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The Journal of Reproduction and Development logoLink to The Journal of Reproduction and Development
. 2025 Jan 25;71(2):85–92. doi: 10.1262/jrd.2024-078

Influences of 5-hydroxytriptamine on sperm hyperactivation and in vitro fertility in rats

Yuki KOYANO 1, Masakatsu FUJINOKI 1
PMCID: PMC11999830  PMID: 39864845

Abstract

The neurotransmitter, 5-hydroxytriptamine (5-HT), is well known. Furthermore, it enhances the acrosome reaction, hyperactivation, and in vitro fertilization (IVF) success in hamsters and mice. In the present study, we examined whether 5-HT enhances hyperactivation and increases IVF success in rats. When rat sperm was exposed to 5-HT, hyperactivation was significantly enhanced. Only the 5-HT4 receptor agonists significantly enhanced hyperactivation. Additionally, both 5-HT and 5-HT4 receptor agonists significantly increase the success of IVF. These results suggested that 5-HT increases IVF success by enhancing hyperactivation and effects of 5-HT are associated with the 5-HT4 receptor. Therefore, in rats, 5-HT enhances capacitation and the 5-HT4 receptor is the key molecule for capacitation.

Keywords: Capacitation, 5-hydroxytriptamine, Hyperactivation, In vitro fertilization, Sperm

After ejaculation, mammalian sperm are capacitated in the oviduct before fertilization. Only capacitated sperm can bind to and fertilize oocytes [1]. Capacitation is a physiological process related to the fertilizing ability of mammalian sperm and includes acrosome reactions and hyperactivation [1, 2]. The acrosome reaction is an exocytotic process that releases hydrolases for the digestion of the oocyte envelope containing the zona pellucida and cumulus cells [1]. Hyperactivation is the specialized motility to move through viscous oviductal fluid and penetrate the oocyte envelope [1,2, 3]. Moreover, the capacity for hyperactivation positively correlates with the in vitro fertilization (IVF) success [4,5,6,7]. In vitro, the sperm cells were artificially capacitated in a specific medium. Albumin, Ca2+, and HCO3– play important roles when sperm are capacitated in vitro [1, 2, 3]. Albumin removes lipids from the sperm plasma membrane [8] and induces Ca2+ influx via activation of the CatSper Ca2+ channel [9], which is essential for hyperactivation [10]. The CatSper Ca2+ channel in mouse sperm is activated by protein kinase A (PKA) [11], whereas the channel in human sperm is not activated by PKA [12]; the human sperm channel is activated by progesterone (P4) [13, 14]. Hamster sperm are not hyperactivated in the absence of albumin or Ca2+ [15, 16]. Ca2+ and HCO3– stimulate soluble adenylate cyclase (sAC), inducing protein phosphorylation and dephosphorylation [1, 3, 17,18,19,20].

Hyperactivation is regulated by hormones and neurotransmitters which are released to the oviduct [21], for example P4 [13,14,15, 22,23,24,25,26,27], estradiol (E2) [25,26,27,28], melatonin (Mel) [16, 24], γ-aminobutyric acid (GABA) [29,30,31], and 5-hydroxytryptamine (5-HT, serotonin) [24, 32,33,34]. In hamsters, humans, mice, and rats, P4 enhances hyperactivation [13,14,15, 22,23,24]. In addition, P4-enhanced hyperactivation of hamster and mouse sperm is suppressed by E2 [25,26,27]. Furthermore, P4 and E2 control the IVF success via regulation of hyperactivation in mice [23, 27]. Mel enhances the hyperactivation of hamster sperm [16, 24], which is suppressed by E2 [28]. In hamsters and mice, 5-HT induces hyperactivation [32,33,34]. Moreover, 5-HT increases IVF success [33]. GABA induces human sperm hyperactivation [29] and suppresses P4-induced and 5-HT2 receptor-associated hyperactivation in hamsters [30, 31].

The functions of diverse tissues are regulated by 5-HT through 5-HT receptors [35, 36]. The 5-HT receptors comprise seven families (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7). The 5-HT1 receptor has five subtypes (5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT1F), whereas the 5-HT2 receptor has three subtypes (5-HT2A, 5-HT2B, and 5-HT2C). The 5-HT1 and 5-HT5 receptors suppress transmembrane adenylate cyclase (tmAC), whereas 5-HT4, 5-HT6, and 5-HT7 stimulate tmAC and activate cAMP signals. The 5-HT2 receptors stimulate phospholipase C (PLC) and activate Ca2+ signaling. The 5-HT3 receptor is a ligand-gated cation channel receptor.

The 5-HT and 5-HT receptors have been detected in mammalian reproductive tissues and cells [34, 36,37,38,39,40,41,42,43,44,45]. Additionally, 5-HT signals are associated with the regulation of steroidogenesis, oocyte maturation, spermatogenesis, capacitation, fertilization, and embryonic development [32,33,34, 36,37,38,39,40,41,42,43,44, 46]. In hamsters [32, 34, 46], 5-HT induces an acrosome reaction and hyperactivation via 5-HT2 and 5-HT4 receptors. In mice, 5-HT enhances hyperactivation via 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptors, whereas it increases IVF success only via the 5-HT4 receptor [33]. Furthermore, 5-HT increases human sperm motility [37].

Therefore, we hypothesized that 5-HT enhances sperm hyperactivation in rats, hamsters, and mice. Moreover, we speculate that 5-HT increases IVF success by enhancing hyperactivation. In this study, we examined whether 5-HT enhances the hyperactivation and affects the IVF success in rats.

Materials and Methods

Chemicals

Bovine serum albumin (BSA), 5-HT, sumatriptan succinate (sumatriptan), α-methylserotonin maleate (methylserotonin), 1-(3-chlorophenyl) biguanide hydrochloride (mCPBG), 5-methoxytryptamine (MT), WAY-208466 dihydrochloride (WAY208466), LP12 hydrochloride hydrate (LP12), and 5-HT4 receptor antagonist (GR113808; GR) were purchased from Merck KGaA (Darmstadt, Germany). Anti 5-HT4 receptor antibody (SR-4 (G-3); sc-376158) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Polyvinylidine difluoride (PVDF) membranes were purchased from Millipore (Bedford, MA, USA). A molecular weight marker set was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). EzWestLumi plus® was purchased from ATTO Corporation (Tokyo, Japan). Pregnant mare serum gonadotropin (PMSG; Serotropin®), and human chorionic gonadotropin (hCG; Gonatropin®) were purchased from ASKA Pharmaceutical (Tokyo, Japan). Other reagent-grade chemicals were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Animals

Wistar-Imamichi rats were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and propagated at the Research Center for Laboratory Animals, Dokkyo Medical University. The rats were housed in light- and temperature-controlled environments (12 h on/off and 25 ± 2°C, respectively) with food and drink available ad libitum. All experiments were approved by the Animal Care and Use Committee of the university (experimental permission number: 0107) and performed in accordance with the university’s guidelines for animal experimentation.

Preparation of hyperactivated sperm

Sperm were collected from the cauda epididymides of male rats (12–24 weeks old). Hyperactivated sperm were prepared as previously described [22]. Modified Tyrode’s albumin lactate pyruvate (mTALP) medium [47] was used as the capacitation medium. The cauda epididymis of each rat was punctured with a 23 G (0.6 mm) needle (Terumo Corporation, Tokyo, Japan), a drop (approximately 10 μl) of epididymal fluid containing sperm was obtained and placed on a culture dish (35 mm in diameter; Iwaki, Asahi Glass Co., Ltd., Tokyo, Japan), and 3 ml of the mTALP medium was added to the dish. The epididymal fluid was incubated for 5 min at 37°C for activation. The supernatant containing the motile sperm was placed in a new dish containing vehicle or GR. After incubation for 5 min, the supernatant was transferred to a new dish containing the vehicle, 5-HT, or agonists. The supernatant was incubated for 5 h at 37°C and 5% CO2 to induce hyperactivation. To prepare the stock solutions, 5-HT (10 mM), sumatriptan (100 μM), methylserotonin (100 pM), mCPBG (100 mM), WAY208466 (7.3 μM), and LP12 (0.13 μM) were dissolved in pure water. MT (10 nM) was dissolved in ethanol (EtOH). GR (1 mM) was dissolved in dimethyl sulfoxide. In all experiments, the maximum concentration of the vehicle was 0.2% (v/v).

Measurements of motility and hyperactivation

Motile and hyperactivated sperm were analyzed as previously described [22]. The motile sperm were recorded on a DVD recorder (RDR-HX50; Sony Corp., Tokyo, Japan) using a CCD camera (Progressive 3CCD, Sony) attached to a microscope (IX70, Olympus Corp., Tokyo, Japan) under phase-contrast illumination in a small CO2 incubator (MI-IBC, Olympus). Sperm cells were recorded for 1 min at 37°C for 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, and 5 h incubations. The movies were used to manually count total sperm, motile sperm, and hyperactivated sperm from two different fields at one observation point. Over 80 sperm were observed at each observation point. The experiments were repeated four times using four rats each. Visual analyses were blindly performed in all experiments. Motile sperm that exhibited a whiplash-like trajectory and bent sickle-like shape of a viper head were considered hyperactivated [21, 48]. The percentages of motility and hyperactivation were calculated as number of motile sperm/total sperm × 100 and number of hyperactivated sperm/total sperm × 100, respectively. If the proportion of motile sperm was ≤ 70%, the experiment was redone.

Motility kinematics evaluation

Motility kinematics were evaluated using a sperm motility analysis system (SMAS) (Ver. 3.18) with the loaded parameter file rat_BM10× 640 nm _bright50_150fps_S200.ini (Ditect Co., Ltd., Tokyo, Japan) as previously described [22]. The SMAS consisted of 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 warm plate (MP10DM; Kitazato Corp., Shizuoka, Japan). The sperm were hyperactivated in the presence or absence of 5-HT according to the methodology described in the section “Preparation of hyperactivated sperm.” After incubation for 2 h, the suspension containing motile sperm (20 μl) was transferred to an observation chamber (0.1 mm deep, 18 mm wide, and 18 mm long) on a glass slide. The SMAS automatically calculated seven parameters: straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF). The wobble coefficient (WOB), manually calculated as VAP/VCL [49]. The SMAS analysis was repeated five times, using one rat each time. In each experiment, ≥ 300 sperm were detected. Only valid motile sperm from the detected sperm were analyzed.

Preparation of sperm protein extracts

Sperm proteins were extracted as described previously [50]. Sperm were homogenized at 30 mg/ml in a urea solution containing 7 M urea and 10% (v/v) 2-mercaptoethanol. The homogenate was 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 the same volume of urea-thiourea solution containing 5 M urea, 1 M thiourea, 10% (v/v) 2-mercaptoethanol, and 2% (v/v) NP-40 as the urea solution. The homogenate was 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-thiourea extract.

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was conducted according to Laemmli [51], using a 10% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS. After staining with Coomassie Brilliant Blue, the gel was scanned using a densitometer (GS-800 densitometer, Bio-Rad Laboratories).

Western blotting (WB)

WB was performed as previously described [34]. After SDS-PAGE, the proteins were transferred onto a PVDF membranes. After transfer, the membrane was washed with Tris-buffered saline (TBS) containing 0.15 M NaCl and 20 mM Tris-HCl (pH 7.5). After one wash, the membrane was incubated for 1 h at 25°C in the BSA-TBS solution containing 5% (w/v) BSA and TBS. After three washes with TBS, the membrane was incubated with the anti 5-HT4 receptor antibody (1:1000 dilution with the BSA-TBS) for 1 h at 25°C. After three washed with TBS, the membrane was incubated with the horseradish peroxidase conjugated anti mouse IgG antibody (1:5000 dilution with BSA-TBS) for 1 h at 25°C. After the membrane was washed three times with 0.05% (w/v) Tween 20 containing TBS, chemical luminescence was conducted using the EzWestLumi plus® (ATTO Corporation). Antibody reactivity was detected using an Ez-Capture MG (ATTO Corporation). WB was repeated four times.

