Progesterone‐enhanced sperm hyperactivation through IP₃–PKC and PKA signals

Abstract

Propose

The present study examined whether regulation of progesterone‐enhanced hyperactivation of spermatozoa is associated with the production of inositol 1,4,5‐trisphosphate (IP₃) and diacylglycerol (DAG) by phospholipase C (PLC) and cyclic adenosine monophosphate (cAMP) by adenylate cyclase (AC), as well as activation of protein kinase C (PKC) and protein kinase A (PKA).

Methods

Hamster spermatozoa were hyperactivated by incubation for 4 h in modified Tyrode's albumin lactate pyruvate (mTALP) medium. In order to examine the effects of IP₃ receptor (IP₃R), PKC and PKA on progesterone‐enhanced hyperactivation, their inhibitors (xestospongin C, bisindolylmaleimide 1 and H‐89) were used.

Results

Progesterone‐enhanced hyperactivation was significantly suppressed by the inhibitors of IP₃R, PKC and PKA.

Conclusions

The results suggest that progesterone‐enhanced sperm hyperactivation occurs through two signal pathways. One is an intracellular Ca²⁺ signal through production of IP₃ and DAG by PLC, binding of IP₃ to IP₃R and activation of PKC by DAG and Ca²⁺. The other is a cAMP–PKA signal through production of cAMP by AC and activation of PKA by cAMP.

Introduction

Hyperactivated spermatazoa exhibit a specialized flagellar movement with a high amplitude and asymmetrical beating pattern [1, 2]. During capacitation, spermatozoa are hyperactivated to create the propulsive force needed to penetrate the zona pellucida (ZP) [1, 3, 4]. After hyperactivation, capacitated spermatozoa undergo the acrosome reaction, which is a modified exocytosis that is required for penetration of the ZP and subsequent fusion of the plasma membranes of the sperm and egg [1, 2, 4, 5].

Spermatozoa can be capacitated in vitro in a culture medium containing albumin, HCO₃ ⁻, and Ca²⁺, of which albumin is an essential component [6, 7, 8] because it removes cholesterol from the plasma membrane and thus changes its fluidity [9]. HCO₃ ⁻ stimulates adenylate cyclase (AC) to increase cyclic adenosine monophosphate (cAMP) concentration [10]. After cAMP actives protein kinase A (PKA), PKA phosphorylates spermatic proteins at their serine/threonine residues and induces activation and capacitation [11, 12, 13, 14, 15, 16]. In many cases, tyrosine phosphorylation also occurs in a cAMP‐dependent manner during activation and capacitation [11, 12, 17, 18]. Ca²⁺ is involved in many intracellular signal transductions, including regulation of AC, phosphodiesterase and protein phosphorylation [11, 19, 20, 21, 22, 23].

Recent studies have demonstrated that hyperactivation is enhanced by ligands such as progesterone, melatonin and serotonin [2, 6, 7, 8, 24, 25, 26]. Moreover, it has been suggested that progesterone‐enhanced hyperactivation is suppressed by 17β‐estradiol [27]. Steroid hormones of these ligands regulate hyperactivation non‐genomically in association with Ca²⁺ signals [2, 4, 6, 28, 29]. Non‐genomic regulation of hyperactivation by progesterone is associated with phospholipase C (PLC) [6]. By activating PLC, inositol 1,4,5‐trisphosphate (IP₃) and diacylglycerol (DAG) are produced from phosphatidylcholine (PC) and phosphatidylinositol (PI), respectively. Ho et al. [20, 22, 30, 31] reported that intracellular Ca²⁺, which is released from an IP₃ receptor (IP₃R)‐gated Ca²⁺‐store located at the base of the sperm flagellum, regulates hyperactivation. It has been also suggested that hyperactivation is regulated by calmodulin‐dependent protein kinase II (CAMK2) [32]. Although DAG is an activator of protein kinase C (PKC), there is no other evidence for its involvement in sperm function. On the other hand, extracellular Ca²⁺ influx is also very important [6], and is induced by progesterone through the CatSper, which is a sperm‐specific Ca²⁺ channel located in the principal piece of the flagellum [33, 34]. When progesterone enhances hyperactivation, tyrosine phosphorylations of spermatic proteins are also increased and/or enhanced [6]. In contrast, many tyrosine phosphorylations are inhibited when progesterone‐enhanced hyperactivation is suppressed by 17β‐estradiol [27]. In general, tyrosine phosphorylation is a very important event during hyperactivation and is regulated by Ca²⁺ signals [1, 2, 11, 19].

