Non‐genomic regulation of mammalian sperm hyperactivation

Abstract

Although it has been suggested that the acrosome reaction is induced through non‐genomic regulation in a ligand‐dependent manner, it is not known whether hyperactivation is similarly regulated. Progesterone and melatonin have been identified as ligands that regulate hyperactivation, the former through non‐genomic regulation with phospholipase C and the latter most likely through a reactive oxygen species‐mitogen activated protein kinase cascade. Both may be involved in spontaneous regulation of hyperactivation via tyrosine phosphorylation. The concentration of many hormones changes according to environmental conditions and biological rhythms, which will modulate ligand‐dependent regulation of hyperactivation.

Introduction

In order to fertilize the egg, mammalian sperm have to be capacitated. Capacitated sperm show the acrosome reaction (AR) and hyperactivation [1, 2]. The AR is a modified exocytotic event [3] that is necessary for penetration of the zona pellucida and for sperm–egg plasma membrane fusion [2]. Hyperactivation is a specialized movement of the sperm flagellum that creates the propulsive force for penetration [2]. Hyperactivated sperm exhibit large bend amplitude, whiplash and frenzied flagellar movements [2, 4, 5, 6].

Capacitation is regulated through several signal pathways associated with activation of protein tyrosine kinase (PTK) and tyrosine phosphorylations [1, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. Protein kinase A (PKA) signals [1, 3, 7, 8, 9, 10, 11], calcium (Ca²⁺) signals with calmodulin‐dependent protein kinase (CAMK) [1, 3, 6, 9, 12, 13, 14, 15, 16], mitogen‐activated protein kinase (MAPK) signals with reactive oxygen species (ROS) [1, 9, 17] and albumin stimulation [15, 16] are the most well known.

Regulation of sperm flagellar movement

Flagellar movement of non‐mammalian sperm has one step, activation [4, 18]. Mammalian sperm flagellar movement, however, has three steps: initiation, activation and hyperactivation [5]. The basic regulatory mechanism of flagellar movement is well known. It depends on cyclic adenosine monophosphate (cAMP) [4, 19, 20, 21, 22, 23, 24], and the energy is derived from hydrolysis of adenosine triphosphate (ATP) by dynein ATPase [4]. Cyclic AMP signals are similar to PKA signals, because in mammalian sperm that are activated and hyperactivated proteins are cAMP‐dependently phosphorylated by PKA signals [4, 7, 8, 9, 10, 11, 23, 24, 25, 26, 27, 28]. PKA is a serine/threonine kinase, so most cAMP‐dependent phosphorylations are serine/threonine phosphorylations [27, 28], but several cAMP‐dependent tyrosine phosphorylations have been detected [7, 8, 9, 23]. Although sperm PTK is not a cAMP‐dependent tyrosine kinase [29], it is widely accepted that sperm PTK is regulated by PKA [4, 10, 11]. Several cAMP‐dependent serine and tyrosine phosphoproteins have been identified. Two cAMP‐dependent serine phosphoproteins are pyruvate dehydrogenases [27, 30], and a cAMP‐dependent tyrosine phosphoprotein is an A‐kinase anchoring protein (AKAP) [12, 31, 32, 33]. Phosphorylation of AKAP is also regulated by Ca²⁺/calmodulin signals [12]. Another cAMP‐dependent tyrosine phosphoprotein, a 10‐kDa protein, has been identified as a neuropeptide‐like protein [23, 34]. The 10‐kDa protein is phosphorylated at tyrosine residues in a cAMP‐dependent manner during activation and at serine/threonine residues during hyperactivation. Non‐cAMP‐dependent protein phosphorylations are also associated with the regulation of sperm activation and hyperactivation [5, 28, 34, 35]. Many of those protein phosphorylations have not been identified, although two have been identified as tubulin and ATP synthase, respectively [36, 37].

Albumin, bicarbonate (HCO₃ ⁻) and Ca²⁺ are widely accepted as extracellular triggers of activation and hyperactivation. Albumin promotes capacitation by removing cholesterol from the sperm plasma membrane [38]; HCO₃ ⁻ stimulates adenylate cyclase to increase cAMP [39]; Ca²⁺ is involved in many intracellular signaling pathways [10, 11, 12, 13, 14, 15, 16]. Stimulation of these essential components leads to many sperm proteins being phosphorylated during capacitation [32]. When sperm begin to move, HCO₃ ⁻ and Ca²⁺ are key molecules that increase the activation of adenylate cyclase to produce cAMP, which in turn induces protein phophorylations. Extracellular Ca²⁺ also plays a key role in sperm hyperactivation, and intracellular Ca²⁺, which is released from an IP₃R (inositol 1,4,5‐trisphoshate receptor) gated Ca²⁺ store, is also very important.

