Progesterone increases the success of in vitro fertilization via enhanced sperm hyperactivation in mice

Abstract

Progesterone (P) enhances spermatozoal hyperactivation, a capacitation event. Hyperactivation is associated with successful in vitro fertilization (IVF). In this study, we examined the effects of P on hyperactivation and IVF in mice. P enhanced spermatozoal hyperactivation and increased IVF success rate in a dose-dependent manner. Moreover, P affected spermatozoal hyperactivation and IVF through the membrane progesterone receptor of the spermatozoal head. These results show that P regulates spermatozoal capacitation and fertilization in mice. The concentration of P changes during the estrous cycle, indicating that spermatozoa are capacitated in response to the oviductal environment and subsequently fertilize the oocyte.

Mammalian spermatozoa bind to oocytes after the process of capacitation [1], during which they undergo hyperactivation of the flagellum and acrosome reaction (AR) at the head. Hyperactivation provides specialized motility for penetrating the oocyte envelope [1,2,3]. The AR is an exocytosis process that releases hydrolases for digestion of the oocyte envelope [1]. Albumin, Ca²⁺, and HCO₃^– play important roles when spermatozoa are capacitated in vitro [1,2,3,4,5]. Spermatozoa cannot be hyperactivated in the absence of albumin and Ca²⁺ [4, 5]. Albumin removes cholesterol from the surface of the spermatozoon [6] and induces an influx of Ca²⁺ by stimulating the CatSper Ca²⁺ channel, which is an important Ca²⁺ channel for hyperactivation [7, 8]. The CatSper Ca²⁺ channel is activated by protein kinase A (PKA) in mouse spermatozoa but not in human spermatozoa [9, 10]. Ca²⁺ and HCO₃^– regulate soluble adenylate cyclases, protein kinases, and protein phosphatases, resulting in protein phosphorylation and dephosphorylation [1, 3, 11,12,13,14,15].

Progesterone (P) and 17β-estradiol (Eβ) are released from the ovary and regulate oviductal physiology and fertilization, thereby changing the oviductal environment [16]. In addition, changes of the oviductal environment affect gamete transport, interactions between the spermatozoon and oviduct, spermatozoal physiology, oocytic physiology, and fertilization [16, 17]. It was reported that some oviductal hormones and neurotransmitters, such as P, melatonin (Mel), serotonin (5-hydroxytryptaine, 5-HT), Eβ, and γ-aminobutyric acid (GABA) regulate spermatozoal capacitation [18,19,20,21,22,23]. Among these, P is a popular effector of AR and hyperactivation in mammalian spermatozoa [18, 19, 24]. As for AR, after P binds to the membrane progesterone receptor (mPR) of the spermatozoal head, it induces the influx of Ca²⁺ via the CatSper Ca²⁺ channel and increases intracellular Ca²⁺ concentration [22, 25, 26]. Phospholipase C (PLC) and PKA are also involved in P-induced AR [27, 28]. Although AR is regulated by common mechanisms in mammalian spermatozoa, there are differences in the regulatory mechanisms of hyperactivation between human spermatozoa and other mammalian (at least rodent) spermatozoa [8,9,10, 19, 29, 30]. In human spermatozoa, P directly stimulates the CatSper Ca²⁺ channel and induces hyperactivation [19, 29, 30], whereas P indirectly stimulates the CatSper Ca²⁺ channel in mice; however, hyperactivation occurs via activation of the CatSper Ca²⁺ channel by PKA [8, 9]. In humans, PKA is not involved in the activation of the CatSper Ca²⁺ channel [10]. In hamsters, P enhances hyperactivation and penetration [5, 31]. Additionally, P stimulates the mPR, resulting in the activation of PLC, inositol 1, 4, 5-trisphosphate receptor-gated Ca²⁺ stores, PKA, and protein kinase C [5, 32]. Although the specific relationship between P and CatSper Ca²⁺ channels remains unknown, the CatSper Ca²⁺ channel is involved in the hyperactivation of hamster spermatozoa [33]. The effective concentration of P for hyperactivation differs from that for AR induction [5, 22, 24, 25, 28,29,30]. AR is induced by a concentration in μg/ml (or μM) of P [22, 24, 25, 28], while hyperactivation is induced by lower concentrations (ng/ml or nM) of P [5, 29, 30]. Moreover, a concentration of 20 ng/ml of P increases penetration; however, it does not induce AR in hamster spermatozoa [31].

