Fertilization of the green tree frog, Rhacophorus arboreus, occurs in the viscous environment of a foam nest, which is laid on vegetation. Their spermatozoa have a characteristic corkscrew-shaped head and a thick tail that extends perpendicularly to its longitudinal axis. However, it is unclear how these corkscrew-shaped spermatozoa move in this highly viscous environment. Here, we found that the spinning of the corkscrew-shaped head, caused by winding and unwinding of the tail, enables the spermatozoa to move through the highly viscous environment of a foam nest, like a corkscrew rotating into a cork. We suggested that dislocations observed in the matrix of satellite microtubules surrounding two axonemes, reflected the planes of sliding of the axonemes, and dyneins on doublets two and six of each axoneme were active during winding and unwinding, respectively. These results provide a novel mechanism for sperm movement that is adapted specifically to a viscous fertilization environment.

Sperm morphology is thought to be shaped by evolutionary pressure from the fertilization environment. Rhacophoridae (Amphibia, Anura) include both foam-nesting and nonfoam-nesting species and exhibit a variety of sperm morphologies. Here, we examine the sperm morphology and motility of a foam-nesting Rhacophoridae frog, Polypedates leucomystax. Their spermatozoa have a sickle-shaped head and a thick tail containing two axonemes with their doublet microtubule ones (Db1s) facing one another. These two axonemes are surrounded by hundreds of satellite microtubules that form a hexagonal lattice structure. The spermatozoa move spirally by directly converting their tail movements into propulsion force, similar to the movement of the sickle-shaped spermatozoa of Xenopus laevis. By comparing the spermatozoa of P. leucomystax to those of other foam-nesting Rhacophoridae frogs, Rhacophorus and Chiromantis, and to the nonfoam-nesting Rhacophoridae frog, Buergeria buergeri, we found the following: (i) Spermatozoa of foam-nesting Rhacophoridae share common morphological features, a pair of axonemes and crystallized satellite microtubules. (ii) Spermatozoa of nonfoam-nesting Rhacophoridae do not exhibit these features. (iii) Sperm motility in foam-nesting Rhacophoridae is adapted to viscous environments. (iv) A diversity of sperm morphology and motility exists even among foam-nesting Rhacophoridae frogs. (v) The spermatozoa of Rhacophorus are more adapted to the foam nest than the spermatozoa of Polypedates. V C 2012 Wiley Periodicals, Inc.

The spermatozoa of the Japanese green tree frog, Rhacophorus arboreus (Amphibia, Anura, Rhacophoridae), have a characteristic corkscrew-shaped head and a thick tail that extends perpendicular to the longitudinal axis of the head. We examined the process of spermatogenesis in Rh. arboreus, particularly spermiogenesis, using light and transmission electron microscopy. Spermiogenesis was categorized into the early, mid, and late stages based on the spermatid morphology and their location within the cyst. Early spermatids had a round nucleus and two independent flagella that elongated from a pair of parallel centrioles. The centrioles became embedded in centriolar adjunct material and attached to the nucleus. Then, the flagella were covered with a mantle-like cytoplasm that contained many microtubules. An acrosome appeared on the pointed side of the slightly elongated nucleus. Mid spermatids had an elongated rod-like head. As the nucleus elongated, the chromatin fibers became thicker and were arranged parallel to the elongation axis. An elongated acrosome was attached helically along the lateral side of the elongated nucleus. The biflagellate spermatids transformed into monoflagellate spermatids with two axonemes through a process in which the plasma membrane of each flagellum expanded. Late spermatids had a coiled or corkscrew-shaped head. An acrosome was located on the inside of the coiled cone composed of a nucleus. Parallel microtubules were connected in rows, and then became crystallized in the tail. The present report contains the first morphological description of spermatogenesis in Rhacophorus and suggests that spermiogenesis evolved to adapt to the fertilization environment.