Deletion of a Seminal Gene Cluster Reinforces a Crucial Role of SVS2 in Male Fertility.

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2-/-) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2-6-/- mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2-/- male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.

This corrects the article DOI: 10.1038/icb.2016.51.

Seminal vesicle protein SVS2 is required for sperm survival in the uterus.

In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.

Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.

The purple sea urchin (Strongylocentrotus purpuratus) genome sequence contains a complex repertoire of genes encoding innate immune recognition proteins and homologs of important vertebrate immune regulatory factors. To characterize how this immune system is deployed within an experimentally tractable, intact animal, we investigate the immune capability of the larval stage. Sea urchin embryos and larvae are morphologically simple and transparent, providing an organism-wide model to view immune response at cellular resolution. Here we present evidence for immune function in five mesenchymal cell types based on morphology, behavior and gene expression. Two cell types are phagocytic; the others interact at sites of microbial detection or injury. We characterize immune-associated gene markers for three cell types, including a perforin-like molecule, a scavenger receptor, a complement-like thioester-containing protein and the echinoderm-specific immune response factor 185/333. We elicit larval immune responses by (1) bacterial injection into the blastocoel and (2) seawater exposure to the marine bacterium Vibrio diazotrophicus to perturb immune state in the gut. Exposure at the epithelium induces a strong response in which pigment cells (one type of immune cell) migrate from the ectoderm to interact with the gut epithelium. Bacteria that accumulate in the gut later invade the blastocoel, where they are cleared by phagocytic and granular immune cells. The complexity of this coordinated, dynamic inflammatory program within the simple larval morphology provides a system in which to characterize processes that direct both aspects of the echinoderm-specific immune response as well as those that are shared with other deuterostomes, including vertebrates.

Identification of an antibacterial polypeptide in mouse seminal vesicle secretions.

In both men and women, pathogenic bacteria enter the reproductive tract and cause harmful symptoms. Intrauterine and oviductal inflammation after copulation may have severe effects, such as infertility, implantation failure, oviduct obstruction, and robust life-threatening bacterial infection. Human seminal plasma is considered to be protective against bacterial infection. Among its components, Semenogelin-I/-II proteins are digested to function as bactericidal factors; however, their sequences are not conserved in mammals. Therefore, alternative antibacterial (bactericidal and/or bacteriostatic) systems may exist across mammals. In this study, we examined the antibacterial activity in the seminal plasma of mice lacking a gene cluster encoding Semenogelin-I/-II counterparts. Even in the absence of the majority of seminal proteins, antibacterial activity remained in the seminal plasma. Moreover, a combination of gel chromatography and liquid chromatography coupled with tandem mass spectrometry revealed that the prostate and testis expressed 4 protein as a novel antibacterial (specifically, bacteriostatic) protein, the sequence of which is broadly conserved across mammals. Our results provide the first evidence of a bacteriostatic protein that is widely present in the mammalian seminal plasma.

Cidaroids, clypeasteroids, and spatangoids: Procurement, culture, and basic methods.

This chapter describes practical methods and key points for using non-camarodont echinoids including cidaroids (Order Cidaroida), clypeasteroids (also known as sand dollars; Order Clypeasteroida), and spatangoids (also known as heart urchins; Order Spatangoida) as experimental subjects for biological studies. The content described here is based on six Japanese species of echinoids (Astriclypeus manni, Clypeaster japonicus, Echinocardium cordatum, Peronella japonica, Prionocidaris baculosa, and Scaphechinus mirabilis). Specific topics addressed in this chapter include the collection and maintenance of adults, embryonic culture, and experimental procedures for micromanipulations, whole mount in situ hybridization, and immunological experiments.

IL17 factors are early regulators in the gut epithelium during inflammatory response to Vibrio in the sea urchin larva.

IL17 cytokines are central mediators of mammalian immunity. In vertebrates, these factors derive from diverse cellular sources. Sea urchins share a molecular heritage with chordates that includes the IL17 system. Here, we characterize the role of epithelial expression of IL17 in the larval gut-associated immune response. The purple sea urchin genome encodes 10 IL17 subfamilies (35 genes) and 2 IL17 receptors. Most of these subfamilies are conserved throughout echinoderms. Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance. IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype, indicating a potential feedback. A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families. The larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism.

Development of ciliary bands in larvae of the living isocrinid sea lily Metacrinus rotundus.

Embryos and larvae of an isocrinid sea lily, Metacrinus rotundus, are described by scanning electron microscopy. Around hatching (35 h after fertilization), the outer surface of the gastrula becomes ubiquitously covered with short cilia. At 40 h, the hatched swimming embryo develops a cilia-free zone of ectoderm on the ventral side. By 3 days, the very early dipleurula larva develops a cilia-free zone ventrally, densely ciliated regions laterally, and a sparsely ciliated region dorsally. At this stage, the posterior and anterior ciliary bands first appear: the former runs along a low ridge separating the densely from the sparsely ciliated epidermal regions, while the latter is visible, at first discontinuously, along the boundary between the densely ciliated lateral regions and the cilia-free ventral zone. In the late dipleurula larva (5 days after fertilization), the anterior and posterior loops of ciliary bands are well defined. The transition from the dipleurula to the semidoliolaria larva occurs at 6 days as the posterior loop becomes rearranged to form incompletely circumferential ciliary bands. The larva becomes competent to settle at this stage. The arrangement of the ciliary bands on the semidoliolaria is maintained during the second week of development, while the larva retains its competence to settle. The larval ciliary patterns described here are compared with those of stalkless crinoids and eleutherozoan echinoderms. The closest morphological similarities are between M. rotundus and the basal eleutherozoan class Asteroidea.