In a previous study [40], abnormally shaped sperm produced by exposure to dibromoacetic acid were unable to be hyperactivated, although they could be activated, so it is likely that the regulatory mechanisms of hyperactivation differ from those of initiation and activation.

Regulation of the AR

The AR is also regulated through protein phosphorylations, such as PKA signals [2, 7, 8, 9, 10, 11], Ca²⁺ and CAMK signals [1, 2, 9, 10, 11, 12], and MAPK signals [1, 17].

It is reported that the AR occurs in a ligand‐dependent manner [1, 41, 42, 43, 44, 45]. Progesterone is a well‐known ligand [2, 41, 42, 43, 44, 45] that has been isolated from follicular fluid [41] and which induces the AR by non‐genomic regulation [42, 43, 44, 45]. There is a progesterone receptor (PR) in the plasma membrane of the human sperm head [42, 43, 44, 45, 46]. Moreover, PLCδ is activated by progesterone and induces the release of Ca²⁺ and the AR [42, 43, 44, 45, 47]. Previous studies of human sperm have suggested that estrogen suppresses the AR induced by progesterone at concentrations in both the nanomolar and micromolar range [45, 48]. It has also been suggested that there is an estrogen receptor (ER) in the human sperm plasma membrane, which affects the function of progesterone via Ca²⁺ oscillation [48].

Non‐genomic regulation of hyperactivation

In non‐mammals, it is well known that sperm motility is changed by ligands, such as steroids and peptides [18], but it is less well known whether mammalian sperm motility is also changed in a ligand‐dependent manner.

It has been suggested that several μg/mL progesterone, which is follicular fluid level, changes the parameters of motility [49] and enhances hyperactivation [50] of human sperm. A previous study in hamsters [51] suggested that 20 ng/mL of progesterone could significantly enhance penetration, but could not induce the AR. Although the effective concentration of progesterone for inducing the AR was several μg/mL, the same concentration did not significantly increase hamster sperm penetration [51]. Libersky and Boatman [52] reported that the progesterone concentration in hamster serum ranged 5.56–12.85 ng/mL, the follicular fluid concentration was 4.2–7.4 μg/mL and oviductal fluid concentration was 44.04–175.06 ng/mL. So, 20 ng/mL progesterone is midway between the concentration in blood and oviductal fluid. In humans, on the other hand, it was reported that the progesterone concentration in human plasma 0–18 ng/mL [53] and the follicular fluid concentration was up to 20 μg/mL [54, 55]. It was recently reported that in hamsters, 20 ng/mL progesterone significantly enhanced hyperactivation through a non‐genomic pathway via PLC [15]. PLC generally produces IP₃ and diacylglycerol from a phosphatidylcholine and/or a phosphatidylinositol. IP₃ controls the intracellular Ca²⁺ concentration and Ca²⁺ signaling via an IP₃ receptor. Intracellular Ca²⁺ regulates hyperactivation and is released from the IP₃‐gated Ca²⁺ store located at the base of the flagellum [13, 14, 56, 57]. Moreover, it has been suggested that hyperactivation is also regulated by CAMK [58]. Diacylglycerol is an activator of protein kinase C, but there is no other evidence for its involvement in sperm function. Therefore, it is likely that progesterone induces an increase in the IP₃ concentration by activating PLC, which releases Ca²⁺ from the IP₃R‐gated Ca²⁺ store and stimulates the regulation of hyperactivation via CAMK.

Where does progesterone bind in sperm? In human sperm, it is suggested that it binds in the acrosome region of the head, where the PR is located [59], and in hamster sperm progesterone also binds in the acrosome region of the head [15]. These findings indicate that stimulation by progesterone starts at the sperm head and travels to the flagellum. Because the IP₃R‐gated Ca²⁺ store is located at the base of the flagellum [13, 57], it seems that Ca²⁺ is the agent that transfers the stimulation of progesterone from the head to the flagellum.

In summary, progesterone induces the AR and hyperactivation in a concentration‐dependent manner [15, 41, 42, 43, 44, 45]. Several μg/mL of progesterone stimulates Ca²⁺ influx, activates PLCδ, releases Ca²⁺ and induces the AR in the sperm of many mammals such as humans and several rodents [15, 41, 42, 43, 44, 45, 47, 48]. Another study found that 20 ng/mL of progesterone activates PLC and enhances penetration [51] and hyperactivation [15] in hamster sperm, although several μg/mL of progesterone enhances hyperactivation through a similar non‐genomic system in human sperm [49, 50].

Enhancement of sperm hyperactivation by melatonin

Melatonin is produced and secreted by the pineal gland and retina; it regulates circadian rhythm and reproduction in seasonal breeders [60, 61]. Hamster reproduction is inhibited by long exposure to melatonin and stimulated by a short exposure, whereas sheep reproduction is stimulated by long exposure and inhibited by short exposure. It has been reported that melatonin does not affect the motility of human sperm [62], but another study [63] has suggested otherwise. Moreover, a melatonin receptor (MR) has been isolated from human sperm [64]. The quality of rat sperm motility was inhibited by melatonin in the micromolar range [65] and it was recently reported that melatonin in the picomolar range enhanced hamster sperm hyperactivation [16]. In a previous human study [66], melatonin concentrations were 38.6 ± 1.8 pM in serum and 98.1 ± 8.9 pM in follicular fluid. Although the melatonin concentration in the follicular fluid of rodents is unknown, it is likely to be similar to that in humans because the serum melatonin concentrations in rodents and humans are similar [62, 66].

Melatonin exerts its effects via specific receptors. MT1 (MR type 1) and MT2 (MR type 2) are seven transmembrane G‐protein coupled receptors [67, 68]. In a recent study using antagonists and agonists of the MR [16], it was suggested that the hamster sperm MR is MT1.

Melatonin inhibits cAMP production and nitric oxide (NO) production in cells [69, 70, 71]. However, because cAMP is a very important molecule for sperm function [2, 4, 6, 10, 11], it seems that melatonin does not inhibit cAMP production in sperm. On the other hand, it has been suggested that low concentration of ROS, which includes NO, are positively involved in the regulation of capacitation [9, 17, 72, 73], although high concentrations of ROS negatively affects sperm function [74]. Generally, mitochondria produce ROS in the cell [17, 72, 73]. Because the MR is located in the sperm midpiece, which contains the mitochondria [75, 76], it is likely that the ROS concentration is controlled by melatonin in the midpiece. Low concentrations of ROS stimulate a MAPK cascade and tyrosine phosphorylation during capacitation [17]. Although it is not well known whether the MAPK cascade regulates hyperactivation, it is widely accepted that tyrosine phosphorylation is associated with hyperactivation [9, 10, 11, 28]. Moreover, it has been suggested that tyrosine phosphorylation is regulated through the MAPK cascade [17]. Therefore, it is likely that a negative effect of melatonin on NO production is involved in modulation of hyperactivation.

Conclusion

Mammalian sperm have to be capacitated in order to fertilize the oocyte [2]. It has been suggested that the AR and hyperactivation occur in a ligand‐dependent manner via non‐genomic regulation [1, 15, 16, 41, 42, 43, 44, 45, 46, 47, 48], the ligands being progesterone and melatonin [1, 15, 16, 41, 42, 43, 44, 45]. Figure 1 shows a proposed ligand‐dependent regulation of hyperactivation. The progesterone signal begins at the sperm head. The melatonin signal begins in the midpiece of the sperm flagellum. Both signals are associated with tyrosine phosphorylation, which is spontaneous regulation of hyperactivation. After tyrosine phosphorylation, sperm are hyperactivated. An important aspect of ligand‐dependent regulation of hyperactivation is that it is not a basic regulatory mechanism. Hormone concentrations are affected by physiological and environmental conditions, such as circadian and seasonal rhythms and the sexual cycle. Therefore, ligand‐dependent regulation is a modulatory mechanism that is activated according to circumstances.

Figure 1.

Hypothesis of the regulatory mechanism of ligand‐induced hyperactivation. AC Adenylate cyclase, G G‐protein, M melatonin, MAPKK mitogen‐activated protein kinase kinase, MAPKKK mitogen‐activated protein kinase kinase kinase, NOS nitric oxide synthase, P progesterone, PC phosphatidylcholine, PI phosphatidylinositol