Distinct Ca²⁺ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt, Cynops pyrrhogaster

Abstract

Activation state of sperm motility named "hyperactivation" enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non-mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L-type and T-type voltage-dependent Ca 2+ channels (VDCCs). Mibefradil, a dominant blocker of Ttype VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L-type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca 2+ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca 2+ level changed especially in the anterior and posterior ends of the midpiece of the blocker-treated sperm. These suggest that different types of Ca 2+ channels mediate the intracellular Ca 2+ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.

A Novel Cysteine Knot Protein for Enhancing Sperm Motility That Might Facilitate the Evolution of Internal Fertilization in Amphibians

“…The binding of the FITC-P3 to the axial rod of the tail was also blocked by the anti-P3 antibody except for that of the principal piece (S2 Fig), thus indicating that the P3 specifically binds to the axial rod of the anterior region of the tail, termed the midpiece. Midpiece is significant for the regulation of sperm motility induced by the SMIS[11]. These results indicate that the obtained cDNA encodes SMIS, whose active site is located at the C-terminal region of the 2nd loop of the CK motif (Figs 1d and 2).…”

In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster

“…A pharmacological study using T-type VDCC blockers, including mibefradil, and L-type VDCC blockers, including nitrendipine, suggests that CaV3.1 or 3.3 of the spermatozoa is involved in the induction of the acrosome reaction by ARIS and CaV3.2 in the initiation of SMIS-induced sperm motility (Takayama-Watanabe et al, 2015). In addition, a nitrendipine-sensitive Ca 2+ channel and a mibefradil-sensitive Ca 2+ channel predominantly mediate the increase of intracellular Ca 2+ to enhance sperm motility in the anterior and posterior midpiece, respectively (Takahashi et al, 2013). The local enhancements are suggested to cause a specific state of motility crucial for spermatozoa to penetrate the jelly matrix.…”

“…This mechanism is crucial for the internal fertilization of C. pyrrhogaster (Takahashi et al, 2006) and contributes to the initiation of sperm motility inside the female body. The motility is pharmacologically suggested to be mediated by a unique molecular mechanism in which L-type and T-type VDCCs act in distinct sites of the sperm midpiece (Takahashi et al, 2013). In addition, C. pyrrhogaster spermatozoa bind to the vitelline envelope in passing through the jelly layer, though Cynops ZPC, unlike X. laevis ZPC, is suggested not to act as a sperm receptor.…”

Acrosome Reaction-Mediated Motility Initiation That Is Critical for the Internal Fertilization of Urodele Amphibians

“…In the sperm of C. pyrrhogaster , intracellular Ca 2+ is increased in both AR induction and motility initiation, as in the sperm of other animal species (Darszon et al 2006 ), and immediately drops down to the initial level (Watanabe et al 2011 ). In addition, we recently found that subsequent infl uxes of Ca 2+ in the motility-initiated sperm cause a high motility state (Takahashi et al 2013 ). Activation of the motility state is needed for sperm to propel through the viscous jelly matrix.…”