Our previous work suggested inherent differences in the physiological response of stallion sperm to pharmacological agents used to ascertain the pathways linked to sperm capacitation [21
]. Moreover, the recent discovery of cAMP-dependent guanine nucleotide exchange factors RAPGEF3 and RAPGEF4 in somatic and germ cells prompted the reevaluation of pathways previously attributed to cAMP-PRKACA activation. Therefore, in this study, we sought to separate the roles of PRKACA and RAPGEF3/RAPGEF4 in the events required to achieve fertilization competency in stallion sperm.
Though it is widely reported that capacitation-dependent protein tyrosine phosphorylation follows a cAMP-PRKACA-driven pathway [6
], we have previously shown that the addition of dbcAMP-IBMX to noncapacitating medium was unable to support time-dependent increases in protein tyrosine phosphorylation in stallion sperm [21
], contrary to what has been shown in all other species studied. This prompted us to further investigate these pathways in stallion sperm. In this study we show that a specific inhibitor of ADCY10, KH7, is able to reduce protein tyrosine phosphorylation to levels similar to those observed in the noncapacitating condition. Notably, the higher concentrations of KH7 required to inhibit protein tyrosine phosphorylation in stallion vs. mouse sperm may reflect a more robust control of these pathways and/or may be a consequence of sperm origin, i.e., ejaculated (stallion) vs. epididymal (mouse). Ejaculated stallion sperm are exposed to bicarbonate in seminal plasma and, thus, this may also explain that some levels of protein tyrosine phosphorylation are observed already at time 0 or in samples incubated in noncapacitating conditions, albeit relatively lower than those in the capacitating condition.
Additionally, we suspected that a highly active phosphodiesterase not inhibited by IBMX may have been responsible for the lack of effect observed when incubating stallion sperm in the presence of dbcAMP-IBMX [21
]. In support of this assumption, in this study cBIMPS, a nondegradable cAMP analogue, was able to support increases in tyrosine phosphorylation levels in sperm incubated in the absence of BSA and/or bicarbonate. However, because cBIMPS is a nonspecific cAMP analogue, we next tested the role of PRKACA in this pathway. Previous reports show that PKI can inhibit protein tyrosine phosphorylation in mouse sperm; however, in our study PKI was unable to completely abolish time-dependent increases in protein tyrosine phosphorylation in stallion sperm incubated under capacitating conditions, and in particular when cBIMPS was also added to the incubation medium.
The above findings and the recent discovery of RAPGEF3/RAPGEF4 as downstream effectors of cAMP prompted us to hypothesize that these proteins might play a role in capacitation-associated protein tyrosine phosphorylation in stallion sperm. In one-dimensional gel electrophoresis, however, we were unable to detect any changes in phosphotyrosine levels when the RAPGEF3/RAPGEF4-specific activator 8pCPT was added to the incubation medium in both stallion or mouse sperm. Under capacitating conditions, any additional effects of 8pCPT may have been masked by the already marked levels of protein tyrosine phosphorylation typical of sperm incubated in this condition. Under noncapacitating conditions, the absence of BSA and/or bicarbonate may have precluded appropriate membrane permeability of the cAMP analogue (particularly in stallion sperm, which are ejaculated and, therefore, coated by seminal plasma components). To be certain that our negative results were not due to membrane permeability issues, we incubated stallion sperm under noncapacitating conditions plus 8pCPT in the presence of BSA. Again, we observed no effect of RAPGEF3/RAPGEF4 activation on tyrosine phosphorylation, and levels remained comparable to the noncapacitating condition (data not shown). Moreover, we reasoned that the observation that PKI does not completely inhibit tyrosine phosphorylation in the presence of cBIMPS might have been likely due to the saturation of cAMP out-competing the inhibition of PRKACA. Altogether, these results suggest that RAPGEF3/RAPGEF4 are not involved in the cAMP-induced increase in tyrosine phosphorylation and support the notion that protein tyrosine phosphorylation follows a cAMP-PRKACA-driven pathway in stallion sperm, as reported for other species.
Most notably, addition of 8pCPT induced rates of acrosomal exocytosis in capacitated stallion (34%) and mouse sperm (45%) comparable to those obtained with progesterone and/or calcium ionophore-treated sperm (P
> 0.05). Because studies in somatic cells report effects of RAPGEF3/RAPGEF4 activation on specific membrane ion channels [31
] and RAPGEF3 localized to the acrosome in this study, we hypothesized a potential link between RAPGEF3 activation, changes in membrane potential, and acrosomal exocytosis.