IVF

IVF was performed as previously described [22]. Superovulation treatment was performed as follows: female rats (4–6 weeks old) were intraperitoneally administered 30 U of PMSG. Then, 48 h after PMSG injection, female rats were intraperitoneally administered 30 U of hCG. IVF was performed 18 h after the hCG injection. One hour before egg collection, one drop (approximately 5 μl) of epidydimal fluid obtained from the cauda epididymides of male rats (12 to 18 weeks-old) was mixed with a 300-μl drop of the mTALP medium with the vehicle, 100 nM 5-HT, 10 pM MT, or 1 μM GR. Sperm at 1.5 × 107 cells/ml were incubated for 1 h at 37°C and 5% CO2. Eighteen hours after hCG injection, eggs were collected from the PMSG/hCG-treated female rats. Cumulus-oocyte complexes (COCs) were collected from the ampulla of both oviducts and incubated with a 300-μl drop of the mTALP medium supplied with the vehicle, 100 nM 5-HT, 10 pM MT, or 1 μM GR. After egg collection and the 1-h incubation of sperm, 50 μl of medium containing pre-incubated sperm was added to the medium containing COCs to start the insemination process. The final sperm concentration was 2.1 × 106 cells/ml. Sperm and COCs were co-incubated for 1, 2, or 6 h at 37°C and 5% CO2. After incubation, eggs and/or COCs were transferred to new mTALP medium supplied with the vehicle, 100 nM 5-HT, 10 pM MT, or 1 μM GR to stop insemination. The eggs and/or COCs were then incubated again. After incubation for 18 h, the cells were observed under a stereoscopic microscope (Stemi 2000-C; Zeiss, Oberkochen, Germany), and eggs and two-cell embryos were manually counted. The percentage of two-cell embryos was calculated as the number of two-cell embryos/number of eggs × 100. All analyses were performed in a blinded manner. Experiments were performed seven times using one female and one male rat each time.

Statistical analysis

Data ware presented as mean ± standard deviation. The data for the two groups (Fig. 2 and Table 1) were analyzed using Student’s t-test in Microsoft Excel (Microsoft Japan, Tokyo, Japan). The data for the four groups (Figs. 1 and 3, and Table 2) were analyzed using one-way analysis of variance and post-hoc test (Student–Newman–Keuls test) in Microsoft Excel ystat2018 (Igakutosho Shuppan, Saitama, Japan). Statistical significance was set at P < 0.05.

Results

Effects of 5-HT on hyperactivation

When rat sperm were exposed to 1 aM to 10 μM 5-HT, 5-HT significantly enhanced hyperactivation at incubation for 1 h, 1.5 h, and 2 h (Fig. 1B), whereas it did not affect the percentage of motile sperm (Fig. 1A). After incubation for 1 h, 1 pM (10–12 M) to 100 pM (10–10 M) 5-HT significantly enhanced hyperactivation compared to that with 1 aM (10–18 M) 5-HT (Fig. 1C). Moreover, over 1 nM (10–11 M) 5-HT significantly enhanced hyperactivation compared to that with the vehicle and 1 aM (10–18 M) 5-HT. After incubation for 1.5 h (Fig. 1D), 100 fM (10–13 M) 5-HT significantly enhanced hyperactivation compared to that with the vehicle and 1 aM (10–18 M) 5-HT. Moreover, 1 pM (10–12 M) and 10 pM (10–11 M) 5-HT significantly enhanced hyperactivation compared with the vehicle and 1 aM to 1 fM (10–18 to 10–15 M) 5-HT. Furthermore, 100 pM to 10 nM (10–10 to 10–8 M) 5-HT significantly enhanced hyperactivation compared with the vehicle and 1 aM to 10 fM (10–18 to 10–14 M) 5-HT. Then, 100 nM (10–7 M) and 1 μM (10–6 M) 5-HT significantly enhanced hyperactivation compared with vehicle, 1 aM to 100 fM (10–18 to 10–13 M) 5-HT, and 10 μM (10–5 M) 5-HT. Although 10 μM (10–5 M) 5-HT significantly enhanced hyperactivation compared with that of the vehicle, and 1 aM to 1 fM (10–18 to 10–15 M) 5-HT, it did not significantly enhance hyperactivation compared to that with 100 nM (10–7 M) and 1 μM (10–6 M) 5-HT. After incubation for 2 h (Fig. 1E), 10 fM (10–14 M) 5-HT significantly enhanced hyperactivation compared to that with 1 aM (10–18 M) 5-HT. More than 100 fM (10–13 M) 5-HT significantly enhanced hyperactivation compared to that with the vehicle and 1 aM to 1 fM (10–18 to 10–15 M) 5-HT. Regarding motility kinematics after incubation for 2 h, 5-HT most significantly affected hyperactivation, but did not affect any other parameters at 2 h (Fig. 1F) or any other incubation time (data not shown).

Fig. 1.

Fig. 1.

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Effects of 5-HT on motility, hyperactivation and motility kinematics. Percentages of motility (A) and hyperactivation (B) are shown as overview of effects after incubation for 1 h (C), 1.5 h (D), and 2 h (E) when sperm were incubated for 5 h in the presence and absence of 5-HT. Experiments were repeated four times using four male rats. (F) Motility kinematics for sperm cultured at 2 h in the presence and absence of 5-HT (100 fM, 100 pM, and 100 nM). Motility kinematics analyses were repeated five times using five male rats. Data represent the mean ± standard deviation. (A–F): (Vehicle) medium with 0.1% pure water as vehicle; (respective concentration of 5-HT) medium with respective concentration of 5-HT and the vehicle. * Significant difference compared with “-18” (P < 0.05). ** Significant difference compared with “Vehicle,” and “-18” (P < 0.05). # Significant differences compared with “Vehicle,” “-18,” “-17,” “-16,” and “-15” (P < 0.05). ## Significant differences compared with “Vehicle,” “-18,” “-17,” “-16,” “-15,” and “-14” (P < 0.05). $ Significant differences compared with “Vehicle,” “-18,” “-17,” “-16,” “-15,” “-14,” and “-13” (P < 0.05). $$ Significant differences compared with “Vehicle,” “-18,” “-17,” “-16,” “-15,” “-7,” and “-6” (P < 0.05).

The 5-HT receptor associated with the 5-HT-enhanced hyperactivation

The 5-HT receptors comprise seven families [35, 36], therefore, we examined which 5-HT receptors were associated with the enhancement of hyperactivation by 5-HT (Fig. 2). After incubation for 2 h, 10 pM MT (5-HT4 receptor agonist) [32,33,34] significantly increased hyperactivation (Fig. 2D). However, sumatriptan (17 nM, 5-HT1B/1D receptor agonist; 100 nM, 5-HT1A receptor agonist) [33, 34, 52], 100 fM methylserotonin (5-HT2 receptor agonist) [32,33,34], 100 μM mCPBG (5-HT3 receptor agonist) [33, 34, 53], 7.3 nM WAY208466 (5-HT6 receptor agonist) [instruction manual] [33, 34], and 0.13 nM LP12 (5-HT7 receptor agonist) [instruction manual] [33, 34] did not impact hyperactivation (Figs. 2A−2C, 2E, and 2F). Furthermore, none of the 5-HT receptor agonists affected motility. As shown in Fig. 3, we examined whether a GR inhibited the effects of 5-HT and MT. In the presence of 5-HT and MT, GR significantly inhibited the enhancement (Figs. 3B and 3D), although it did not affect motility (Figs. 3A and 3C). In addition, the 5-HT4 receptor was detected as an approximately 40-kDa band in the urea extract of rat sperm (Fig. 4).

Fig. 2.

Fig. 2.

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Effects of 5-HT receptor agonists on hyperactivation. Percentages of motility and hyperactivation were determined after 2 h of culture when sperm were cultured for 5 h with 17 nM or 100 nM sumatriptan (A), 100 fM methylserotonin (B), 100 μM mCPBG (C), 10 pM MT (D), 7.3 nM WAY-208466 (E), and 0.13 nM LP12 (F). Experiments were repeated four times using four male rats. Data represent the mean ± standard deviation. (A) (Vehicle) medium with 0.1% (v/v) pure water as vehicle; (respective concentrations of Sumatriptan) medium with indicated concentrations of Sumatriptan and the vehicle. (B) (Vehicle) same as above; (100 fM Methylserotonin) the medium with 100 fM methylserotonin and the vehicle. (C) (Vehicle) same as above; (100 μM mCPBG) the medium with 100 μM mCPBG and the vehicle. (D) (Vehicle) medium with 0.1% (v/v) ethanol as vehicle; (10 pM MT) medium with 10 pM MT and the vehicle. (E) (Vehicle) medium with 0.1% (v/v) pure water as vehicle; (7.3 nM WAY208466) medium with 7.3 nM WAY208466 and the vehicle. (F) (Vehicle) same as above; (0.13 nM LP12) medium with 0.13 nM LP12 and the vehicle. * Significant difference compared with “Vehicle” (P < 0.05). mCPBG, 1-(3-chlorophenyl) biguanide hydrochloride; MT, 5-methoxytryptamine.

Fig. 3.

Fig. 3.

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Relation of the 5-HT4 receptor on the enhancement of hyperactivation by 5-HT. Percentages of motility (A and C) and hyperactivation (B and D) are shown when sperm were incubated for 5 h in the presence and absence of 5-HT (100 fM, 100 pM, and 100 nM) (A and B), and MT (C and D). Experiments were repeated four times using four male rats. Data represent 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; (GR) medium with 1 μM GR and the vehicle; (respective concentration of 5-HT) medium with respective concentration of 5-HT and the vehicle; (respective concentrations of 5-HT + GR) medium with respective concentrations of 5-HT, 1 μM GR and the vehicle. (C and D) (Vehicle) medium with 0.1% (v/v) ethanol and 0.1% (v/v) dimethyl sulfoxide as vehicle; (MT) medium with 10 pM MT and the vehicle; (GR) medium with 1 μM GR and the vehicle; (MT + GR) medium with 10 pM MT, 1 μM GR and the vehicle. * Significant difference compared with “Vehicle,” “GR,” and “respective concentration of 5-HT + GR” (P < 0.05). ** Significant difference compared with “GR” (P < 0.05). # Significant differences compared with “Vehicle,” “GR,” and “MT + GR” (P < 0.05). GR, 5-HT4 receptor antagonist (GR113808); MT, 5-methoxytryptamine.

Fig. 4.

Fig. 4.

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Detection of the 5-HT4 receptor from rat sperm. Lanes a and b show CBB stained gel. Lanes c and d show western blotting against the anti-5-HT4 receptor antibody. Lanes a and c show the urea-extract. Lanes b and d show the urea-thiourea extract. The numbers on the left side of lane a show molecular weight marker. The extracts were applied at 10 μl in each lane. Arrow indicates the antibody reaction. CBB, Coomassie Brilliant Blue.

Effects of 5-HT and MT on the IVF success

The 5-HT enhances hyperactivation via the 5-HT4 receptor (Figs. 1–3), therefore, we examined the effects of 5-HT and MT on IVF success (Table 1). When sperm and COCs were co-incubated for 6 h in the presence of 100 nM 5-HT, which is the midway concentration used to enhance hyperactivation, 5-HT did not affect IVF success. MT did not affect IVF success when sperm and COCs were co-incubated for 6 h. However, when sperm and COCs were co-incubated for 2 h in the presence of 5-HT and MT, they significantly increased. When sperm and COCs were co-incubated for 1 h in the presence of 5-HT and MT, only MT significantly increased the IVF success. Moreover, the increase in IVF success by 5-HT was inhibited by GR (Table 2).

Table 1. Effects of 5-HT and MT on the IVF success.

No. of eggs No. of two-cell embryos Two-cell embryos (%)
6 h insemination
Vehicle (H2O) 239 144 62.23 ± 18.43
100 nM 5-HT 199 113 56.18 ± 16.13
Vehicle (DMSO) 154 72 47.02 ± 12.98
MT 136 66 51.28 ± 11.66
2 h insemination
Vehicle (H2O) 208 84 41.68 ± 10.28 *
100 nM 5-HT 168 86 51.79 ± 9.57
Vehicle (DMSO) 133 44 31.99 ± 9.57 *
MT 165 71 47.15 ± 14.29
1 h insemination
Vehicle (H2O) 152 60 41.05 ± 13.04
100 nM 5-HT 147 57 37.60 ± 11.50
Vehicle (DMSO) 144 50 41.96 ± 20.86 *
MT 114 74 66.90 ± 19.82
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Each value indicates the IVF success in the presence of 5-HT or MT. Sperm and COCs were co-incubated for 1, 2, and 6 h. Data represent the mean ± standard deviation. (Vehicle [H2O]): medium with 0.1% (v/v) pure water as the vehicle; (Vehicle [DMSO]): medium with 0.1% (v/v) DMSO as the vehicle; (100 nM 5-HT): medium with 100 nM 5-HT and 0.1% (v/v) pure water as the vehicle; (MT): medium with 10 pM MT and 0.1% (v/v) DMSO as the vehicle. * Significant differences compared with “Vehicle (H2O)” or “Vehicle (DMSO)” (P < 0.05). COC, cumulus-oocyte complex; IVF, in vitro fertilization; MT, 5-methoxytryptamine.