Because the ability of hyperactivation is correlated with in vitro fertilization (IVF) success in humans [34], the success rate of IVF can be increased by amplifying the number of hyperactivated spermatozoa using oviductal hormones and neurotransmitters, such as P, among others. However, P does not increase the success rate of IVF in humans [34]. Recently, 5-HT has been shown to enhance sperm hyperactivation and increase IVF success in mice [35]. As the effect of P on mouse spermatozoal hyperactivation differs from that of P on human spermatozoal hyperactivation [8,9,10, 19, 29, 30], P may enhance sperm hyperactivation and thus increase the success rate of IVF in mice. In the present study, we examined whether P enhances spermatozoal hyperactivation and increases the success rate of IVF.

Materials and Methods

Chemicals

Bovine serum albumin (BSA), P, fluorescein isothiocyanate (FITC), BSA-conjugated P (FITC/BAS-P), and RU486 (mifepristone) were purchased from Merck KGaA (Darmstadt, Germany). Pregnant mare serum gonadotropin (PMSG; Serotropin®) and human chorionic gonadotropin (hCG; Gonatropin®) were purchased from ASKA Pharmaceutical Co., Ltd. (Tokyo, Japan). Other reagent-grade chemicals were purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Animals

ICR mice were bred at the Research Center for Laboratory Animals of the Dokkyo Medical University. The mice were maintained in an environment of 25°C under a 12-h light/dark cycle. This study was approved by the Animal Care and Use Committee of the university (experimental permission number: 0107) and was performed in accordance with the university guidelines for animal experimentation.

Preparation of hyperactivated spermatozoa

Spermatozoa were collected from the cauda epididymis of male mice (10–20-week-old). Hyperactivated spermatozoa were prepared as previously described [12] with some modifications. Modified Tyrode’s albumin lactate pyruvate (mTALP) medium [36] was used as the capacitation medium. The cauda epididymis was pricked with a 26 G (0.45 mm) needle (Terumo Corporation, Tokyo, Japan), and a drop of sperm was obtained. A drop (approximately 3 μl) of cauda epididymis spermatozoa was placed on a culture dish (35 mm diameter; Iwaki, Asahi Glass Co., Ltd., Tokyo, Japan), and 3 ml of medium was added to the dish. The spermatozoa were incubated for 5 min at 37°C for activation. The supernatant containing motile sperm was placed in a new dish containing vehicle or RU486. After incubation for 5 min, the supernatant was transferred to a new dish containing the vehicle, P, or FITC/BSA-P. Spermatozoa were incubated for 4 h at 37°C and 5% CO₂ to induce hyperactivation. To prepare the stock solutions, P (20 μg/ml) and RU486 (23.4 mM) were dissolved in ethanol. The FITC/BSA-P (7 μM) was dissolved in pure water. For all the experiments, the maximum concentration of the vehicle (ethanol and pure water) was 0.2%.

Measurements of motility and hyperactivation

The motility and hyperactivation were measured as previously described [35]. The movement of motile spermatozoa was 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) with phase-contrast illumination in a small CO₂ incubator (MI-IBC; Olympus). Observations were performed at 37°C for 1 min. A visual analysis of the movie included manual counts of the number of total, motile, and hyperactivated spermatozoa in 10 different fields. For all the experiments, visual analyses were performed in a blinded manner. Motile spermatozoa exhibiting asymmetric and whiplash-like flagellar movements were considered to be hyperactivated [18, 35]. The percentages of motility and hyperactivation were defined as the number of motile spermatozoa/total spermatozoa × 100 and the number of hyperactivated spermatozoa/total spermatozoa × 100, respectively. Each experiment was repeated four times using four mice. If the proportion of motile spermatozoa was ≤ 80%, the experiment was repeated.

