The regulation role of calcium channels in mammalian sperm function: a narrative review with a focus on humans and mice

Search strategy

Firstly, the databases searched in this study include PubMed, Google Scholar, Cochrane Library, and CNKI. The search terms are “calcium channels”; “mammalian sperm”; “CatSper”; “male infertility”; “voltage-gated calcium channels”; “store-operated calcium channels”; “cyclic nucleotide-gated channels”; “fertilization”; “sperm capacitation”; “sperm hyperactivation”; and “sperm acrosome reaction.” Secondly, the retrieval process is not limited by date or language, with the retrieval date set as June 31, 2024. A total of 457 articles were collected, primarily consisting of original research articles. Furthermore, the reference lists of primary articles were examined for additional relevant publications. Subsequently, the abstracts and main content of the articles were analyzed and screened for inclusion in the review.

Inclusion and exclusion criteria

This study employed the following criteria to determine the inclusion of research: (1) a clearly defined timeframe for the studies; (2) specification of sample size to ensure generalizability and validity; (3) clarity in the criteria for sperm sample selection and processing, as well as intervention and control measures; (4) research that provides mean, standard deviation, and sample size for result quantification. The exclusion criteria included: (1) duplicate studies; (2) unreliable research data; (3) the use of incorrect statistical methods or instances where the experimental data did not indicate the mean and standard deviation.

Capacitation

In the testis, spermatozoa are derived from primordial germ cells and subsequently attain their fertilizing capability during their passage through the epididymis. The activation of sperm motility predominantly occurs at the time of ejaculation. Upon entry into the female reproductive tract, sperm undergo a process termed ‘capacitation’, which is essential for oocyte fertilization (Gervasi & Visconti, 2017). This capacitation process involves three Ca²⁺-mediated stages: initial activation, the initiation of a protein kinase cascade and hyperactivation of motility. When human spermatozoa are incubated in media that facilitate capacitation, a notable increase in protein kinase A (PKA) activity is observed within the initial 10 min. Conversely, processes such as protein tyrosine phosphorylation, cholesterol efflux, and the removal of decapacitation factors transpire over a prolonged period, spanning several hours (Stival et al., 2016).

These processes result in downstream physiological changes in sperm, specifically sperm hyperactivation, which facilitates detachment from the oviductal epithelium within the sperm reservoir. The increase in membrane fluidity facilitates lipid raft reorganization at the apical ridge regions and acrosomal remodeling, both of which are essential for acrosomal exocytosis (Sáez-Espinosa et al., 2020). In general, mammalian sperm capacitation is regulated by external and internal factors, primarily involving sterol acceptors (like albumin), bicarbonate, and calcium (Bailey, 2010; Macías-García et al., 2015). These molecules activate the protein kinase A (PKA) and protein tyrosine kinase (PTK) pathways, which are essential for capacitation. During the capacitation process, intracellular Ca²⁺ levels in mammalian sperm increase, primarily due to the activation of voltage-gated Ca²⁺ channels (Ca_V), CatSper channels, and CNG channels (Darszon et al., 2011). Inhibition of these channels by utilizing antagonists can effectively impede the mammalian sperm capacitation process. The inhibition of these channels through the use of antagonists can effectively hinder the process of mammalian sperm capacitation. Moreover, an elevation in intracellular Ca²⁺ levels and pH_i within sperm cells can directly activate soluble adenylate cyclase (sAC), thereby promoting the production of intracellular cyclic adenosine monophosphate (cAMP). This, in turn, modulates the phosphorylation of sperm functional proteins via the cAMP/PKA pathway, and ultimately affect sperm capacitation (Vicente-Carrillo, Álvarez-Rodríguez & Rodríguez-Martínez, 2017; Alonso et al., 2017).

Hyperactivation

In general, the initiation of sperm capacitation is inhibited within the male reproductive tract due to the presence of seminal plasma. Upon entry into the female reproductive tract, sperm undergo capacitation and acquire hyperactivated motility (HAM), which is characterized by high-amplitude flagellar beats and vigorous motility. This enhanced motility enables the sperm to pass the lumen filled with mucus with escaping the oviductal reservoir, bind to, and penetrate the zona pellucida, followed by fertilizing the oocyte after their arrival to the fertilizing site (Zaferani, Suarez & Abbaspourrad, 2021).