In non‐genomic regulation, progesterone also activates AC to increase cAMP concentration [4, 28, 29, 35, 36, 37]. Cyclic AMP is an essential molecule for hyperactivation, and regulates tyrosine phosphorylation of spermatic proteins through PKA signals [1, 11, 12, 17]. However, it is unclear if progesterone enhances hyperactivation through cAMP–PKA signals.

Therefore, the present study examined whether progesterone‐enhanced hyperactivation is regulated through IP₃–PKC signals and/or cAMP–PKA signals.

Materials and methods

Chemicals

Progesterone was purchased from Sigma Chemical Company (St. Louis, MO, USA). 2‐[1‐(3‐Dimethylaminopropyl)‐1H‐indol‐3‐yl]‐3‐(1H‐indole‐3‐yl)‐maleimide (bisindolylmaleimide 1) and bovine serum albumin (BSA) fraction V were purchased from Merck KGaA (Darmstadt, Germany). N‐[2‐(N‐formyl‐p‐chlorocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide (H‐85) and N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide (H‐89) were purchased from Seikagaku Corporation (Tokyo, Japan). Xestospongin C and other reagent‐grade chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Preparation of hyperactivated spermatozoa

Spermatozoa were obtained from the caudal epididymis of sexually mature male golden hamsters (Mesocricetus auratus), which were housed in accordance with the guidelines of the Dokkyo Medical University and the Laboratory Animal Research Center in Dokkyo Medical University for the care and use of laboratory animals.

Hyperactivated spermatozoa were prepared according to the method described previously [16], using a modified Tyrode's albumin lactate pyruvate (mTALP) medium containing 101.02 mM NaCl, 2.68 mM KCl, 2 mM CaCl₂, 1.5 mM MgCl₂‐6H₂O, 0.36 mM NaH₂PO₄‐2H₂O, 35.70 mM NaHCO₃, 4.5 mM d‐glucose, 0.09 mM sodium pyruvate, 9 mM sodium lactate, 0.5 mM hypotaurine, 0.05 mM (‐)epinephrine, 0.2 mM sodium taurochoric acid, 5.26 μM sodium metabisulfite, 0.05 % (w/v) streptomycine sulfate, 0.05 % (w/v) potassium penicillin G and 15 mg/ml BSA (pH 7.4 at 37 °C under 5 % (v/v) CO₂ in air). An aliquot of caudal epididymal spermatozoa was placed at a culture plate (35‐mm dish), and 3 ml of the mTALP medium were carefully added before incubation for 5 min to allow the spermatozoa to swim up. The supernatant containing motile spermatozoa was collected, placed on a culture plate and incubated for 4 h at 37 °C under 5 % CO₂ in air to accomplish hyperactivation. Progesterone and inhibitors were added to the medium after placing motile spermatozoa on the culture plate. For examination of the effects of inhibitors, spermatozoa were exposed to progesterone after exposure to each inhibitor for 5 min. Progesterone was dissolved in methanol (MeOH). Bisindolylmaleimide 1, H‐85, H‐89 and xestospongin C were dissolved in dimethyl sulfoxide (DMSO). In all experiments, the maximal concentration of vehicle was 0.2 % by volume.