Motility kinetic analysis using the sperm motility analysis system (SMAS)

Motility kinetics were evaluated using SMAS for animals (Ver. 3.18) with the loaded parameter file mouse_BM10×_640 nm_Bright59_150fps-shutter200.ini (Ditect Co., Ltd., Tokyo, Japan) as previously described [35]. The spermatozoa were hyperactivated in the presence or absence of P according to the methodology provided in the section “preparation of hyperactivated spermatozoa”. After incubation for 2 h, the suspension containing motile spermatozoa (15 μl) was transferred to an observation chamber (0.1 mm deep, 18 mm wide, and 18 mm long) made of mending tape attached to the glass slide in two parallel strips, which were covered with a cover glass. Spermatozoal 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 E2000; Nikon Corp., Tokyo, Japan) with phase-contrast illumination, 650 nm band-pass filter, and warm plate (MP10DM; Kitazato Corp., Shizuoka, Japan). SMAS analyzed 150 consecutive images obtained from a single field at 10 × magnification with a negative phase contrast. SMAS automatically calculated the straight-line velocity (VSL; μm/sec), curvilinear velocity (VCL; μm/sec), average-path velocity (VAP; μm/sec), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH; μm), and beat-cross frequency (BCF; Hz) using the manually calculated wobbler coefficient (WOB; defined as VAP/VCL) [37]. The SMAS analysis was repeated four times using four different mice. In each experiment, more than 300 spermatozoa were detected. Only motile spermatozoa judged to be significant were analyzed.

IVF

IVF was performed according to a previously described method [35] with some modifications. The superovulation treatment was carried out as follows: female mice (age: 8–12 weeks) were intraperitoneally (i.p.) administered 10 units of PMSG at 1600 h three days before IVF was conducted, followed by 10 units of hCG (i.p.) at 1630 h one day before IVF was performed. At 0950 h on the day IVF was performed, one drop (approximately 3 μl) of the dense mass of spermatozoa obtained from the cauda epididymis of male mice (age: 10–20 weeks) was mixed with 300 μl drops of mTALP medium in the presence or absence of the vehicle, P, and RU486. The spermatozoa (approximately 2 × 10⁷ cells/ml) were incubated for 1 h at 37°C and 5% CO₂. The eggs were collected at 1030 h on the day of IVF (18 h after hCG injection) from the PMSG/hCG-treated female mice. Cumulus-oocyte complexes (COCs) were dissected from the ampullae of both oviducts and incubated with 300 μl drops of mTALP medium in the presence or absence of the vehicle, P, and RU486. At 1050 h on the day of IVF, 10 μl of the suspension containing pre-incubated spermatozoa was added to the medium containing COCs to start the insemination process. Notably, the final spermatozoal concentration was 6.7 × 10⁵ cell/ml. Spermatozoa and COCs were incubated together for 0.5 or 5 h at 37°C and 5% CO₂. After incubation, the eggs and COCs were collected and washed with mTALP medium in the presence or absence of the vehicle, P, and RU486 to remove the spermatozoa and cumulus cells from the medium, and insemination was terminated. After washing, the eggs and COCs were re-incubated, observed at 1600 h on the day of IVF using a stereoscopic microscope (SMZ-10; Nikon Solutions Co., Ltd., Tokyo, Japan), and the total number of eggs was manually counted. The eggs and COCs were re-observed at 1600 h on the day after IVF using a stereoscopic microscope. The two-cell embryos were counted manually. The percentage of two-cell embryos was defined as follows: number of two-cell embryos/total number of eggs × 100. The analyses were performed in a blinded manner. The experiments were performed four times with four different male and female mice.