Table 2. Involvement of 5-HT4 receptor on the 5-HT induced IVF success.

No. of eggs No. of two-cell embryos Two-cell embryos (%)
Vehicle 208 92 43.39 ± 3.42 *
100 nM 5-HT 110 71 64.15 ± 5.02
GR 125 49 38.27 ± 7.38
100 nM 5-HT + GR 96 40 43.16 ± 8.76
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Each value indicated successful IVF in the presence of 5-HT and GR. Sperm and COCs were co-incubated for 2 h, and the data represent the mean ± standard deviation. (Vehicle) the medium with 0.1% (v/v) pure water and 0.1% (v/v) dimethyl sulfoxide as the vehicle; (100 nM5-HT) the medium with 100 nM 5-HT and the vehicle; (GR) the medium with 1

μM GR and the vehicle; (100 nM 5-HT + GR) the medium with 100 nM 5-HT and 1 μM GR and the vehicle. * Significant differences compared with “Vehicle” (P < 0.05). COC, cumulus-oocyte complex; GR, 5-HT4 receptor antagonist (GR113808); IVF, in vitro fertilization.

Discussion

In the present study, we examined whether 5-HT enhances hyperactivation and increases IVF success in rats. The results showed that 5-HT enhanced hyperactivation and increased IVF success (see Fig. 1 and Table 1). These effects of 5-HT were associated with the 5-HT4 receptor only (see Figs. 2 and 3, and Table 2). In addition, 5-HT4 receptors were detected in rat sperm (see Fig. 4). In hamster sperm, stimulation of the 5-HT4 receptor activates the CatSper Ca2+ channel and sAC which are associated with the basic regulatory mechanisms of hyperactivation [3, 10, 34, 54, 55]. Therefore, it is likely that 5-HT stimulated the basic regulatory mechanism of hyperactivation via the 5-HT4 receptor and accelerates hyperactivation. However, 5-HT did not affect motility and motility kinematics (see Fig. 1). In mice, 5-HT enhances hyperactivation via 5-HT2, 5-HT3, 5-HT4, and 5-HT7 [33]. Moreover, 5-HT increases IVF success via the 5-HT4 receptor [33]. In hamsters, 5-HT dose-dependently enhanced hyperactivation via 5-HT2 and 5-HT4 receptors [32, 34]. In mice, hamsters, and rats, 5-HT does not affect motility and motility kinematics [24, 33]. However, in humans, 5-HT slightly increases the motility, such as VSL, VCL, and VAP [37]. From the results of the present study and previous studies [24, 32, 33, 34, 37], it is suggested that 5-HT affects hyperactivation and the IVF success, but slightly affects motility and motility kinematics.

In rodents, it is likely that the 5-HT4 receptor is a common receptor to enhance sperm capacitation because 5-HT significantly affects hyperactivation and the IVF success [32, 33, 34] (see Figs. 1–3). In neurons, the 5-HT4 receptor stimulates tmAC, produces cAMP, and activates PKA [35, 36]. In hamster sperm, the 5-HT4 receptor stimulates tmAC and activated PKA [34]. Moreover, in hamsters [34] and mice [11], PKA stimulates the CatSper Ca2+ channel and activates sAC. PKA is activated after sAC activation. The CatSper Ca2+ channel and sAC play important roles in sperm hyperactivation [3, 10, 22, 34, 54, 55]. Therefore, in rat sperm, it is likely that 5-HT enhances hyperactivation through the 5-HT4 receptor and cAMP-signals, and increases IVF success.

Hyperactivation is also regulated by Ca2+ signaling [1]. In hamster sperm, P4 enhances hyperactivation via the membrane progesterone receptor (mPR) [15]. After P4 binds to mPR, it activates PLC, stimulating the inositol 1,4,5-trisphosphate (IP3) receptor and Ca2+ signals [15, 56]. In rat sperm, P4 enhances hyperactivation through the mPR, PLC, IP3 receptors, and Ca2+-signals [22]. P4 also stimulates tmAC, but does not affect CatSper Ca2+ channels [22]. Moreover, P4 affects IVF success [22]. In mouse sperm, P4 binds to mPR, stimulates Ca2+ signals, and enhances hyperactivation but does not affect the CatSper Ca2+ channel [10, 13, 23]. Furthermore, P4 increases IVF success [23]. In human sperm, P4 stimulates the CatSper Ca2+ channel and Ca2+-signals and induces hyperactivation, although it does not affect the IVF success [6, 13, 14]. In contrast, in mice, stimulation of the 5-HT2 receptor enhances hyperactivation but does not affect IVF success [33]. For example, in neurons, the 5-HT2 receptor activates PLC, produces IP3 affecting the IP3 receptor, and stimulates Ca2+-signals [35, 36]. In hamsters, 5-HT binds to the 5-HT2 receptor and stimulats PLC/IP3-Ca2+-signals, such as P4-stimulated Ca2+-signals [34]. Therefore, it is likely that cAMP signals in mice are suitable to increase IVF success via the enhancement of hyperactivation, rather than Ca2+-signals. The present results and those of previous reports suggest that 5-HT is a common enhancer of hyperactivation and IVF success, and the 5-HT4 receptor and cAMP signals are important signals. It is unknown whether 5-HT affects the hyperactivation of human sperm, although 5-HT increased velocities of human sperm [37]. Another study [57] showed that 5-HT regulates human sperm motility via the 5-HT2 receptor.

