Fig. 4). These results suggest that Trp and 5-HTP enhance hamster sperm hyperactivation via sperm biosynthesis of 5-HT. In the reproductive organs, cumulus cells have been reported to secrete 5-HT [3]. The findings of this study suggest that sperm may also be a novel source of 5-HT.

In humans, 5-HT was found to increase sperm velocity, VSL, VCL, and VAP [34]. On the other hand 5-HT had no effect on sperm velocity in hamsters, mice, or rats [10,11,12]. However, in mice, a 5-HT₂ receptor agonist decreased VSL and VCL, whereas a 5-HT₄ receptor agonist decreased VCL [10]. In hamsters, agonists of 5-HT₂ receptor reduced VSL, whereas a 5-HT₄ receptor agonist did not affect sperm velocity [33]. In hamsters, mice, and rats, 5-HT exhibited no effect on parameters such as LIN, STR, WOB, ALH, and BCF [10,11,12]. However, in hamsters and mice, an agonist of the 5-HT₄ receptor decreased ALH [10, 33]. In the present study, Trp did not affect the motility kinematics (Supplementary Tables 1 and 2). As 5-HT was previously found to have failed to affect the motility kinematics of hamster sperm [10], we speculate that this failure may have compromised the effect of Trp on motility kinematics via the autocrine action of 5-HT.

A previous study [9] suggested that 5-HT enhances the hyperactivation of hamster sperm in a dose-dependent manner. High concentrations of 5-HT (nM or μM level) stimulated the 5-HT₄ receptor, whereas low concentrations (fM or pM level) stimulated the 5-HT₂ receptor. Given that Trp and 5-HTP stimulated the 5-HT₄ receptor (Fig. 2; Supplementary Fig. 3), 5-HT (secreted via the autocrine pathway) may also stimulate the 5-HT₄ receptor. Therefore, 5-HT secreted by COCs [3] stimulates 5-HT₂ or 5-HT₄ receptors in a dose-dependent manner, whereas 5-HT secreted by autocrine cells stimulates only the 5-HT₄ receptor (Supplementary Fig. 5). In hamsters, stimulation of 5-HT₄ receptor by 5-HT activates tmAC [33], which in turn activates PKA, ultimately leading to the activation of CatSper Ca²⁺ channels. During the enhancement of hyperactivation, Trp stimulates the 5-HT₄ receptor, which is associated with the tmAC, sAC, PKA, and CatSper Ca²⁺ channels (Figs. 2 and 4, 5, 6).

In mice and rats, 5-HT increased IVF success by enhancing hyperactivation [10, 12]. In doing so, 5-HT stimulated 5-HT₂, 5-HT₃, 5-HT₄, and 5-HT₇ receptors, although the increase in IVF success was solely associated with the 5-HT₄ receptor in mice [10]. On the other hand, 5-HT-associated hyperactivation and IVF success in rats was associated with 5-HT₄ receptor stimulation alone [12]. Moreover, in hamster sperm, 5-HT synthesized from Trp primarily stimulated the 5-HT₄ receptor and enhanced hyperactivation (Figs. 1 and 2). These results indicate that 5-HT₄ is a key receptor in the 5-HT-mediated regulation of sperm capacitation .

Hormones and neurotransmitters secreted into the oviduct regulate sperm capacitation events, including hyperactivation and AR [5, 46,47,48,49]. Progesterone enhanced hyperactivation in hamsters, humans, mice, and rats [22, 31, 43, 44, 50], whereas it induced the AR in hamsters and humans [46, 51]. The progesterone-induced enhancement of hyperactivation and the AR in hamsters and humans was suppressed by estradiol [52,53,54,55,56]. In hamsters, γ-aminobutyric acid suppressed the effects of progesterone on hyperactivation [57]. Melatonin and 5-HT enhanced hyperactivation [7, 9] and 5-HT induced the AR in hamster sperm [6]. The enhanced hyperactivation by melatonin was suppressed by estradiol [58]. Meanwhile, the 5-HT₂ receptor-mediated enhancement of hyperactivation by 5-HT was suppressed by γ-aminobutyric acid [59]. In humans, γ-aminobutyric acid induced hyperactivation [60], whereas, in mice and pigs, it induced the AR [61, 62]. Dopamine induced the hyperactivation of human sperm [63]. These hormones and neurotransmitters are present in the follicular and oviductal fluids [5, 46,47,48,49]. This study showed that sperm contained TPH and AADC, produced 5-HT from Trp, and secreted 5-HT (Fig. 3; Supplementary Fig. 4). As sperm contain AADC, an enzyme associated with dopamine biosynthesis, dopamine is produced from tyrosine or DOPA. Therefore, investigations into the regulation of sperm capacitation events should factor in the effects of hormones and neurotransmitters present in follicular and oviductal fluids, as well as autocrine hormones and neurotransmitters within sperm.

In conclusion, Trp enhanced sperm hyperactivation via the 5-HT₄ receptor in hamster sperm, which exhibited a 5-HT biosynthesis system that synthesizes and secretes 5-HT from Trp. Given that 5-HT is also secreted by COCs, sperm hyperactivation is potentially regulated by two sources of 5-HT: one secreted by COCs and the other by sperm.

Conflict of interests

The authors declare no conflicts of interest.