It has been shown that capacitation-dependent hyperpolarization of the sperm plasma membrane is required for fertility [38
]. In the mouse, this represents a change in membrane potential (Em
) from approximately −30 mV to approximately −60 mV, is dependent on the activation of various ion channels, and prepares the sperm for acrosomal exocytosis [39
]. We report herein the resting membrane potential of stallion sperm to be −37.11 mV and show that capacitated stallion sperm hyperpolarize to −53.74 mV (P
= 0.002). In the mouse this hyperpolarization of the plasma membrane is postulated to occur via cAMP-PRKACA-mediated activation [41
] followed by phosphorylation of the cystic fibrosis transmembrane conductor (CFTR). In turn, CFTR activation allows the influx of negatively charged Cl−
ions as well as the inhibition of the constitutively active epithelial sodium channels (ENaC), thus preventing the influx of Na+
]. Additionally, capacitation-associated increases in intracellular pH activate K+ATP
channels, also contributing to membrane hyperpolarization [45
Though RAPGEF3/RAPGEF4 activation with 8pCPT will induce hyperpolarization in cerebellar neurons [43
], in various other excitable and nonexcitable somatic cells, RAPGEF3/RAPGEF4 activation has been observed to have effects on the abovementioned ion channels such that a depolarization of the plasma membrane would be expected. For instance, in pancreatic beta cells, Kang et al. [31
] observed a RAPGEF3/RAPGEF4-mediated inhibition of K+ATP
channels; in rat pulmonary epithelial cells, 8pCPT increased the channel open probability of ENaC [47
]; and, in rat hepatocytes, 8pCPT stimulated an outwardly rectifying Cl−
]. In accordance, we demonstrate herein that incubation of stallion sperm under capacitating conditions in the presence of the RAPGEF3/RAPGEF4-selective agonist 8pCPT maintained stallion sperm Em
in the depolarized state, thus precluding membrane hyperpolarization as observed under the capacitating conditions.
Interestingly, previous work suggests that zona pellucida (ZP3)-induced acrosomal exocytosis results from plasma membrane depolarization [42
]. In this model, capacitation-dependent hyperpolarization results in the recruitment of low voltage-activated (LVA) T-type ion channels, which are then available for activation. Contact with ZP3 stimulates membrane depolarization and opening of LVA channels, thus resulting in calcium influx and the initiation of acrosomal exocytosis [42
]. In our study, 1) RAPGEF3 localized to the acrosomal region of stallion and mouse sperm, 2) 8pCPT induced acrosomal exocytosis in capacitated stallion and mouse sperm, 3) 8pCPT did not induce acrosomal exocytosis in noncapacitated stallion sperm, and 4) 8pCPT maintained the stallion sperm membrane in the depolarized state. Based on these observations, we hypothesize that ZP3-directed RAPGEF3 activation is involved in the acrosome reaction-associated membrane depolarization possibly through its effects upon yet undetermined ion channels. This, in turn, may lead to the activation of LVA channels previously recruited as a result of capacitation-dependent hyperpolarization, followed by a sudden calcium influx and acrosomal exocytosis. Previous studies have demonstrated the presence of ENaC-δ in the acrosomal region of mouse sperm [38
], colocalizing this ion channel with RAPGEF3. Additionally, ENaC-α localized to the midpiece of mouse sperm [38
] and CFTR to the midpiece of both human and mouse sperm [41
], colocalizing these ion channels with RAPGEF4. We therefore speculate that these ion channels could contribute to RAPGEF3-mediated changes in Em
and acrosomal exocytosis; future studies should aim at identifying the particular ion channels through which RAPGEF3 and/or RAPGEF4 may facilitate sperm membrane depolarization. Moreover, a voltage-gated proton channel (Hv1), an H+
extrusion channel, has been recently identified as a major player in the alkalinization of CatSper channels and the subsequent Ca2+
influx required for sperm hyperactivation. Interestingly, this Hv1 channel is activated by membrane depolarization. Therefore, we cannot disregard the possibility that this and other ion channels may be identified as playing a role in our hypothesized pathway in future research [49
Altogether, the results of this study present a novel role for cAMP-activated RAPGEF3/RAPGEF4 in mammalian sperm physiology, as it pertains to a role in acrosomal exocytosis and its effects on membrane potential. These results highlight the importance of contrasting the effects of RAPGEF3/RAPGEF4 and PRKACA, as both are cAMP-dependent proteins potentially playing specific and diverging roles in the events that lead to the acquisition of fertilization competency. Moreover, the dichotomy of different pathways (PRKACA vs. RAPGEF3/RAPGEF4) stemming from a common activator (cAMP) directing two different events—capacitation vs. acrosomal exocytosis—emphasizes the notion that sperm are highly specialized and compartmentalized cells. Future studies should be directed toward investigating the channels affected by RAPGEF3/RAPGEF4 activation and the resulting sperm membrane depolarization.