Bicarbonate, cAMP, and Ca²⁺ present in follicular fluids are recognized as pivotal elements in the induction of sperm hyperactivation. Both bicarbonate and cAMP promote the influx of extracellular Ca²⁺ by activating calcium channels in sperm, thereby establishing Ca²⁺ as an essential trigger for sperm hyperactivation in mammals. In-vitro studies have demonstrated that the presence of extracellular Ca²⁺ is crucial for the initiation of sperm hyperactivated motility (Bailey, 2010; Iwamatsu et al., 1985; Fraser, 1987). When hyperactivated hamster sperm were washed with a Ca²⁺-free medium, they maintained hyperactivation for 5 min but exhibited reduced motility after 30 min. The re-introduction of 1.8 mM Ca²⁺ in bath solution successfully restored hyperactivated motility (Iwamatsu et al., 1985). Furthermore, the Ca²⁺ ionophore A23187 transiently induces hyperactivated motility in boar and golden hamster sperm (Fraser, 1987). Notably, progesterone, which is prevalent throughout the female genital tract, facilitates extracellular calcium influx via activation of sperm-specific calcium channels (CatSper) (Lishko, Botchkina & Kirichok, 2011; Strünker et al., 2011). This process is critical for the induction of hyperactivated motility in sperm. Sperm deficient in CatSper channels are incapable of achieving hyperactivated motility post-capacitation, ultimately resulting in male infertility (Wennemuth et al., 2000; Darszon et al., 1999; Treviño et al., 2004; Lishko et al., 2012). In boar and bull, sperm hyperactivation can be activated by the influx of extracellular Ca²⁺. More significantly, akin to humans and mice, the expression of the CatSper channel protein is detectable in both boar and bull sperm. Concurrently, progesterone can activate the CatSper channels in these species, inducing hyperactivated sperm motility. Conversely, the CatSper inhibitors NNC 55-0396 and mibefradil markedly inhibit the progesterone-induced activation of sperm (Vicente-Carrillo, Álvarez-Rodríguez & Rodríguez-Martínez, 2017; Miki & Clapham, 2013; Romero-Aguirregomezcorta et al., 2019; Suarez et al., 1992; Holt & Fazeli, 2016; Song et al., 2011).

Acrosome reaction

The acrosome, a structure resembling a cap situated in the anterior two-thirds of the sperm head, originates from Golgi vesicles. Enclosed within a membrane, the acrosome functions as a lysosome-like organelle housing various enzymes such as hyaluronidase, acrosin, acid phosphatase and so on. It is widely acknowledged that the acrosome reaction occurs when spermatozoa bind to the zona pellucida surrounding the oocyte. However, certain study propose that mouse sperm may not need to be acrosome-intact to facilitate this binding (Stival et al., 2016). In addition, it had been observed that rabbit sperm in the perivitelline space lacked acrosomes, yet these acrosome-reacted sperm could still penetrate the zona pellucida of other unfertilized oocytes (Kuzan, Fleming & Seidel, 1984).

During the acrosome reaction, the plasma membrane of the sperm fuses with the outer acrosomal membrane, facilitating the formation of a channel for the release of intra-acrosomal complex enzymes. Upon the completion of acrosomal exocytosis, the vesicles formed as a result of membrane fusion are entirely dispersed, resulting in the full exposure of the acrosomal matrix. This release enables the sperm cell recognize and bind to the ZP, ultimately fertilize the oocyte (Liu, Garrett & Baker, 2004; Liu et al., 2007; Abou-Haila & Tulsiani, 2000; Ramalho-Santos et al., 2001). Notably, the exposure of the acrosomal matrix is induced by an influx of extracellular calcium via channels, such as CatSper, store-operated Ca²⁺ channels and voltage-dependent Ca²⁺ channels (Gupta & Bhandari, 2011; Hirohashi & Yanagimachi, 2018).

In most mammalian except for murine and rabbit, the acrosomal exocytosis was induced upon contact with the ZP, and this process is primarily controlled by following signaling pathways (Inoue et al., 2011; Jin et al., 2011; Kuzan, Fleming & Seidel, 1984; Buffone, Hirohashi & Gerton, 2014). After the activation of receptors on the sperm surface such as ZP3R that are coupled with G-proteins, the Gi protein-coupled phospholipase C β1 (PLC β1) receptor initiates a signaling cascade that involves the activation of adenylyl cyclase (AC), leading to an increase in cyclic adenosine monophosphate (cAMP) levels in the cytosol (Breitbart, Rubinstein & Lax, 1997). This process further activates protein kinases and related proteins in the plasma membrane, ultimately facilitating the fusion of the plasma membrane with the outer acrosomal membrane of the sperm. Additionally, PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors (IP3R) located in the acrosome and the redundant nuclear envelope (RNE) in the sperm’s neck region, which open IP3-gated channel and releases Ca²⁺ from internal store such as the acrosome or RNE into the cytoplasm (Walensky & Snyder, 1995). Upon the depletion of Ca²⁺ in intracellular stores, store-operated calcium channels (SOCCs) are activated. Furthermore, both DAG and Ca²⁺ can activate protein kinase C (PKC) (Gonzalez-Garcia et al., 2013), which will increase cytoplasmic calcium concentration by opening voltage-dependent calcium channel in plasma membrane. Ultimately, a sustained elevation in intracellular calcium concentration ([Ca²⁺]_i) will promote the occurrence of the acrosome reaction (AR). In human sperm, the exchange protein directly activated by cAMP (EPAC), a Rap-specific guanine nucleotide exchange factor, has been demonstrated to mobilize Ca²⁺ from the sperm acrosome preceding the AR (Mata-Martínez et al., 2021).