The behavior and the morphology of sea lilies with shortened stalks: implications on the evolution of feather stars.

Extant crinoids can be divided into two groups, stalked sea lilies and stalkless feather stars. Feather stars are considered to have evolved from stalked ancestors by losing most of the stalk, but other differences are present between the two groups. The unsegmented centrodorsal, long and curved cirri near the crown, small calyx, and the ability to swim are all feather star features not found in the sea lilies. To figure out which of the above features evolved directly correlating with loss of the stalk in feather stars, we cut off the stalk from the sea lily Metacrinus rotundus and kept them alive in an aquarium. The specimens with shortened stalks were able to stand and crawl with their arms without the support of their stalks, but swimming was not observed for any of the animals. Morphologically, neither fusion of the remaining segments nor the reduction of the size of the calyx were observed, but the cirri became long and curved near the crown. Therefore, the extant sea lilies possess a potential to adapt to incidents of stalk loss. Specimens autotomizing most of their stalks were observed, suggesting that the potential is actually employed in nature. This mechanism linking the reduction of the stalk and the changes in the morphology of cirri may have played an important role in the evolution of the feather stars, if the stalked ancestors of feather stars also possessed this potential. Experimental zoological approaches as this study may provide new insights to the questions of evolution.

Sp185/333: a novel family of genes and proteins involved in the purple sea urchin immune response.

The Sp185/333 system of genes, messages and proteins are expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, and is an extraordinary example of diversification of a putative innate immune response system in an invertebrate. Reviewed here, is the current understanding of this complex system as illustrated by sequence comparisons of the genes, messages and deduced proteins with descriptions of diversity, including preliminary results on genomic organization and descriptions of 185/333 in other echinoids. Sp185/333 gene expression in adults and embryos occurs in response to immune challenge and includes changes in the frequencies of Sp185/333-positive coelomocytes in the adults. The diversity of the Sp185/333 protein repertoire in coelomocytes is far greater than the sequence diversity encoded in the genes, which may be the result of rapid gene recombination, RNA editing and/or low-fidelity transcription, plus post-translational modifications. This review concludes with preliminary results and speculations on protein function.

Phylogenetic correspondence of the body axes in bilaterians is revealed by the right-sided expression of Pitx genes in echinoderm larvae.

Chordates and echinoderms are two of the three major deuterostome phyla and show conspicuous left-right (LR) asymmetry. The establishment of LR asymmetry has been explored in vertebrates, but is largely unknown in echinoderms. Here, we report the expression pattern of genes that are orthologous to the chordate left-side specific gene Pitx, cloned from the sea urchin Hemicentrotus pulcherrimus (HpPitx) and the starfish Asterina pectinifera (ApPitx). HpPitx transcripts were first detected bilaterally in one cell of the ventrolateral primary mesenchyme-cell aggregate of early prism larvae. New expression was detected asymmetrically in the right counterpart of a bilateral pair of mesodermal coelomic pouches and in the right ectoderm. In starfish bipinnaria larvae, the ApPitx signal was detected in the right coelomic pouch and in the right half of the ectoderm along the ciliary bands. These results suggest that the function of Pitx in establishing LR asymmetry was introduced in the last common ancestor of echinoderms and chordates. However, the right-side specific expression in echinoderm larvae is inverted compared to chordate embryos. This indicates that the LR axis is inversely represented between echinoderms and chordates, which supports the scenario that dorsoventral axis inversion was introduced into the chordate lineage by turning upside down.

Ion flow regulates left-right asymmetry in sea urchin development.

The degree of conservation among phyla of early mechanisms that pattern the left-right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H(+)/K(+)-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H(+)/K(+)-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H(+) and/or K(+), and also of Ca(2+), functions in the establishment of LR asymmetry in sea urchin development.

Genomic insights into the immune system of the sea urchin.

Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.

RTK and TGF-beta signaling pathways genes in the sea urchin genome.