The ability of hyperactivation is related to the IVF success [4,5,6,7]. Although P4 induces hyperactivation of human sperm [13, 14], P4-induced hyperactivation of human sperm did not increase the IVF success [6]. In rats, P4 enhances hyperactivation but does not increase IVF success [22]. However, P4 enhanced hyperactivation and increased IVF success [23]. In mice and rats, 5-HT enhanced hyperactivation and increased IVF success [33] (see Fig. 1 and Table 1). Both P4 and 5-HT also induced an acrosome reaction in humans and hamsters, although their concentrations to induce an acrosome reaction are higher than their concentrations to enhance hyperactivation [13, 14, 15, 46, 58, 59]. Therefore, the P4 and 5-HT concentrations used in the present and previous studies were unable to induce an acrosome reaction. It was previously reported that the zona pellucida induced acrosome reaction [60, 61, 62]. However, it was recently reported that sperm showed acrosome reaction in cumulus layers and the oviduct before sperm were bound to the zona pellucida [63, 64, 65, 66, 67]. When sperm show an acrosome reaction before binding to the zona pellucida, they may be exhibiting an autonomous acrosome reaction. Therefore, under the conditions of our experiments, it is likely that the sperm autonomously induced the acrosome reaction after the enhancement of hyperactivation by P4 and 5-HT.

The 5-HT and 5-HT receptors are found in Leydig cells, oocytes, COC, follicular fluid, and embryos [36, 42, 43]. In rats, the concentration of 5-HT in oviducts is between 2.06 and 3.34 μg/g fresh tissue [38]. In humans, concentrations of 5-HT in the preovulatory follicles and cystically degenerated follicles are 14.3 ± 8.9 μg/100 ml and 12.2 ± 6.2 μg/100 ml [40]. In the female reproductive organs, 5-HT is mainly released from cumulus cells, and associated with the regulation of steroidogenesis and oocyte maturation [36]. The 5-HT induced P4-release from human granulose cells [41]. However, in testis, 5-HT is released from Leydig cells and binds to the 5-HT2 receptor, inducing suppression of androgen secretion [42, 43, 44]. Moreover, 5-HT is associated with the regulation of seasonal breeding through gonadotropin-induced cAMP signaling in hamsters [43]. In hamsters, mice, and rats, 5-HT enhanced hyperactivation and induced acrosome reaction [24, 32, 33, 34, 46] (see Fig. 1). In addition, 5-HT increases the success rate of IVF in mice and rats [33] (see Table 1). In humans, 5-HT increases the rate of motility and protein phosphorylation associated with capacitation [37]. Therefore, 5-HT regulates various reproductive functions, from gametes to individuals.

In this study, we showed that 5-HT enhanced hyperactivation and increased the IVF success in rats through the 5-HT4 receptor P4 enhanced rat sperm hyperactivation but did not increase IVF success [22], therefore, it is likely that 5-HT is an important molecule that regulates sperm capacitation and fertilization in rats. In mice, 5-HT enhanced sperm hyperactivation and increased IVF success via the 5-HT4 receptor [33]. Moreover, 5-HT4 receptors are associated with induction of the acrosome reaction and hyperactivation of hamster sperm [24, 32, 34, 46]. Therefore, it is likely that 5-HT and 5-HT4 receptors play key roles in rodent sperm function.

Conflict of interests

The authors declare no conflicts of interest.

Acknowledgments

This work was partially supported by the Research Grant Award 2024 of Dokkyo International Medical Education and Research Foundation (to MF) and JPSP KAKENHI Grant Number JP21K09435 (to MF).

