Factors and pathways involved in capacitation: how are they regulated?

Abstract

In mammals, fertilization occurs via a comprehensive progression of events. Freshly ejaculated sperm have yet to acquire progressive motility or fertilization ability. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation. Capacitation is a significant prerequisite to fertilization. During the process of capacitation, changes in membrane properties, intracellular ion concentration and the activities of enzymes, together with other protein modifications, induce multiple signaling events and pathways in defined media in vitro or in the female reproductive tract in vivo. These, in turn, stimulate the acrosome reaction and prepare spermatozoa for penetration of the egg zona pellucida prior to fertilization. In the present review, we conclude all mainstream factors and pathways regulate capacitation and highlight their crosstalk. We also summarize the relationship between capacitation and assisted reproductive technology or human disease. In the end, we sum up the open questions and future avenues in this field.

Keywords: cAMP-PKA; calcium ion; capacitation; protein phosphorylation; signaling pathway.

Figure 1. The molecular basis of events associated with capacitation

Once ejaculated, removal of cholesterol from the sperm plasma membrane by albumin occurs first. Then the Ca2+ and HCO3- present in the seminal fluid enter the sperm through different channels include Catsper and NBC, respectively. Heparin and progesterone can also help take up Ca2+ into cell, increase intracellular free calcium and pH. The transmembrane movement of HCO3- has been associated with the increase in pHi as observed during capacitation. One of another early happenings during capacitation is the production of ROS, which will trigger and regulate a series of events including protein phosphorylation in a time-dependent fashion. Then spermatoza response to ROS generate the different signaling includes reversible redox signaling and activation of PKA signaling pathway by induce cAMP in a dose-dependent way. Cytosolic and mitochondrial glucose and other lectate are important to supply NADH for the sperm oxidase that produces O2·- for bull sperm capacitation. The factors which activate secondary messenger systems like sAC, then activates PKA. The function of endocannabinoid family to cAMP are controversial, some support it can induce sAC and cAMP production while others hold opposite opinion. PKA through the activation of tyrosine kinases and/or the inhibition of protein phosphatases, then allows an increase in protein phosphorylation and stimulate capacitation or acrosomal exocytosis. Other researches reveal HDL and other substances in the oviduct fluid also induce the capacitation. The function of glucose involved in capacitation remains elusive. Some papers report its absence impair the capacitation while others show glucose per se do not influence capacitation in vitro. EGF stimulates gene transcription during capacitation by activate Ras/Rho, but further mechanisms also remain unknown. The beginning of these events seems to be initiated by the elimination of cholesterol from the sperm membrane. However, all these events are necessary for the sperm to acquire fertilization capacity.

Figure 2. Crosstalk of mammalian sperm pH

i and other ions regulation during capacitation. Appropriate sperm intracellular pH is required for sperm capacitation. Protons in sperm may accumulate via different ion exchange, ATP hydrolysis as well as glycolysis. The influx of HCO3- into the sperm is mediated by Na+/HCO3- cotransporters (NBC) and Cl-/HCO3- exchangers (SLC26) so Na+ and Cl- may act in indirect way. Na, K-ATPase alpha 4 is another pump which enhance the sperm motility and hyperactivity during capacitation. Possibly, intracellular Ca2+ may regulate the Cl- exchange, but remain some further confirmation. Ca2+ influx in spermatozoa is principally regulated by CatSper channels. CatSper is regulated by many factors such as progesterone, and its crosstalk with other channels are also intricate. With Ca2+, HCO3- activates sAC, then increase cAMP and leads to PKA and protein tyrosine phosphorylation. Sperm specific Na+/H+ exchanger (sNHE) directly induce the pH increase in sperm accompany with other ion channels include Hv1. On the one hand, rapid proton extrusion is carried out by Hv1 in human sperm, which induce capacitation in indirect manner. On the other hand, Slo3 is activated by intracellular alkalinization and Ca2+ in human sperm, maintaining pHi and contributing to the hyperpolarization that occurs during capacitation. cAMP and cGMP are produced from ATP in mature spermatozoa, induce many channels via G-protein, activate sAC that ultimately induce capacitation. In the mitochondria, glycolysis produce H2O and CO2, which then transfer to HCO3- and H+, maintain the sperm pH hemostasis. Cellular HCO3- are equilibrated by the CO2/HCO3- conversion in mitochondria, thus regulate pH in sperm.

Figure 3. Different models of activation of tyrosine phosphorylation during capacitation

A schematic representation of the different molecular models of the activation of tyrosine phosphorylation during sperm capacitation. The removal of cholesterol from the plasma membrane increases membrane fluidity, results in an influx of HCO3- and Ca2+ ions through NBC and calcium channels. The increased intracellular concentration of HCO3-, Ca2+ and ROS, activates the sAC/PKA pathway. However, several papers also report Ca2+ negatively regulates protein tyrosine phosphorylation in human sperm. These phosphorylation of PKA substrates might be directly or indirectly involved in the phosphorylation of MEK-like proteins and subsequently tyrosine residues in fibrous sheath proteins. Also, Src family kinases can inactivate phosphatase, then change the PKA phosphorylation status. Interestingly, cold shock can stimulate and enhance the permeability of the membrane, which also stimulates the ROS pathway and causes an increase in protein tyrosine phosphorylation (PTP). The onset of tyrosine kinases activation is followed by tyrosine phosphorylation. The binding of EGF activates the ERK pathway which increases PTP. NO· can activate the ERK pathway intermediate Ras/Rho protein, while a high NO· can also up act to regulate the cGMP/PKG pathway. The PI3K/Akt axis always modulates the phosphorylation of the Thr-Glu-Tyr motif and tyrosine. It is possible that the downstream effectors such as PDK1 and Akt activate NOS, which then stimulate Ras and the ERK pathway and later cause the increase in PTP. ROS may involve in this pathway as well. Besides, another important factor is glucose, transported by GLUTs into the sperm cell, are useful for ATP generation by glycolysis. ATP is then used for sperm hyperactivation motility and PTP.