The Receptor Tyrosine kinase (RTK) and TGF-beta signaling pathways play essential roles during development in many organisms and regulate a plethora of cellular responses. From the genome sequence of Strongylocentrotus purpuratus, we have made an inventory of the genes encoding receptor tyrosine kinases and their ligands, and of the genes encoding cytokines of the TGF-beta superfamily and their downstream components. The sea urchin genome contains at least 20 genes coding for canonical receptor tyrosine kinases. Seventeen of the nineteen vertebrate RTK families are represented in the sea urchin. Fourteen of these RTK among which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR, ROS, RYK, TIE and TRK are present as single copy genes while pairs of related genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the subfamilies of TGF-beta ligands identified in vertebrates are present in the sea urchin genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and Lefty, as well as the TGF-beta sensu stricto that had not been characterized in invertebrates so far. Expression analysis indicates that the early expression of nodal, BMP2/4 and lefty is restricted to the oral ectoderm reflecting their role in providing positional information along the oral-aboral axis of the embryo. The coincidence between the emergence of TGF-beta-related factors such as Nodal and Lefty and the emergence of the deuterostome lineage strongly suggests that the ancestral function of Nodal could have been related to the secondary opening of the mouth which characterizes this clade, a hypothesis supported by functional data in the extant species. The sea urchin genome contains 6 genes encoding TGF-beta receptors and 4 genes encoding prototypical Smad proteins. Furthermore, most of the transcriptional activators and repressors shown to interact with Smads in vertebrates have orthologues in echinoderms. Finally, the sea urchin genome contains an almost complete repertoire of genes encoding extracellular modulators of BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN and Twisted gastrulation. Taken together, these findings indicate that the sea urchin complement of genes of the RTK and TGF-beta signaling pathways is qualitatively very similar to the repertoire present in vertebrates, and that these genes are part of the common genetool kit for intercellular signaling of deuterostomes.

The immune gene repertoire encoded in the purple sea urchin genome.

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology.

Regrowth of the stalk of the sea lily, Metacrinus rotundus (Echinodermata: Crinoidea).

Sea lilies are critical to understanding the evolution of the echinoderm body plan, because they are the only extant group whose adults possess a stalk, a prevalent feature in the radiation of a number of primitive echinoderm lineages. Extensive crown regeneration ability has been reported in Metacrinus rotundus, but the regenerative potential of the stalk has never been determined in any species of sea lilies. In this study, we show that M. rotundus whose stalks have been completely excised are capable of stalk regeneration. The process is similar to the growth of the original stalk, but much slower, and the regenerated stalks are not morphologically identical to the original stalk. Since stalk regeneration, in contrast to well-studied regeneration events, probably requires little additional activation of morphogenetic programs, we refer to the stalk regeneration phenomenon as "stalk regrowth" to distinguish it as a special form of regeneration. Since specimens whose entire stalk below the basal plates had been removed were able to regrow, the basal plates, and probably the aboral nerve center within them, are essential for stalk regrowth. Sea lily stalk regrowth is described in detail, and the evolution of feather stars is discussed in light of the growth pattern of the sea lily stalk.

Molecular heterotopy in the expression of Brachyury orthologs in order Clypeasteroida (irregular sea urchins) and order Echinoida (regular sea urchins).

The expression patterns of Brachyury (Bra) orthologs in the development of four species of sand dollars (order: Clypeasteroida), including a direct-developing species, and of a sea urchin species (order: Echinoida) were investigated during the period from blastula to the pluteus stage, with special attention paid to the relationship between the expression pattern and the mode of gastrulation. The sand dollar species shared two expression domains of the Bra orthologs with the Echinoida species, in the vegetal ring (the first domain) and the oral ectoderm (the second domain). The following heterotopic changes in the expression of the Bra genes were found among the sand dollar species and between the sand dollars and the Echinoida species. (1) The vegetal ring expressing Bra in the sand dollars was much wider and was located at a higher position along the AV axis, compared with that in the Echinoida species. The characteristic Bra expression in the vegetal ring of the sand dollar embryos was thought to be involved in the mode of gastrulation, in which involution continues from the beginning of invagination until the end of gastrulation. (2) Two of the three indirect-developing sand dollar species that were examined exhibited a third domain, in which Bra was expressed on the oral side of the archenteron. (3) In the direct-developing sand dollar embryos, Bra was expressed with an oral-aboral asymmetry in the vegetal ring and with a left-right asymmetry in the oral ectoderm. In the Echinoida species, Bra was expressed in the vestibule at the six-armed pluteus stage.

Larval stages of a living sea lily (stalked crinoid echinoderm).

The embryos and larvae of stalked crinoids, which are considered the most basal group of extant echinoderms, have not previously been described. In contrast, much is known about the development of the more accessible stalkless crinoids (feather stars), which are phylogenetically derived from stalked forms. Here we describe the development of a sea lily from fertilization to larval settlement. There are two successive larval stages: the first is a non-feeding auricularia stage with partly longitudinal ciliary bands (similar to the auricularia and bipinnaria larvae of holothurian and asteroid echinoderms, respectively); the second is a doliolaria larva with circumferential ciliary bands (similar to the earliest larval stage of stalkless crinoids). We suggest that a dipleurula-type larva is primitive for echinoderms and is the starting point for the evolution of additional larval forms within the phylum. From a wider evolutionary viewpoint, the demonstration that the most basal kind of echinoderm larva is a dipleurula is consistent with Garstang's auricularia theory for the phylogenetic origin of the chordate neural tube.