References

  • 1.Florman HM, Fissore RA. Fertilization in mammals. In: Plant TM, Zeleznik AJ (ed.), Knobil and Neill’s Physiology of Reproduction. Vol 1. London: Elsevier Inc.; 2015: 149–196. [Google Scholar]
  • 2.Toshimori K, Eddy EM. The spermatozoon. In: Plant TM, Zeleznik AJ (ed.), Knobil and Neill’s Physiology of Reproduction. Vol 1. London: Elsevier Inc.; 2015: 99–148. [Google Scholar]
  • 3.Harayama H. Flagellar hyperactivation of bull and boar spermatozoa. Reprod Med Biol 2018; 17: 442–448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wang C, Lee GS, Leung A, Surrey ES, Chan SY. Human sperm hyperactivation and acrosome reaction and their relationships to human in vitro fertilization. Fertil Steril 1993; 59: 1221–1227. [PubMed] [Google Scholar]
  • 5.McPartlin LA, Suarez SS, Czaya CA, Hinrichs K, Bedford-Guaus SJ. Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes. Biol Reprod 2009; 81: 199–206. [DOI] [PubMed] [Google Scholar]
  • 6.Alasmari W, Barratt CLR, Publicover SJ, Whalley KM, Foster E, Kay V, Martins da Silva S, Oxenham SK. The clinical significance of calcium-signalling pathways mediating human sperm hyperactivation. Hum Reprod 2013; 28: 866–876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Pregl Breznik B, Kovačič B, Vlaisavljević V. Are sperm DNA fragmentation, hyperactivation, and hyaluronan-binding ability predictive for fertilization and embryo development in in vitro fertilization and intracytoplasmic sperm injection? Fertil Steril 2013; 99: 1233–1241. [DOI] [PubMed] [Google Scholar]
  • 8.Langlais J, Roberts KD. A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 1985; 12: 183–224. [Google Scholar]
  • 9.Xia J, Ren D. The BSA-induced Ca2+ influx during sperm capacitation is CATSPER channel-dependent. Reprod Biol Endocrinol 2009; 7: 119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q, Tilly JL, Clapham DE. A sperm ion channel required for sperm motility and male fertility. Nature 2001; 413: 603–609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Orta G, de la Vega-Beltran JL, Martín-Hidalgo D, Santi CM, Visconti PE, Darszon A. CatSper channels are regulated by protein kinase A. J Biol Chem 2018; 293: 16830–16841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wang T, Young S, Krenz H, Tüttelmann F, Röpke A, Krallmann C, Kliesch S, Zeng XH, Brenker C, Strünker T. The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA. J Biol Chem 2020; 295: 13181–13193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lishko PV, Botchkina IL, Kirichok Y. Progesterone activates the principal Ca2+ channel of human sperm. Nature 2011; 471: 387–391. [DOI] [PubMed] [Google Scholar]
  • 14.Strünker T, Goodwin N, Brenker C, Kashikar ND, Weyand I, Seifert R, Kaupp UB. The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm. Nature 2011; 471: 382–386. [DOI] [PubMed] [Google Scholar]
  • 15.Noguchi T, Fujinoki M, Kitazawa M, Inaba N. Regulation of hyperactivation of hamster spermatozoa by progesterone. Reprod Med Biol 2008; 7: 63–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fujinoki M. Melatonin-enhanced hyperactivation of hamster sperm. Reproduction 2008; 136: 533–541. [DOI] [PubMed] [Google Scholar]
  • 17.Fujinoki M, Suzuki T, Takayama T, Shibahara H, Ohtake H. Profiling of proteins phosphorylated or dephosphorylated during hyperactivation via activation on hamster spermatozoa. Reprod Med Biol 2006; 5: 123–135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Xie F, Garcia MA, Carlson AE, Schuh SM, Babcock DF, Jaiswal BS, Gossen JA, Esposito G, van Duin M, Conti M. Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization. Dev Biol 2006; 296: 353–362. [DOI] [PubMed] [Google Scholar]
  • 19.Suzuki T, Fujinoki M, Shibahara H, Suzuki M. Regulation of hyperactivation by PPP2 in hamster spermatozoa. Reproduction 2010; 139: 847–856. [DOI] [PubMed] [Google Scholar]
  • 20.Wertheimer E, Krapf D, de la Vega-Beltran JL, Sánchez-Cárdenas C, Navarrete F, Haddad D, Escoffier J, Salicioni AM, Levin LR, Buck J, Mager J, Darszon A, Visconti PE. Compartmentalization of distinct cAMP signaling pathways in mammalian sperm. J Biol Chem 2013; 288: 35307–35320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Fujinoki M, Takei GL, Kon H. Non-genomic regulation and disruption of spermatozoal in vitro hyperactivation by oviductal hormones. J Physiol Sci 2016; 66: 207–212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Miyazawa Y, Fujinoki M. Enhancement of rat spermatozoal hyperactivation by progesterone. J Reprod Dev 2023; 69: 279–290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Suzuki R, Fujinoki M. Progesterone increases the success of in vitro fertilization via enhanced sperm hyperactivation in mice. J Reprod Dev 2023; 69: 147–153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Miyashita M, Fujinoki M. Effects of aging and oviductal hormones on testes, epididymides, and sperm of hamster. Reprod Med Biol 2022; 21: e12474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Fujinoki M. Suppression of progesterone-enhanced hyperactivation in hamster spermatozoa by estrogen. Reproduction 2010; 140: 453–464. [DOI] [PubMed] [Google Scholar]
  • 26.Fujinoki M. Regulation and disruption of hamster sperm hyperactivation by progesterone, 17β-estradiol and diethylstilbestrol. Reprod Med Biol 2014; 13: 143–152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Fujikura M, Fujinoki M. Progesterone and estradiol regulate sperm hyperactivation and in vitro fertilization success in mice. J Reprod Dev 2024; 70: 96–103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Fujinoki M, Takei GL. Estrogen suppresses melatonin-enhanced hyperactivation of hamster spermatozoa. J Reprod Dev 2015; 61: 287–295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Calogero AE, Hall J, Fishel S, Green S, Hunter A, D’Agata R. Effects of γ-aminobutyric acid on human sperm motility and hyperactivation. Mol Hum Reprod 1996; 2: 733–738. [DOI] [PubMed] [Google Scholar]
  • 30.Kon H, Takei GL, Fujinoki M, Shinoda M. Suppression of progesterone-enhanced hyperactivation in hamster spermatozoa by γ-aminobutyric acid. J Reprod Dev 2014; 60: 202–209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Fujinoki M, Takei GL. γ-Aminobutyric acid suppresses enhancement of hamster sperm hyperactivation by 5-hydroxytryptamine. J Reprod Dev 2017; 63: 67–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Fujinoki M. Serotonin-enhanced hyperactivation of hamster sperm. Reproduction 2011; 142: 255–266. [DOI] [PubMed] [Google Scholar]
  • 33.Sugiyama Y, Fujinoki M, Shibahara H. Effects of 5-hydroxytryptamine on spermatozoal hyperactivation and in vitro fertilization in mice. J Reprod Dev 2019; 65: 541–550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sakamoto C, Fujinoki M, Kitazawa M, Obayashi S. Serotonergic signals enhanced hamster sperm hyperactivation. J Reprod Dev 2021; 67: 241–250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PPA. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 1994; 46: 157–203. [PubMed] [Google Scholar]