Ligand binding assays of P

Ligand binding assays of P were performed as previously described [5, 38], with some modifications. A drop (approximately 3 μl) of cauda epididymis spermatozoa was placed on the culture dish, to which 3 ml of medium was added, followed by incubation for 5 min to allow the spermatozoa to swim up. The sperm suspension was placed in a new dish containing either the vehicle or RU486. After incubation for 5 min, the sperm suspension was replaced with a new dish containing the vehicle or FITC/BSA-P. After incubation for 5 min, several microliters of the supernatant were placed on a glass slide without fluorescence and observed using a CCD camera (WARYCAM-CIX2000; WAYMER INC., Osaka, Japan) attached to a light microscope (IX70; Olympus) with phase-contrast illumination and a fluorescence unit.

Statistical Analysis

The data consisting of two groups, as shown in Fig. 1, were statistically analyzed via the Student’s t-test using Microsoft Excel (Microsoft Japan, Tokyo, Japan). When the data consisted of four groups, as shown in Figs. 2, 3, and 4, they were statistically analyzed via a one-way analysis of variance (ANOVA) and post-hoc test (Student-Newman-Keuls (SNK) test) using Microsoft Excel with the add-on: ystat2018 (Igakutosho Shuppan, Saitama, Japan). A P-value of < 0.05 was considered as statistically significant.

Fig. 1.

Effects of progesterone (P) on spermatozoal motility, hyperactivation, motility kinetics, and in vitro fertilization (IVF). The percentages of motility (A) and hyperactivation (B) of spermatozoa when cultured for 4 h in the presence and absence of 20 ng/ml of P. (C) The motility kinetics of spermatozoa when cultured at 2 h in the presence and absence of 20 ng/ml of P. (D) The percentages of two-cell embryos when IVF was performed in the medium with 20 ng/ml of P. The data represent the mean ± standard deviation. In (A), (B), (C), and (D), (Vehicle) medium with 0.1% ethanol as vehicle; (20 ng/ml P) medium with 20 ng/ml of P and vehicle. * Significant difference compared with “Vehicle” (P < 0.05).

Results

Effects of P on hyperactivation and IVF

A concentration of 20 ng/ml of P enhanced the hyperactivation of hamster spermatozoa [5]; therefore, we examined whether the concentration of 20 ng/ml of P enhanced the hyperactivation of mouse spermatozoa in this study. As shown in Figs. 1A and B, 20 ng/ml of P significantly increased the percentage of hyperactivated spermatozoa after incubation for 1, 1.5, 2, and 2.5 h but did not affect the percentage of motile spermatozoa. As for the motility kinetics, after incubation for 2 h, 20 ng/ml of P significantly decreased VSL, LIN, and ALH but did not affect the VCL, VAP, STR, BCF, and WOB (Fig. 1C).

As 5-HT increased the success rate of IVF via the enhancement of hyperactivation in mouse spermatozoa [35], we examined the effects of 20 ng/ml of P on the success of IVF in mice (Fig. 1D). When spermatozoa and COCs were co-incubated for 5 h, 20 ng/ml of P did not affect IVF success. However, 20 ng/ml of P significantly increased the success rate of IVF when spermatozoa and COCs were co-incubated for 0.5 h.

Dose-dependent effects of P on spermatozoa hyperactivation and IVF

In hamsters, P enhances spermatozoal hyperactivation in a dose-dependent manner [5]. As shown in Figs. 2A and B, P increased the percentage of hyperactivated spermatozoa after incubation for 1, 1.5, 2, and 2.5 h in a dose-dependent manner; however, it did not affect the percentage of motile spermatozoa. Specifically, 10 ng/ml of P did not affect the percentage of hyperactivated spermatozoa, while 20 ng/ml of P significantly increased the percentage of hyperactivated spermatozoa after incubation for 1, 1.5, 2, and 2.5 h. Moreover, 40 ng/ml of P significantly increased the percentage of hyperactivated spermatozoa after incubation for 1.5 and 2 h. The effect of 20 ng/ml of P was consistently observed to be higher than that of 40 ng/ml of P, although there was no significant difference between the effects of 20 ng/ml and 40 ng/ml of P.