The sperm-specific calcium channel CatSper

Voltage-gated calcium channels (VGCCs)

Voltage-gated Ca²⁺ channels (VGCCs) constitute a class of transmembrane proteins. Under physiological conditions or at resting membrane potentials, VGCCs typically remain in a closed state. Membrane depolarization activates VGCCs, leading to Ca²⁺ influx, which elevates intracellular Ca²⁺ levels and subsequently initiates a cascade of alterations in cellular physiological processes (Hwang & Chung, 2023). In mammalian sperm, VGCCs are comprosed of α and β subunits, with the α subunits serving as the functional unit. Each α subunit contains four homologous transmembrane structural domains, each comprising six transmembrane hydrophobic α-helices (S1–S6). The voltage sensor is constituted by segments S1–S4, while the P-loop connecting segments S5 and S6 contains a conserved α-helix sequence and a glutamate residue responsible for ion selectivity (Fig. 1) (Yao, Gao & Yan, 2024).

VGCCs are categorized into three subfamilies: Cav1, Cav2, and Cav3. Cav1 and Cav2 are high-voltage-activated channels that require significant membrane depolarization for activation. These channels can be further classified into L-type, N-type, P/Q-type, R-type and T-type, based on their current characteristics. The Cav3 gene encodes T-type calcium channels, which include the α1G (Cav3.1), α1H (Cav3.2), and α1I (Cav3.3) subunits. These channels are characterized by their activation at low membrane potentials and exhibit faster inactivation at more negative potentials compared to high voltage-activated (HVA) channels. The expression of these channels at protein level could be detected in both murine and human sperm (Fox, Nowycky & Tsien, 1987). Cav3.1 predominantly localized in the principal piece and mid-piece of the sperm flagellum. Additionally, Cav3.3 predominantly situated in the mid-piece of the flagellum in human sperm, which are similar to their localization in mouse sperm (Treviño et al., 2004). The functional regulation of Cav channels is crucial for the normal development of spermatocytes. The inhibition of either Cav1 or Cav3 channels by their respective blockers, nifedipine and ethosuximide, may lead to the disruption of normal spermatogenesis and steroidogenesis during prepubertal testicular maturation. These blockers are effective in inducing a premature arrest of developing spermatids and reducing Leydig cell abundance by inhibiting StAR protein expression, thereby diminishing testosterone production (Lee et al., 2011). During sperm capacitation, the influx of a small amount of Ca²⁺ through Cav3 channels into the cytoplasm significantly affects sperm physiological functions (Rehfeld, 2020). More importantly, Cav1 and Cav3 are additionally implicated in the regulation of the human sperm acrosome reaction (Wennemuth et al., 2000).

Previous research demonstrated that Ca²⁺ and calmodulin regulate the interaction between Cav3.2 and calcineurin. The binding of Cav3.2 to calcineurin results in a reduction of the enzyme’s phosphatase activity and subsequently decrease the current density of Cav3.2, which has been identified as a key regulator of sperm acrosome reaction (Camello-Almaraz et al., 2006; Lissabet et al., 2020; Albrizio et al., 2015). Furthermore, mice deficient in the α1E subunit of Cav2.3 exhibit significant subfertility in natural mating scenarios and demonstrate pronounced abnormalities in acrosome exocytosis (AE) and in vitro fertilization. These findings suggest that both Cav3.2 and Cav2.3 are involved in the regulation of acrosome reaction (AR) in murine sperm (Huang, Chen & Chen, 2013).

Store-operated calcium channels (SOCCs)