Measurement of the motility and hyperactivation of spermatozoa

Motility and hyperactivation measurements were performed according to the method described previously [16], with some modifications. Motility and hyperactivation were recorded on VHS via a CCD camera (Progressive 3CCD, Sony Corp., Tokyo, Japan) attached to a microscope (IX70, Olympus Corp., Tokyo, Japan) with phase‐contrast illumination and a small CO₂ incubator (MI‐IBC, Olympus). Each observation was performed at 37 °C, recorded for 2 min, and analyzed by manually counting the numbers of total spermatozoa, motile spermatozoa and hyperactivated spermatozoa in 10 different fields. Motile spermatozoa that exhibited asymmetric and whiplash flagellar movement and a circular and/or octagonal swimming locus were defined as hyperactivated [38]. The percentages of motile and hyperactivated spermatozoa were respectively defined as the number of motile spermatozoa/number of total spermatozoa × 100, and the number of hyperactivated spermatozoa/number of total spermatozoa × 100. Experiments were performed four times using four hamsters. Statistical analysis was carried out using post hoc analysis of the variance (ANOVA) test. A value of P < 0.05 was considered significant.

Results

Effects of IP₃R and PKC inhibitors on progesterone‐enhanced hyperactivation

In order to examine whether progesterone‐enhanced hyperactivation is regulated through IP₃R, hyperactivated hamster spermatozoa were exposed to xestospongin C (IP₃R inhibitor) in mTALP medium or mTALP medium with 20 ng/ml progesterone (Fig. 1). The percentage of motile spermatozoa was not inhibited by xestospongin C under either condition (Fig. 1a, c). Although neither 500 nM nor 1 μM xestospongin C suppressed hyperactivation, they significantly inhibited progesterone‐enhanced hyperactivation (Fig. 1b, d). As shown in Fig. 1d, enhancement of hyperactivation by progesterone was significantly inhibited by 500 nM xestospongin C after incubation for 1, 1.5 and 2 h. However, progesterone weakly but significantly enhanced hyperactivation under exposure to 500 nM xestospongin C after incubation for 1.5 and 2 h. On the other hand, enhancement of sperm hyperactivation by progesterone was strongly inhibited by 1 μM xestospongin C (Fig. 1d).

Figure 1.

Effects of xestospongin C on progesterone‐enhanced hyperactivation. After exposure to xestospongin C for 5 min, spermatozoa were exposed to progesterone. The percentages of motile spermatozoa (a) and hyperactivated spermatozoa (b) are shown when 500 nM or 1 μM xestospongin C were added to the mTALP medium. The percentages of motile spermatozoa (c) and hyperactivated spermatozoa (d) are shown when 500 nM or 1 μM xestospongin C and 20 ng/ml progesterone were added to the mTALP medium. Data are expressed as mean ± SD. In a and b (Vehicle), mTALP + 0.1 % (v/v) DMSO; (500 nM Xestospongin C), mTALP + 500 nM xestospongin C + 0.1 % (v/v) DMSO; (1 μM xestospongin C), mTALP + 1 μM xestospongin C + 0.1 % (v/v) DMSO. In c and d (Vehicle), mTALP + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P), mTALP + 20 ng/ml progesterone + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P + 500 nM Xestospongin C), mTALP + 20 ng/ml progesterone + 500 nM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P + 1 μM Xestospongin C), mTALP + 20 ng/ml progesterone + 1 μM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO. ^aSignificant difference compared with “Vehicle” and “P + 1 μM Xestospongin C” (P < 0.05); ^bSignificant difference compared with “Vehicle”, “P + 500 nM Xestospongin C” and “P + 1 μM Xestospongin C” (P < 0.05); ^cSignificant difference compared with “Vehicle” (P < 0.05)

The next step in examining whether progesterone‐enhanced hyperactivation is associated with PKC used bisindolylmaleimide 1 as a non‐specific PKC inhibitor (Fig. 2). The percentage of motile spermatozoa was not inhibited by bisindolylmaleimide 1 under any conditions (Fig. 2a, c). Although 10 nM bisindolylmaleimide 1 did not inhibit hyperactivation at all (Fig. 2b), it significantly inhibited progesterone‐enhanced hyperactivation (Fig. 2d).