Fig. 2.

Dose-dependent effects of P on spermatozoal motility, hyperactivation, and IVF. The percentages of motility (A) and hyperactivation (B) of spermatozoa when cultured for 4 h in the presence and absence of P (10, 20, and 40 ng/ml). (C) The percentages of two-cell embryos when IVF was performed in the medium with 10, 20, and 40 ng/ml of P. Spermatozoa and COCs were co-incubated for 0.5 h. The data represent the mean ± standard deviation. In (A), (B), and (C), (Vehicle) medium with 0.2% ethanol as vehicle; (10 ng/ml P) medium with 10 ng/ml of P and vehicle; (20 ng/ml P) medium with 20 ng/ml of P and vehicle; (40 ng/ml P) medium with 40 ng/ml of P and vehicle. * Significant difference compared with “Vehicle” and “10 ng/ml P” (P < 0.05). ** Significant difference compared with “Vehicle” (P < 0.05). *** Significant difference compared with “Vehicle”, “10 ng/ml P”, and “40 ng/mL P” (P < 0.05).

Notably, 20 ng/ml of P significantly increased the success rate of IVF, while 10 and 40 ng/ml of P did not affect the success rate of IVF (Fig. 2C).

Involvement of the progesterone receptor (PR) in P-enhanced spermatozoa hyperactivation and IVF

Generally, P enters the cell and binds to an intracellular PR to perform various functions [19,20,21]. In hamster spermatozoa, P enhances hyperactivation via PR [5], although in human spermatozoa, P induces hyperactivation without PR [19, 29, 30]. Therefore, we examined whether P enhances hyperactivation via PR in mouse spermatozoa. As shown in Figs. 3A and B, the increase in the percentage of hyperactivated spermatozoa by P was significantly inhibited by RU486 (a PR antagonist); however, RU486 did not affect the percentage of motile spermatozoa. Moreover, the increase in IVF success induced by P was significantly inhibited by RU486 treatment (Fig. 3C).

Fig. 3.

Involvement of the progesterone receptor (PR) in the regulation of hyperactivation and IVF. The percentages of motility (A) and hyperactivation (B) of spermatozoa when cultured for 4 h in the presence of 20 ng/ml of P and 23.4 μM of RU486. (C) The percentages of two-cell embryos when IVF was performed in the medium with 20 ng/ml of P and 23.4 μM of RU486. Spermatozoa and COCs were co-incubated for 0.5 h. The data represent the mean ± standard deviation. In (A), (B), and (C), (Vehicle) medium with 0.2% ethanol as vehicle; (P) medium with 20 ng/ml of P and vehicle; (RU486) medium with 23.4 μM RU486 and vehicle; (P + RU486) medium with 20 ng/ml of P, 23.4 μM of RU486, and vehicle. * Significant difference compared with “Vehicle”, “RU486”, and “P + RU486” (P < 0.05). ** Significant difference compared with “RU486” and “P + RU486” (P < 0.05).

In mammalian spermatozoa, P binds to the mPR [5, 19, 22]. P enters the cell, binds to intracellular PR, and induces gene expression [20, 21], although FITC/BSA-P cannot enter the cell because BSA blocks the cellular entry of P [39]. Therefore, the effects of FITC/BSA-P occur via non-genomic regulation without gene expression [20, 21, 25, 40, 41]. FITC/BSA-P at 7 nM, which is equivalent to 20 ng/ml of P, significantly increased the percentage of hyperactivated spermatozoa after incubation for 1, 1.5, 2, and 2.5 h but did not affect the percentage of motile spermatozoa (Figs. 4A and B). The increase in the percentage of hyperactivated spermatozoa induced by FITC/BSA-P was significantly inhibited by RU486 treatment (Fig. 4B).

