A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na /H exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt . Reverse-transcription PCR failed to detect sNHE in the newt sperm.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster.

Differences of Extracellular Cues and Ca Permeable Channels in the Signaling Pathways for Inducing Amphibian Sperm Motility.

Low osmolality of freshwater and/or sperm motility-initiating substance (SMIS) induce amphibian sperm motility through increases in intracellular Ca. In the internally fertilizing newt , the sperm motility-initiating substance engages T type voltage-dependent Ca + channels and N-methyl D-aspartate-type glutamate receptors to initiate sperm motility and L type voltage-dependent Ca channels to enhance motility. In the present study, differences in the usages of SMIS and Ca permeable channels for sperm motility regulation were examined in amphibians that undergo different reproductive modes.

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens.

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg's salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg's salt solution were examined.

NMDA-type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm.

The N-methyl d-aspartate type glutamate receptor (NMDAR) is a ligand-gated cation channel that causes Ca influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA-seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster.

Acrosome reaction-inducing substance triggers two different pathways of sperm intracellular signaling in newt fertilization.

The acrosome reaction is induced in the sperm of Cynops pyrrhogaster immediately in response to a ligand protein called acrosome reaction-inducing substance (ARIS) in the egg jelly at fertilization, whereas a spontaneous acrosome reaction occurs time-dependently in correlation with the decline of sperm quality for fertilization. The ARIS-induced acrosome reaction was recently found to be mediated by TRPV4 in association with the NMDA type glutamate receptor, although the intracellular mediators for the acrosome reaction are largely unclear. In the present study, spontaneous acrosome reaction was significantly inhibited by Ni, RN1734, and diltiazem, which blocks Cav3.

Sperm storage influences the potential for spontaneous acrosome reaction of the sperm in the newt Cynops pyrrhogaster.

Sperm storage is supposed to influence sperm quality, although the details remain unclear. In the present study, we found that sperm stored in a sperm storage site, the vas deferens of Cynops pyrrhogaster, spontaneously undergo acrosome reaction following incubation in Steinberg's salt solution (ST). Percentages of acrosome-reacted sperm increased time-dependently to about 60% in 24 hr.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm.

Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C.

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

Internal fertilization ensures successful reproduction of tetrapod vertebrates on land, although how this mode of reproduction evolved is unknown. Here, we identified a novel gene encoding sperm motility-initiating substance (SMIS), a key protein for the internal fertilization of the urodele Cynops pyrrhogaster by Edman degradation of an isolated protein and subsequent reverse transcription polymerase chain reaction. The SMIS gene encoded a 150 amino-acid sequence including the cysteine knot (CK) motif.

Sperm proteases that may be involved in the initiation of sperm motility in the newt, Cynops pyrrhogaster.

A protease of sperm in the newt Cynops pyrrhogaster that is released after the acrosome reaction (AR) is proposed to lyse the sheet structure on the outer surface of egg jelly and release sperm motility-initiating substance (SMIS). Here, we found that protease activity in the sperm head was potent to widely digest substrates beneath the sperm. The protease activity measured by fluorescein thiocarbamoyl-casein digestion was detected in the supernatant of the sperm after the AR and the activity was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), an inhibitor for serine or cysteine protease, suggesting the release of serine and/or cysteine proteases by AR.

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

A specific sperm-egg interaction in the oviductal matrix is crucial for internal fertilization of the red-bellied newt, Cynops pyrrhogaster. An understanding of the molecular basis of this interaction is expected to elucidate the evolutionary history of internal fertilization in amphibians. Recently, deep sequencing technology has provided global gene information even in nonmodel animals, allowing us to understand specific features of the molecular mechanisms underlying fertilization in C.

Contribution of different Ca²⁺ channels to the acrosome reaction-mediated initiation of sperm motility in the newt Cynops pyrrhogaster.

Initiation of sperm motility in urodeles, which is induced by a sperm motility-initiating substance (SMIS) in the sequestered granules on the surface of egg jelly, is mediated by the acrosome reaction (AR), which is triggered by an AR-inducing substance (ARIS) on a sheet-like structure. Details of the unique process of the interaction between egg jelly and sperm in these species is still unclear. The current study showed the fine structure of egg jelly in the newt Cynops pyrrhogaster, a urodele species, revealing that its outer surface was covered by a sheet-like structure of approximately 0.

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.

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.

Sperm motility initiation by egg jelly of the anuran, Discoglossus pictus may be mediated by sperm motility-initiating substance of the internally-fertilizing newt, Cynops pyrrhogaster.

The egg jelly of Discoglossus pictus contains sperm motility-activating activity, the molecular basis of which has not been studied. Discoglossus pictus sperm initiated motility immediately after immersion in egg-jelly extract, as well as after immersion in hyposmotic solution, which initiates sperm motility in the external fertilization of anuran amphibians. Sequential treatment of the D.

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels.

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca²(+)](i)). In quiescent non-motile sperm loaded with the Ca²(+) indicator Fluo-4, intracellular free Ca²(+) was observed around mitochondria using confocal scanning laser microscopy.

Identification of the sperm motility-initiating substance in the newt, Cynops pyrrhogaster, and its possible relationship with the acrosome reaction during internal fertilization.

Motility initiation is a key event during internal fertilization of female-stored sperm, although the underlying mechanisms remain unclear. In internally fertilizing urodeles, quiescent sperm initiate motility on the surface of the egg-jelly, a thick extracellular matrix that accumulates around the egg in oviduct. By immunizing mice with egg-jelly extracts, we successfully generated an alpha34 monoclonal antibody (mAb) which neutralized sperm motility-initiating activity in the egg-jelly of the newt, Cynops pyrrhogaster, in a dose-dependent manner.

Two states of active spermatogenesis switch between reproductive and non-reproductive seasons in the testes of the medaka, Oryzias latipes.

Seasonal change in spermatogenesis was examined in the restricted spermatogonium-type testes of a teleost, Oryzias latipes. Histological observation revealed that the number of each stage of germ cells during most of the non-reproductive season, from October to January (O-J period) was nearly half of that during the reproductive season, from May to July (M-J period), except for type B spermatogonia (B-gonia), which was actually equal. As a result, the ratio of primary spermatocytes (P-cytes) to B-gonia was remarkably small in the O-J period.

Identification of egg-jelly substances triggering sperm acrosome reaction in the newt, Cynops pyrrhogaster.

Our previous studies have shown that the acrosome reaction (AR) occurs in egg-jelly of the Japanese newt, Cynops pyrrhogaster. This is analogous to the substances of echinoderms but distinct from those of many other vertebrates derived from the egg envelope or its derivative, the zona pellucida. To identify the AR-inducing substances in newt egg jelly, a monoclonal antibody (mAb) was generated against the jelly by screening the culture supernatants to find the one that best neutralized the AR-inducing activity of the jelly substance.

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope.

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+).

Ordered progress of spermiogenesis to the fertilizable sperm of the medaka fish, Oryzias latipes, in cell culture.

Spermiogenesis is significant for producing sperm with equipment for achieving fertilization. Although multiple events occur in a particular order during spermiogenesis, it is unclear how the timing of those events is controlled. In the present study, we found that primary spermatocytes obtained from the spermatogenic testes of Oryzias latipes synchronously differentiated into sperm without contact with somatic cells in culture.