Figure 2.

Effects of bisindolylmaleimide 1 on progesterone‐enhanced hyperactivation. After exposure to bisindolylmaleimide 1 for 5 min, spermatozoa were exposed to progesterone. The percentages of motile spermatozoa (a) and hyperactivated spermatozoa (b) are shown when 10 nM bisindolylmaleimide 1 was added to mTALP medium. The percentages of motile spermatozoa (c) and hyperactivated spermatozoa (d) are shown when 10 nM bisindolylmaleimide 1 and 20 ng/ml progesterone were added to mTALP medium. Data are expressed as mean ± SD. In a and b (Vehicle), mTALP + 0.1 % (v/v) DMSO; (10 nM bisindolylmaleimide 1), mTALP + 10 nM bisindolylmaleimide 1 + 0.1 % (v/v) DMSO. In c and d (Vehicle), mTALP + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P), mTALP + 20 ng/ml progesterone + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P + 10 nM bisindolylmaleimide 1), mTALP + 20 ng/ml progesterone + 10 nM bisindolylmaleimide 1 + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO. ^aSignificant difference compared with “Vehicle” and “P + 10 nM bisindolylmaleimide 1” (P < 0.05)

Effects of PKA inhibitors on progesterone‐enhanced hyperactivation

Neither H‐89 nor H‐85 at 1 μM affected the percentage of motile spermatozoa in both the mTALP medium and mTALP medium with 20 ng/ml progesterone (Fig. 3a, c). As for hyperactivation, they did not affect the percentage of hyperactivated spermatozoa in the mTALP medium (Fig. 3b). As for progesterone‐enhanced hyperactivation, 1 μM H‐89 significantly inhibited it, but 1 μM H‐85 did not affect progesterone‐enhanced hyperactivation (Fig. 3d).

Figure 3.

Effects of H‐89 and H‐85 on progesterone‐enhanced hyperactivation. After exposure to H‐89 or H‐85 for 5 min, spermatozoa were exposed to progesterone. The percentages of motile spermatozoa (a) and hyperactivated spermatozoa (b) are shown when 1 μM H‐89 or 1 μM H‐85 were added to mTALP medium. The percentages of motile spermatozoa (c) and hyperactivated spermatozoa (d) are shown when 1 μM H‐89 or 1 μM H‐85 and 20 ng/ml progesterone were added to mTALP medium. Data are expressed as mean ± SD. In a and b (Vehicle), mTALP + 0.1 % (v/v) DMSO; (1 μM H‐89), mTALP + 1 μM H‐89 + 0.1 % (v/v) DMSO; (1 μM H‐85), mTALP + 1 μM H‐85 + 0.1 % (v/v) DMSO. In c and d (Vehicle), mTALP + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P), mTALP + 20 ng/ml progesterone + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P + 1 μM H‐89), mTALP + 20 ng/ml progesterone + 1 μM H‐89 + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P + 1 μM H‐85), mTALP + 20 ng/ml progesterone + 1 μM H‐85 + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO. ^aSignificant difference compared with “Vehicle” and “P + 1 μM H‐89” (P < 0.05)

Discussion

Hyperactivation is a special flagellar movement exhibited by capacitated spermatozoa [1, 2]. Hyperactivation is spontaneously regulated by albumin, cAMP–PKA signals and Ca²⁺ signals [1, 2, 11, 12, 19, 20, 21, 22], but recent studies have demonstrated that hyperactivation can be modulated by hormones such as progesterone, 17β‐estradiol, melatonin and serotonin [6, 7, 8, 24, 27]. Progesterone enhances hyperactivation through extracellular Ca²⁺, the membrane progesterone receptor and PLC [6], whereas 17β‐estradiol suppresses progesterone‐enhanced hyperactivation through the membrane estrogen receptor [27]. Serotonin enhances hyperactivation through extracellular Ca²⁺ and two types of serotonin receptor (5HT₂ and 5HT₄) [8], whereby hyperactivation is enhanced through PLC–IP₃ signals when serotonin stimulates the 5HT₂ receptor, but is enhanced through AC–cAMP–PKA signals when the 5HT₄ receptor is stimulated. Melatonin also enhances hyperactivation by stimulating melatonin receptor type 1 [7], which suppresses nitric oxide synthase (NOS) and leads to a low concentration of NO. Low concentrations of NO stimulate a MAP kinase cascade and tyrosine phosphorylations, which are associated with sperm capacitation [39].