Fig. 4.

Involvement of the membrane PR in the enhancement of hyperactivation by P. The percentages of motility (A) and hyperactivation (B) of spermatozoa when cultured for 4 h in the presence of 7 nM of fluorescein isothiocyanate (FITC)/bovine serum albumin (BSA)-P and 23.4 μM of RU486. FITC/BSA-P (7 nM) is equivalent to approximately 20 ng/ml of P. The data represent the mean ± standard deviation. In (A) and (B), (Vehicle) medium with 0.1% pure water and 0.1% ethanol as vehicle; (FITC/BSA-P) medium with 7 nM of FITC/BSA-P and vehicle; (RU486) medium with 23.4 μM RU486 and vehicle; (FITC/BSA-P + RU486) medium with 7 nM of FITC/BSA-P, 23.4 μM of RU486, and vehicle. * Significant difference compared with “Vehicle”, “RU486”, and “FITC/BSA-P + RU486” (P < 0.05). ** Significant difference compared with “RU486” and “FITC/BSA-P + RU486” (P < 0.05).

In hamsters and humans, P binds to the spermatozoal head [5, 38, 40]. As shown in Figs. 5A, 5A’, 5B, and 5B’, FITC/BSA-P bound to the head of mouse spermatozoa. Moreover, the binding of FITC/BSA-P was inhibited by RU486 (Figs. 5C, 5C’, 5D, and 5D’).

Fig. 5.

Binding of P to the spermatozoal head. (A, A’, B, and B’) Mouse spermatozoa were incubated in the modified Tyrode’s albumin lactate pyruvate (mTALP) medium with 7 nM of FITC/BSA-P, which is equivalent to approximately 20 ng/mL of P, and 0.1% ethanol. (C, C’, D, and D’) Mouse spermatozoa were incubated in the mTALP medium with 7 nM of FITC/BSA-P, 23.4 μM RU486, and 0.1% ethanol. (E and E’) Mouse spermatozoa were incubated in the mTALP medium with 23.4 μM of RU486, 0.1% pure water, and 0.1% ethanol. (F and F’) Mouse spermatozoa were incubated in the mTALP medium with 0.1% pure water and 0.1% ethanol. (A–F) Observed under a light field; (A’–F’) Observed under a fluorescent field. The arrow heads indicate the spermatozoal heads. Bar represents 100 μm.

Discussion

Hyperactivation is a capacitation event related to fertilization [1, 3]. In addition, some oviductal hormones and neurotransmitters regulate hyperactivation [18, 19, 23]. P, Mel, and 5-HT can induce and dose-dependently enhance sperm hyperactivation [4, 5, 29, 30, 33, 35, 42, 43]. In contrast, Eβ dose-dependently suppresses the enhancement of hyperactivation by P and Mel [38, 44, 45]. Moreover, the enhancement rate of hyperactivation depends on the P and Eβ concentrations in hamsters [44]. Although GABA acts as an inducer in human spermatozoa [46], it also suppresses the enhancement of hyperactivation by P and 5-HT in hamster spermatozoa [47, 48]. In hamsters, P increases spermatozoal penetration [31]. Moreover, 5-HT increases the success of IVF in mice [35].