  • 36.Dubé F, Amireault P. Local serotonergic signaling in mammalian follicles, oocytes and early embryos. Life Sci 2007; 81: 1627–1637. [DOI] [PubMed] [Google Scholar]
  • 37.Jiménez-Trejo F, Tapia-Rodríguez M, Cerbón M, Kuhn DM, Manjarrez-Gutiérrez G, Mendoza-Rodríguez CA, Picazo O. Evidence of 5-HT components in human sperm: implications for protein tyrosine phosphorylation and the physiology of motility. Reproduction 2012; 144: 677–685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Juorio AV, Chedrese PJ, Li XM. The influence of ovarian hormones on the rat oviductal and uterine concentration of noradrenaline and 5-hydroxytryptamine. Neurochem Res 1989; 14: 821–827. [DOI] [PubMed] [Google Scholar]
  • 39.Campos MB, Vitale ML, Calandra RS, Chiocchio SR. Serotonergic innervation of the rat testis. J Reprod Fertil 1990; 88: 475–479. [DOI] [PubMed] [Google Scholar]
  • 40.Bòdis J, Bognàr Z, Hartmann G, Török A, Csaba IF. Measurement of noradrenaline, dopamine and serotonin contents in follicular fluid of human graafian follicles after superovulation treatment. Gynecol Obstet Invest 1992; 33: 165–167. [DOI] [PubMed] [Google Scholar]
  • 41.Bódis J, Török A, Tinneberg HR, Hanf V, Papenfuss F, Schwarz H. Serotonin induces progesterone release from human granulosa cells in a superfused granulosa cell system. Arch Gynecol Obstet 1993; 253: 59–64. [DOI] [PubMed] [Google Scholar]
  • 42.Tinajero JC, Fabbri A, Ciocca DR, Dufau ML. Serotonin secretion from rat Leydig cells. Endocrinology 1993; 133: 3026–3029. [DOI] [PubMed] [Google Scholar]
  • 43.Frungieri MB, Gonzalez-Calvar SI, Rubio M, Ozu M, Lustig L, Calandra RS. Serotonin in golden hamster testes: testicular levels, immunolocalization and role during sexual development and photoperiodic regression-recrudescence transition. Neuroendocrinology 1999; 69: 299–308. [DOI] [PubMed] [Google Scholar]
  • 44.Gerendai I, Banczerowski P, Csernus V, Halász B. Innervation and serotoninergic receptors of the testis interact with local action of interleukin-1beta on steroidogenesis. Auton Neurosci 2007; 131: 21–27. [DOI] [PubMed] [Google Scholar]
  • 45.Jiménez-Trejo F, Coronado-Mares I, Boeta M, González-Santoyo I, Vigueras-Villaseñor R, Arriaga-Canon C, Herrera LA, Tapia-Rodríguez M. Identification of serotoninergic system components in stallion sperm. Histol Histopathol 2018; 33: 951–958. [DOI] [PubMed] [Google Scholar]
  • 46.Meizel S, Turner KO. Serotonin or its agonist 5-methoxytryptamine can stimulate hamster sperm acrosome reactions in a more direct manner than catecholamines. J Exp Zool 1983; 226: 171–174. [DOI] [PubMed] [Google Scholar]
  • 47.Maleszewski M, Kline D, Yanagimachi R. Activation of hamster zona-free oocytes by homologous and heterologous spermatozoa. J Reprod Fertil 1995; 105: 99–107. [DOI] [PubMed] [Google Scholar]
  • 48.Shalgi R, Phillips DM. Motility of rat spermatozoa at the site of fertilization. Biol Reprod 1988; 39: 1207–1213. [DOI] [PubMed] [Google Scholar]
  • 49.Mortimer ST. A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 1997; 3: 403–439. [DOI] [PubMed] [Google Scholar]
  • 50.Fujinoki M, Suzuki T, Takayama T, Shibahara H, Ohtake H. Profiling of proteins phosphorylated or dephosphorylated during hyperactivation via activation on hamster spermatozoa. Reprod Med Biol 2006; 5: 123–135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685. [DOI] [PubMed] [Google Scholar]
  • 52.McCarthy BG, Peroutka SJ. Comparative neuropharmacology of dihydroergotamine and sumatriptan (GR 43175). Headache 1989; 29: 420–422. [DOI] [PubMed] [Google Scholar]
  • 53.Zhang LN, Su SW, Guo F, Guo HC, Shi XL, Li WY, Liu X, Wang YL. Serotonin-mediated modulation of Na+/K+ pump current in rat hippocampal CA1 pyramidal neurons. BMC Neurosci 2012; 13: 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Xie F, Garcia MA, Carlson AE, Schuh SM, Babcock DF, Jaiswal BS, Gossen JA, Esposito G, van Duin M, Conti M. Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization. Dev Biol 2006; 296: 353–362. [DOI] [PubMed] [Google Scholar]
  • 55.Wertheimer E, Krapf D, de la Vega-Beltran JL, Sánchez-Cárdenas C, Navarrete F, Haddad D, Escoffier J, Salicioni AM, Levin LR, Buck J, Mager J, Darszon A, Visconti PE. Compartmentalization of distinct cAMP signaling pathways in mammalian sperm. J Biol Chem 2013; 288: 35307–35320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Fujinoki M. Progesterone-enhanced sperm hyperactivation through IP3-PKC and PKA signals. Reprod Med Biol 2012; 12: 27–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Omote M, Wakimoto Y, Shibahara H. Possible role of 5-hydroxytryptamine (5-HT) receptor on human sperm motility regulation. Cureus 2023; 15: e49530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Osman RA, Andria ML, Jones AD, Meizel S. Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 1989; 160: 828–833. [DOI] [PubMed] [Google Scholar]
  • 59.Libersky EA, Boatman DE. Effects of progesterone on in vitro sperm capacitation and egg penetration in the golden hamster. Biol Reprod 1995; 53: 483–487. [DOI] [PubMed] [Google Scholar]
  • 60.Florman HM, Storey BT. Mouse gamete interactions: the zona pellucida is the site of the acrosome reaction leading to fertilization in vitro. Dev Biol 1982; 91: 121–130. [DOI] [PubMed] [Google Scholar]
  • 61.Gupta SK, Bansal P, Ganguly A, Bhandari B, Chakrabarti K. Human zona pellucida glycoproteins: functional relevance during fertilization. J Reprod Immunol 2009; 83: 50–55. [DOI] [PubMed] [Google Scholar]
  • 62.Hatakeyama S, Araki Y, Ohgi S, Yanaihara A, Araki Y. Fertilization with human sperm bound to zona pellucida by pressing onto the oocyte membrane. Hum Cell 2020; 33: 521–527. [DOI] [PubMed] [Google Scholar]
  • 63.Cummins JM, Yanagimachi R. Sperm-egg ratios and the site of the acrosome reaction during in vitro fertilization in the hamster. Gamete Res 1982; 5: 239–256. [Google Scholar]
  • 64.Nakanishi T, Ikawa M, Yamada S, Parvinen M, Baba T, Nishimune Y, Okabe M. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett 1999; 449: 277–283. [DOI] [PubMed] [Google Scholar]
  • 65.Sun TT, Chung CM, Chan HC. Acrosome reaction in the cumulus oophorus revisited: involvement of a novel sperm-released factor NYD-SP8. Protein Cell 2011; 2: 92–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, Chiba K, Hirohashi N. Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci USA 2011; 108: 4892–4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.La Spina FA, Puga Molina LC, Romarowski A, Vitale AM, Falzone TL, Krapf D, Hirohashi N, Buffone MG. Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct. Dev Biol 2016; 411: 172–182. [DOI] [PMC free article] [PubMed] [Google Scholar]

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