The intracellular signal transductions associated with the regulation of progesterone‐enhanced hyperactivation were examined in the present study. Because progesterone enhances hyperactivation through PLC [6], it was examined whether IP₃ and DAG are associated with the enhancement of hyperactivation by progesterone, using both IP₃R and PKC inhibitors (Figs. 1, 2). Progesterone‐enhanced hyperactivation was significantly inhibited by both inhibitors, although hyperactivation itself was not inhibited by them. On the other hand, it is reported that intracellular Ca²⁺ is released from an IP₃R‐gated Ca²⁺‐store and regulates hyperactivation through CAMK2 [20, 22, 30, 31, 32], so it is likely that progesterone enhances hyperactivation through activation of PLC, production of IP₃, binding of IP₃ to the IP₃R‐gated Ca²⁺‐store, release of intracellular Ca²⁺ and activation of CAMK2. Because it is reported that CAMK2 regulates tyrosine phosphorylations [40], it seems that non‐genomic regulation by progesterone includes the spontaneous regulatory mechanism of hyperactivation. In the previous study, progesterone increased and enhanced tyrosine phosphorylations when it enhanced hyperactivation [6]. Because progesterone stimulates activation of PLC, moreover, it is likely that progesterone induces the production of DAG and activates PKC. After activation of PKC, however, the regulatory mechanisms are not clear.

In the non‐genomic regulation of hyperactivation, progesterone also activates AC to increase cAMP concentration [4, 28, 29, 35]. Because cAMP activates PKA, which regulates sperm hyperactivation and tyrosine phosphorylations [1, 11, 12, 17], the present study examined the effects of PKA inhibitors on progesterone‐enhanced hyperactivation (Fig. 3). H‐89 at 1 μM significantly inhibited progesterone‐enhanced hyperactivation but H‐85 at 1 μM did not have effect (Fig. 3d). Therefore, it is likely that PKA also regulates progesterone‐enhanced hyperactivation through AC. Because AC and PKA are essentially related to the spontaneous regulatory mechanism of hyperactivation [1, 2, 11, 12], it seems that stimulation by progesterone includes the spontaneous regulatory mechanism of hyperactivation through the activation of AC. Does the same PKA regulate all sperm functions such activation, hyperactivation and progesterone‐enhanced hyperactivation? The responses of spermatozoa to PKA inhibitors were multiple in the present study. Although 1 μM H‐89 did not affect the percentage of motile spermatozoa and hyperactivated spermatozoa at all, it suppressed progesterone‐enhanced hyperactivation (Fig. 3). Thus, it seems that PKA regulation of progesterone‐enhanced hyperactivation differs from PKA regulation of motility or hyperactivation.

It is known that non‐genomic progesterone‐regulated hyperactivation is associated with extra‐ and/or intra‐cellular Ca²⁺ and PLC [2, 4, 28, 29]. The results of the present study suggest that hyperactivation is enhanced through IP₃R and PKC signaling after spermatozoa are exposed to progesterone. Moreover, it is also suggested that progesterone activates PKA during progesterone‐enhanced hyperactivation. Because progesterone increases and enhances tyrosine phosphorylations [6], it is proposed that aforementioned signals also increase and enhance tyrosine phosphorylations.

Acknowledgments

This work was partially supported by a Grants‐in‐Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 15790860 and no. 18791135).