In this study, we examined the effects of P on hyperactivation and IVF success in mice. P significantly enhanced hyperactivation in a dose-dependent manner (Figs. 1 and 2). Moreover, 20 ng/ml of P was the most effective concentration. P affected hyperactivation via mPR at the spermatozoal head (Figs. 3, 4, 5). These results suggest that the effects of P on spermatozoa are due to non-genomic regulation by mPR. Additionally, 20 ng/ml of P increased the rate of IVF success (Figs. 1 and 2), and 20 ng/ml of P increased penetration but did not induce AR in hamsters [31], suggesting that P increases the hyperactivation, penetration, and success rate of IVF in mice. As AR is generally induced by the zona pellucida [28], it may have been induced by the zone pellucida in the present study. In contrast, although 40 ng/ml of P enhanced hyperactivation, it did not affect the success rate of IVF (Fig. 2). The enhancement of hyperactivation by 40 ng/ml of P had a large fluctuation in rates. Therefore, the increase in IVF success rate may be unstable.

Generally, the concentrations of P in serum, follicular fluids, and oviductal fluids change according to the estrous cycle. In hamsters, the concentrations of P in serum and follicular fluids ranged between 5.64–12.85 ng/ml and 4.2–7.4 μg/ml during the periovulatory period [49]. Moreover, the concentration of P in oviductal fluids ranged between 44.01–175.06 ng/ml during the periovulatory period [49]. Additionally, in rodents, P dose-dependently affected the spermatozoal physiology involved in capacitation and fertilization in vitro. In hamsters, the concentration of 20 ng/ml of P increased spermatozoal penetration and enhanced hyperactivation [5, 31]. Similarly, in mice, the concentration of 20 ng/ml of P enhanced spermatozoal hyperactivation and increased IVF success (Figs. 1 and 2). These findings suggest that spermatozoal function and fertilization are controlled by the oviductal environment of P in rodents. The oviductal environment consists of several chemicals, including P, Mel, 5-HT, Eβ, and GABA, which change according to the estrous cycles and affect fertilization-associated events of spermatozoa and oocytes [16,17,18,19, 31]. Moreover, Mel enhances spermatozoal hyperactivation [4]. The enhancement of spermatozoal hyperactivation by P and Mel is suppressed by Eβ in a dose dependent manner [38, 44, 45] and that by P and 5-HT is suppressed by GABA in the dose dependent manner [47, 48]. However, the detailed effects and regulatory mechanisms of these hormones and neurotransmitters on spermatozoal hyperactivation and IVF success are unknown. Therefore, these issues should be examined in future studies.

In a human study [34], although hyperactivation was involved in the success rate of IVF, P-induced hyperactivation did not affect the rate of IVF success. In mice, P- and 5-HT-enhanced hyperactivation affected the rate of IVF success (Fig. 1) [35]. P and 5-HT did not affect the success rate of IVF when mouse spermatozoa and COCs were co-incubated for 5 h but increased the success rate when they were co-incubated for 0.5 h (Fig. 1) [35]. Since mouse spermatozoa were incubated for 1 h before co-incubation with COCs [Materials and Methods], the 0.5-h co-incubation of spermatozoa and COCs corresponded to the 1.5-h incubation of spermatozoa. When mouse spermatozoa were incubated for 1.5 h in the presence of P or 5-HT, the percentage of hyperactivation was significantly increased (Fig. 1) [35]. Therefore, P and 5-HT could potentially increase the success rate of IVF and enhance spermatozoal hyperactivation.

Upon the hyperactivation of the spermatozoon, there is a change in the motility kinetics because the motion trajectory of the spermatozoon changes significantly [37, 50]. When mouse spermatozoa were incubated for 2 h, P-enhanced hyperactivation was associated with a decrease in VSL, LIN, and ALH (Fig. 1C). In a previous study using hamsters [51], P-enhanced hyperactivation was related to a decrease in VSL when spermatozoa were incubated for 2 h. A decrease in VSL may be a common response to P-enhanced hyperactivation in rodent spermatozoa.

In conclusion, P enhances hyperactivation via the mPR in mice. In addition, the success of mouse IVF is increased by artificial spermatozoal hyperactivation induced by P. Notably, P shortened the co-incubation times of spermatozoa and COCs compared to the normal IVF method.

Conflict of interests

The authors declare that there are no conflicts of interest.