In mammalian sperm, the release of Ca²⁺ from intracellular stores is frequently succeeded by the influx of extracellular Ca²⁺ through specific channels localized in the plasma membrane (Herrick et al., 2005). A plausible regulatory mechanism for this phenomenon involves the release of Ca²⁺ from intracellular stores, which induces the repositioning of the sensor stromal interaction molecule (STIM) protein, located on the membrane of the calcium store, into close proximity to the plasma membrane. This repositioning enables STIM to interact with store-operated calcium channels (SOCCs) and transient receptor potential canonical channels (TRPC), thereby facilitating the influx of extracellular Ca²⁺. SOCCs are composed of ORAI proteins (ORAI1-3), which possess four transmembrane segments (TMs) that form a pore between TM2 and TM3. Additionally, a Ca²⁺-binding domain (CBD) is located at the core of the pore (Fig. 1). This domain plays a crucial role in modulating channel function by interacting with either calcium ions or calmodulin (CaM), leading to the inhibition of SOCCs. Calcium ions entering the cytoplasm via SOCCs are stored in sperm mitochondria. Additionally, the distribution of STIM1, ORAI and TRPC have already been identified in mice and human sperm (Krasznai et al., 2006; Lefièvre et al., 2012; Darszon et al., 2012). STIM1 demonstrates a localization pattern analogous to that of the Ca²⁺ pool, being primarily concentrated in the acrosomal region, the neck, and the midpiece of the flagellum. The outer acrosomal membrane of sperm contains a Ca²⁺-pump (Ca²⁺-ATPase) (Schuh et al., 2004) and inhibition of this pump by thapsigargin results in a reduction of intra-acrosomal [Ca²⁺], subsequently triggering the activation of SOCCs (Parrish, Susko-Parrish & Graham, 1999). The influx of Ca²⁺ into the cytoplasm through SOCCs resulted in a sustained elevation of intracellular Ca²⁺ concentrations, subsequently triggering the acrosome reaction in mammalian sperm, encompassing species such as humans, mice, cattle, and goats (Lawson et al., 2007).

Currently, members of the TRPC family of transient receptor potentials are recognized as the most likely candidates for SOCCs in murine and human sperm (Castellano et al., 2003). The mRNA expression of TRPCs (TRPC1-7) could be detected in mouse spermatogonia, while the level of protein expression of TRPC1, 3, 6 proteins have been detected in mature mouse sperm (Trevino et al., 2001). Previous report proposed that TRPC2 may serve as a critical channel protein for sperm SOCCs. Immunohistochemical analyses revealed that TRPC2 is predominantly localized in the anterior region of the head, with a lesser extent in the posterior region of the head in mouse sperm. Upon stimulation by ZP3, TRPC2 emerged as a critical regulator of the sustained elevation in intracellular Ca²⁺ levels, consequently facilitating the acrosome reaction in murine spermatozoa. Subsequent investigations have demonstrated the extensive distribution of TRP channels in human spermatozoa, with expression localized to both the head and tail regions, thereby implicating a potential role in the modulation of human sperm motility (Jungnickel et al., 2001).

Cyclic nucleotide-gated (CNG) channels

The cyclic nucleotide-gated (CNG) channel is constituted by four distinct A-type subunits, namely CNGA1, CNGA2, CNGA3, and CNGA4, as well as two B-type subunits, CNGB1 and CNGB3. Each subunit is typified by six transmembrane α-helices and a C-terminal cyclic nucleotide binding domain (CNBD) (Kaupp & Seifert, 2002). In mammalian, CNG channel is a heterotetramer that composed of A and B subunits, both of which belongs to the voltage-gated ion channel superfamily. Moreover, the functionality and permeability of cyclic nucleotide-gated (CNG) channels are modulated by cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). Genetic anomalies in the genes encoding CNG subunits can precipitate aberrant channel operation and instigate the emergence of clinical and pathological manifestations. Previous research has revealed disparate expression patterns of CNG subunits within the male reproductive system. In bovine, the B1 subunit expression is identifiable in bovine testis, with both the A3 and B1 subunits being distributed in the flagellum of mature bovine sperm. In murine, the B3 subunit is primarily expressed in the testis of mice and the A1 subunit is predominantly localized within spermatid remnants in the rat testis (Gerstner et al., 2000; Wiesner et al., 1998; Shi et al., 2005). Functional investigations have demonstrated that CNG channels in mouse sperm facilitate the influx of extracellular calcium, thereby triggering sperm capacitation (Cisneros-Mejorado & Sanchez Herrera, 2012). Notably, activity of CNG channel is mainly be regulated by intracellular cGMP or cAMP levels. The influence of cGMP on the permeability of CNG channels to Ca²⁺ was observed to be more substantial than cAMP.

In murine sperm, both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) can phosphorylate serine residues S557 and S579 within the cGMP/cAMP-binding domain of CNG channels, facilitate the binding of cGMP to cyclic nucleotide-binding domain (CNBD) and subsequent modulate CNG channel permeability (Trudeau & Zagotta, 2003). Following the elevation of intracellular Ca²⁺ levels, calmodulin are activated, which suppresses the activity of cyclic nucleotide-gated (CNG) channels. Simultaneously, in order to maintain calcium homeostasis, the calcium pump Ca²⁺-ATPase isoform 4 (PMCA4) continues to export intracellular Ca²⁺ to the extracellular environment. The reduction of intracellular calcium levels induces the activation of guanylate cyclase (GC), which consequently amplifies the production of cyclic guanosine monophosphate (cGMP) as depicted in Fig. 1. This sequence of events subsequently stimulates CNG channels, thereby facilitating the preservation of calcium equilibrium between intracellular and extracellular compartments, which is a critical factor in the regulation of sperm motility (Darszon et al., 2008).