Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

BACKGROUND: Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. RESULTS: Large consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. CONCLUSIONS: Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size

High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice. Morohoshi A, Miyata H, Shimada K, Nozawa K, Matsumura T, et al. PLOS Genetics. 2020. 16(1) doi:10.1371/journal.pgen.100858

Positive selection on ADAM10 builds species recognition in the synchronous spawning coral Acropora

The reef-building coral Acropora is a broadcast spawning hermaphrodite including more than 110 species in the Indo-Pacific. In addition, many sympatric species show synchronous spawning. The released gametes need to mate with conspecifics in the mixture of the gametes of many species for their species boundaries. However, the mechanism underlying the species recognition of conspecifics at fertilization remains unknown. We hypothesized that rapid molecular evolution (positive selection) in genes encoding gamete-composing proteins generates polymorphic regions that recognize conspecifics in the mixture of gametes from many species. We identified gamete proteins of Acropora digitifera using mass spectrometry and screened the genes that support branch site models that set the “foreground” branches showing strict fertilization specificity. ADAM10, ADAM17, Integrin α9, and Tetraspanin4 supported branch-site model and had positively selected site(s) that produced polymorphic regions. Therefore, we prepared antibodies against the proteins of A. digitifera that contained positively selected site(s) to analyze their functions in fertilization. The ADAM10 antibody reacted only with egg proteins of A. digitifera, and immunohistochemistry showed ADAM10 localized around the egg surface. Moreover, the ADAM10 antibody inhibited only A. digitifera fertilization but not the relative synchronous spawning species A. papillare. This study indicates that ADAM10 has evolved to gain fertilization specificity during speciation and contributes to species boundaries in this multi-species, synchronous-spawning, and species-rich genus

Ca2+ efflux via plasma membrane Ca2+-ATPase mediates chemotaxis in ascidian sperm

When a spermatozoon shows chemotactic behavior, transient [Ca2+]i increases in the spermatozoon are induced by an attractant gradient. The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa

Calaxin is required for cilia-driven determination of vertebrate laterality

Sasaki, K., Shiba, K., Nakamura, A. et al. Calaxin is required for cilia-driven determination of vertebrate laterality. Commun Biol 2, 226 (2019). https://doi.org/10.1038/s42003-019-0462-

Calaxin establishes basal body orientation and coordinates movement of monocilia in sea urchin embryos

Through their coordinated alignment and beating, motile cilia generate directional fluid flow and organismal movement. While the mechanisms used by multiciliated epithelial tissues to achieve this coordination have been widely studied, much less is known about regulation of monociliated tissues such as those found in the vertebrate node and swimming planktonic larvae. Here, we show that a calcium sensor protein associated with outer arm dynein, calaxin, is a critical regulator for the coordinated movements of monocilia. Knockdown of calaxin gene in sea urchin embryos results in uncoordinated ciliary beating and defective directional movement of the embryos, but no apparent abnormality in axoneme ultrastructure. Examination of the beating cycle of individual calaxin-deficient cilia revealed a marked effect on the waveform and spatial range of ciliary bending. These findings indicate that calaxin-mediated regulation of ciliary beating is responsible for proper basal body orientation and ciliary alignment in fields of monociliated cells

Microtubule stabilizer reveals requirement of Ca2+-dependent conformational changes of microtubules for rapid coiling of haptonema in haptophyte algae

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is ‘coiling’, which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca2+, suggesting changes in the mechanical properties of microtubules. Addition of Ca2+ resulted in the conversion of haptonematal twist into the planar bends near the proximal region. These results indicate that switching microtubule conformation, possibly with the aid of Ca2+-binding microtubule-associated proteins is responsible for rapid haptonematal coiling

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems