Microscopic anatomy of gonadal area in the deep-sea clam Calyptogena pacifica (Bivalvia: Vesicomyidae) with emphasis on somatic cells.

Somatic cells in the gonadal area of male and female deep-sea clams, Calyptogena pacifica, were examined using light and transmission electron microscopy. Acini both at the pre-spawning stage and at the stage of active spermatogenesis were observed to be simultaneously present in sections through a male gonad. Oocytes of various degrees of maturity were simultaneously present in female acini. No storage tissue and cells similar to adipogranular cells or vesicular connective tissue of other mollusks were found in the gonadal area of C. pacifica. Instead, in both males and females, numerous hemocytes surround the acini. Among hemocytes, two types of granulocytes and erythrocytes were identified. Bundles of muscle cells were also found in the interacinar space. Male intraacinar accessory cells were rather large, glycogen-rich, with lipid inclusions, and phagosomes contained spermatogenic cells. Female accessory cells had well-developed endoplasmic reticulum, but they did not form any follicles around oocytes, being in their basal part, closer to basal lamina. Such a specific pattern of gonad organization can presumably be explained by both symbiosis with sulfide-oxidizing bacteria and phylogenetic aspects that should be further studied. Some evidence for continuous gametogenesis are discussed.

Ultrastructural aspects of spermatogenesis in Calyptogena pacifica Dall 1891 (Vesicomyidae; Bivalvia).

The process of spermatogenesis and spermatozoon morphology was characterized from a deep-sea bivalve, Calyptogena pacifica (Vesicomyidae, Pliocardiinae), a member of the superfamily Glossoidea, using light and electron microscopy. Spermatogenesis in C. pacifica is generally similar to that in shallow-water bivalves but, the development of spermatogenic cells in this species has also some distinguishing features. First proacrosomal vesicles are observed in early spermatocytes I. Although, early appearance of proacrosomal vesicles is well known for bivalves, in C. pacifica, these vesicles are associated with electron-dense material, which is located outside the limiting membrane of the proacrosomal vesicles and disappears in late spermatids. Another feature of spermatogenesis in C. pacifica is the localization of the axoneme and flagellum development. Early spermatogenic cells lack typical flagellum, while in spermatogonia, spermatocytes, and early spermatids, the axoneme is observed in the cytoplasm. In late spermatids, the axoneme is located along the nucleus, and the flagellum is oriented anteriorly. During sperm maturation, the bent flagellum is transformed into the typical posteriorly oriented tail. Spermatozoa of C. pacifica are of ect-aqua sperm type with a bullet-like head of about 5.8 µm in length and 1.8 µm in width, consisting of a well-developed dome-shaped acrosomal complex, an elongated barrel-shaped nucleus filled with granular chromatin, and a midpiece with mainly four rounded mitochondria. A comparative analysis has shown a number of common traits in C. pacifica and Neotrapezium sublaevigatum.

Integrated assessment of the acclimation capacity of the marine bivalve Crenomytilus grayanus under naturally highly contaminated conditions: Subcellular distribution of trace metals and structural alterations of nephrocytes.

The aim of our work was to assess whether the cellular processes in the nephrocytes of the long-lived mussel Crenomytilus grayanus tend to acclimation or destruction under trace metal contamination. Mussels were collected from three sites in the north-western Pacific Ocean: reference site, upwelling site, and a site highly contaminated with trace metals. Concentration, subcellular distribution of trace metals (Cd, Cu, Zn, and Pb) in the mussel kidneys, and ultrastructural alterations of the nephrocytes were studied. To assess the total load of accumulated trace metals, the total concentration coefficient (∑СС) was determined. In the kidneys of the reference C. grayanus, trace metals were eliminated from cell metabolism mainly by lysosomal granules or residue bodies. Under high levels of contamination, the defense mechanisms of C. grayanus are practically suppressed (no metallothionein-like protein peak, decreased content of granules) by the total effect of accumulated pollutants that leads to the destruction of cellular structures. Under natural conditions (upwelling site), increased accumulation of trace metals in the mussel kidneys did not lead to an increase in the number or size of lysosomal granules. However, abnormal high Cd accumulation in the kidneys caused the synthesis of high levels of metallothionein-like proteins that sequester most of the studied trace metals. To quickly lower the metal levels in nephrocytes under these conditions, a unique long-term acclimatory response - apocrine-like secretion in nephrocytes, which provides rapid elimination of me-MTLP complexes from the cell arose. Thus, our integrated study of the subcellular distribution of trace metals and ultrastructural alterations in nephrocytes allowed us to characterize the features of the structural and functional alterations in mussel cells under the field conditions tested.

Spermatozoa ultrastructure of two basal pilidiophoran nemerteans, Hubrechtella juliae and Sonnenemertes cantelli (Nemertea, Pilidiophora).

Nemertea is a phylum of worms with a simple internal morphology; nemerteans' spermatozoon morphology can be used for their classification and phylogenetic analyses. The aim of the present study was to describe spermatozoa of the nemerteans Hubrechtella juliae and Sonnenemertes cantelli from the basal groups of the class Pilidiophora at the ultrastructure level. Both species have primitive ('compact-head' sensu Stricker and Folsom, 1998) spermatozoa with ovoid head and five mitochondria in the midpiece, but differ in the structure of acrosomal complex: in Hubrechtella juliae, the single lens-shaped acrosomal vesicle contains an area of moderate electron density not enclosed by a separate membrane; in Sonnenemertes cantelli, the acrosome shows a unique morphology and contains a few electron-dense vesicles with irregular shapes and positions and one more electron-lucent elongated vesicle. Such a pattern of the acrosomal complex organization is described for Nemertea for the first time. An assumption is made that the states "two or more mitochondria" and "posterior acrosomal ring component" may be synapomorphies of Hubrechtiiformes+Heteronemertea (class Pilidiophora), whereas "the posterior margin of the acrosomes forms an acrosomal ring component" is presumably an autapomorphy of the family Lineidae s.l. The results suggest that spermatozoa provide a useful source of characters for nemertean systematics.

Autophagy in nutrient storage cells of the Pacific oyster, Crassostrea gigas.

In oysters, nutrients are stored in a special type of cells referred to as vesicular-connective tissue cells (VCT-cells). These cells accumulate and provide nutrient to satisfy various needs of the organism, including gametogenesis. During the annual reproductive cycle, VCT-cells pass through a series of changes in their morphology associated with nutrients mobilization for developing germ cells. The results presented here show an approximately 33-35% increase in the number of autophagic vesicles in cytoplasm of VCT-cells in the gonadal area of C. gigas during the stage of active gametogenesis as compared to the resting stage of reproductive cycle. No destruction of VCT-cells due to autophagy or any other factors was observed, both in males and females. Our results indicate that autophagy does increase in VCT-cells of C. gigas and plays a certain role in nutrient mobilization from these cells.

Peripheral sensory neurons govern development of the nervous system in bivalve larvae.

Recent findings regarding early lophotrochozoan development have altered the conventional model of neurogenesis and revealed that peripheral sensory elements play a key role in the initial organization of the larval nervous system. Here, we describe the main neurogenetic events in bivalve mollusks in comparison with other Lophotrochozoa, emphasizing a novel role for early neurons in establishing larval nervous systems and speculating about the morphogenetic function of the apical organ. We demonstrate that during bivalve development, peripheral sensory neurons utilizing various transmitters differentiate before the apical organ emerges. The first neurons and their neurites serve as a scaffold for the development of the nervous system. During veliger stage, cerebral, pleural, and visceral ganglia form along the lateral (visceral) nerve cords in anterior-to-posterior axis. The pedal ganglia and corresponding ventral (pedal) nerve cords develop much later, after larval settlement and metamorphosis. Pharmacological abolishment of the serotonin gradient within the larval body disrupts the navigation of "pioneer" axons resulting in malformation of the whole nervous system architecture. Comparative morphological data on neurogenetic events in bivalve mollusks shed new light on the origin of the nervous system, mechanisms of early axon navigation, and sequence of the tetraneurous nervous system formation. Furthermore, this information improves our understanding of the basic nervous system architecture in larval Bivalvia and Mollusca.

Morphology of nutrient storage cells in the gonadal area of the Pacific oyster, Crassostrea gigas (Thunberg, 1793).

Successful gametogenesis in invertebrates is tightly associated with functioning of specific nutrient-storing cells. In oysters, cells of vesicular connective tissue (VCT-cells), also referred to as storage cells, which form a meshwork around gonadal acini, are the major population of cells that accumulate and provide nutrients for developing gametes. During the annual reproductive cycle, populations of developing germ cells and VCT-cells demonstrate the inversely proportional size dynamics: the larger the acini, the smaller the VCT-cells. In the present study, the morphology of VCT-cells in the Pacific oyster, Crassostrea gigas, at the active gametogenesis stage of reproductive cycle has been studied using light and transmission electron microscopy. At this stage, VCT-cells are big, irregularly shaped cells containing large nucleus with a single large nucleolus. The cytoplasm contains weakly developed endoplasmic reticulum, mitochondria in the perinuclear area and at periphery of the cell, numerous lipid droplets, and glycogen particles. Ultrastructure of VCT-cells is similar to the organization of brown adipocytes in mammals. The surface of cells has numerous cytoplasmic processes that are presumably associated with the transport function and provide close interaction with adjacent cells. The spatial relationship between VCT-cells and myoepithelial elements of the gonad area is demonstrated and discussed.

Macroautophagy is involved in residual bodies formation during spermatogenesis in sea urchins, Strongylocentrotus intermedius.

An ultrastructural study of developing spermatids in sea urchins, Strongylocentrotus intermedius, showed that macroautophagy is involved in formation of residual bodies and removal of excessive cytoplasm by spermatids during spermatogenesis in this species. During late stages of spermatogenesis spermatids sequester excessive cytoplasm into vesicles, surrounded by a double membrane. Subsequently, these vesicles fused to one another into larger vacuoles, up to 1.5 µm in diameter. Finally, the vacuoles transformed into residual bodies by condensing their content into finely granular material of varying electron density, separated from cytoplasm by a single membrane. An immunoelectron microscopic study of late spermatids with the antibodies, raised against microtubule-associated protein 1 A/1B-light chain 3 (LC3), which is a marker of autophagosomes, showed that residual bodies in late spermatids of S. intermedius were LC3-positive.

Fine structure of the gametes and spermiogenesis in Spiophanes uschakowi (Annelida: Spionidae) from the Sea of Japan, with comments on fertilization biology in broadcast-spawning spionids.

Spiophanes uschakowi is a common polychaete living in tubes in sandy sediments in shallow waters of the Sea of Japan. Females and males release their gametes into the water where fertilization and holopelagic, planktotrophic larval development occur. In females, oogenesis is intraovarian: vitellogenesis occurs when the oocytes grow in paired ovaries attached to genital blood vessels in fertile segments. The developed oocytes are accumulated in the coelomic cavity prior to spawning. The newly released oocytes are lentiform, 185-200 µm in diameter, with honey-combed envelopes 5-7 µm thick. Each oocyte has 41-49 cortical alveoli regularly arranged in a peripheral circle, a nucleus 80-83 µm in diameter, and a single nucleolus about 30 µm in diameter. In males, spermatogonia proliferate in testes and the rest of spermatogenesis occurs in the coelomic cavity. During spermiogenesis, the acrosomal vesicle migrates from the posterior to the anterior part of the spermatid. The spermatozoa are ect-aquasperm with a plate-like acrosome 0.58 ± 0.06 µm thick and 2.14 ± 0.13 µm in diameter, barrel-shaped nucleus 2.23 ± 0.13 µm long and 3.18 ± 0.13 µm in diameter, short midpiece 0.93 ± 0.09 µm long with five spherical mitochondria, two centrioles and one small lipid droplet, and a flagellum 62-63 µm long with 9 × 2 + 2 organization of microtubules. The acrosome is a complex heterogeneous structure with 4-6 subspherical apical bodies, and numerous small branched basal cisternae. The anterior end of the nucleus is truncate, while its posterior end has wide shallow depressions accommodating the mitochondria. The centrioles are situated in the center of the midpiece between mitochondria and oriented obliquely to each other. The structure of the gametes of broadcast-spawning spionids is reviewed and the roles of surface granules in species-specific attraction of sperm toward eggs by releasing chemical signals (sperm chemotaxis), and cortical alveoli as a place of penetration of spermatozoa into oocytes (micropyle) are suggested. The lentiform oocytes of Spiophanes spp. are unique among Spionidae by their shape, while spermatozoa are unique by their plate-like acrosomes.

Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia).

BACKGROUND: Bivalves comprise a large, highly diverse taxon of invertebrate species. Developmental studies of neurogenesis among species of Bivalvia are limited. Due to a lack of neurogenesis information, it is difficult to infer a ground pattern for Bivalvia. To provide more comprehensive morphogenetic data on bivalve molluscs and relationships among molluscan clades, we investigated neurogenesis in the Pacific oyster, Crassostrea gigas, from the appearance of the first sensory cells to the formation of the larval ganglionic nervous system by co-immunocytochemistry of the neuronal markers FMRFamide or 5-HT and vesicular acetylcholine transporter (VAChT). RESULTS: Neurogenesis begins with the emergence of the apical serotonin-immunoreactive (5-HT-ir) sensory cells and paired sensory posttrochal dorsal and ventral FMRFamide-immunoreactive (FMRFamide-ir) cells at the early trochophore stage. Later, at the early veliger stage, the apical organ (AO) includes 5-HT-ir, FMRFamide-ir, and VAChT-ir cells. At the same stage, VAChT-ir cells appear in the posterior region of larvae and send axons towards the AO. Thus, FMRFamide-ir neurites and VAChT-ir processes form scaffolds for longitudinal neurite bundles develop into the paired ventral nerve cords (VNC). Later-appearing axons from the AO/CG neurons join the neurite bundles comprising the VNC. All larval ganglia appear along the VNC as paired or fused (epiathroid) clusters in late veliger and pediveliger larvae. We observed the transformation of the AO into the cerebral ganglia, which abundantly innervated the velum, and the transformation of ventral neurons into the pedal ganglia, innervating the foot, gills, and anterior adductor muscle. The visceral ganglia appear last in the pediveliger oyster and innervate the visceral mass and posterior adductor of premetamorphic larvae. In addition, a local FMRFamide-ir network was detected in the digestive system of pediveliger larvae. We identified VAChT-ir nervous elements in oyster larvae, which have not been observed previously in molluscs. Finally, we performed a morphology-based comparative analysis of neuronal structures among bivalve, conchiferan, and aculiferan species. CONCLUSIONS: We described the development of the nervous system during the larval development in Crassostrea gigas. These data greatly advance the currently limited understanding of neurodevelopment in bivalves and mollusks, which has hampered the generation of a ground pattern reconstruction of the last common ancestor of Mollusca. Our morphological data support phylogenomic data indicating a closer Bivalvia-Gastropoda sister group relationship than the Bivalvia-Scaphopoda (Diasoma) group relationship.

Microautophagy in nutritive phagocytes of sea urchins.

Two types of cells were observed in germinative epithelium of male and female sea urchins: germ cells and somatic accessory cells; the latter referred to as nutritive phagocytes. At the onset of gametogenesis, nutritive phagocytes accumulate nutrients and greatly increase in their size. As gametogenesis progresses, the accumulated nutrients are transferred from nutritive phagocytes into developing gametes, and size of the nutritive phagocytes decreases. An electron microscopic study of nutritive phagocytes in sea urchins, Strongylocentrotus intermedius, at different stages of annual reproductive cycle showed for the first time that both macro- and microautophagy take place in nutritive phagocytes. Both processes occur simultaneously and regulate size and composition of nutritive phagocytes in male and female sea urchins. Nutritive phagocytes consume redundant cytoplasm via macroautophagy. Microautophagy is probably involved in consumption of redundant membranes that appear within nutritive phagocytes due to destruction of nutrient-storing globules, macroautophagy, and phagocytosis of germ cells or their remnants.

Spermatogenesis and spermatozoa ultrastructure of two Dipolydora species (Annelida: Spionidae) from the Sea of Japan.

Spermatogenesis and the structure of the spermatozoa of two spionid polychaetes Dipolydora bidentata and Dipolydora carunculata are described by light and transmission electron microscopy. Both species are gonochoristic borers in shells of various molluscs. Proliferation of spermatogonia occurs in paired testes regularly arranged in fertile segments, and the rest of spermatogenesis occurs in the coelomic cavity. Early spermatogenesis occurs quite similarly in the two species but results in formation of tetrads of interconnected spermatids in D. bidentata and octads of spermatids in D. carunculata. Three consecutive stages of spermiogenesis are recognized according to the condensation of chromatin in nucleus: (1) early spermatids with heterogeneous, partly clumped chromatin, (2) middle spermatids with homogeneous, coarsely granular chromatin, and (3) late spermatids with homogeneous fibrillar chromatin. Moreover, late stage of spermatids is further classified into two stages, I and II, according to the position of the acrosome and shape of the nucleus. In late spermatids I, the acrosome is situated in the anterior invagination of the funnel-shaped to oval nucleus, whereas in late spermatids II the acrosome is situated on top of the elongated nucleus. Ultrastructural composition of cells at each stage of spermatogenesis is described and illustrated. The possible process of morphogenesis of organelles during spermato- and spermiogenesis is reconstructed for both species. The proacrosomal vesicle first appears in early spermatids near the Golgi complex and then migrates anteriorly; in the middle spermatids, the acrosome comes to lie in a deep anterior nuclear fossa. In late spermatids I, this fossa evaginates and a posterior fossa develops in the nucleus housing basal body and the anterior part of the axoneme. In late spermatids II, the mitochondria elongate and probably reduce in number due to fusion of some of them. The mature spermatozoa in both species are introsperm with the conical acrosome, subacrosomal plate, long nucleus with short posterior fossa, long midpiece with elongated mitochondria, and long flagellum with 9×2+2 organization of microtubules. Numerous flat rounded platelets with putative glycogen are present throughout most part of the nucleus and the midpiece. The process of spermatogenesis in D. bidentata and D. carunculata is similar to that in other Dipolydora, Polydora and Pseudopolydora species. Spermatozoa in these polydorin spionids have similar composition and differ mainly in size of the nucleus and the midpiece. Elongated spermatozoa are adapted for transfer in spermatophores and an internal fertilization which is characteristic for brooding species. Diversely modified spermatozoa among spionids may be signs of the diversity of fertilization biology within the Spionidae. The exact places where fertilization occurs in brooding spionids however remains unknown.

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast.

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 µm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 µm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.

MicroRNA-mediated drug resistance in breast cancer.

Chemoresistance is one of the major hurdles to overcome for the successful treatment of breast cancer. At present, there are several mechanisms proposed to explain drug resistance to chemotherapeutic agents, including decreased intracellular drug concentrations, mediated by drug transporters and metabolic enzymes; impaired cellular responses that affect cell cycle arrest, apoptosis, and DNA repair; the induction of signaling pathways that promote the progression of cancer cell populations; perturbations in DNA methylation and histone modifications; and alterations in the availability of drug targets. Both genetic and epigenetic theories have been put forward to explain the mechanisms of drug resistance. Recently, a small non-coding class of RNAs, known as microRNAs, has been identified as master regulators of key genes implicated in mechanisms of chemoresistance. This article reviews the role of microRNAs in regulating chemoresistance and highlights potential therapeutic targets for reversing miRNA-mediated drug resistance. In the future, microRNA-based treatments, in combination with traditional chemotherapy, may be a new strategy for the clinical management of drug-resistant breast cancers.

Morphology of spermatogenic and accessory cells in the mussel Modiolus kurilensis under environmental pollution.

A comparative light- and electron microscopic study of the male gonads of the bivalve mollusk Modiolus kurilensis from the reference and polluted sites in Amursky Bay (Sea of Japan) was conducted. Testicular acini in the mussels from the reference site had well-ordered structure (vertical spermatogenic columns located among the accessory cells bodies) whereas in the testes of the mollusks from the polluted site, the accessory and spermatogenic cell populations were disarranged. Mussels from the polluted station had about 26% of spermatogenic cells with marginal localization of nuclear chromatin, swollen outer nuclear membrane and heavily vacuolated cytoplasm and about 8% of spermatozoa with transformed or destructed acrosome; in mussels from the reference station, these values were close to zero. The accessory cells in the mussels from the polluted site were underdeveloped, and their phagocytic activity was inhibited. Our ultrastructural observations provide evidence that both spermatogenic and accessory cells are targets of environmental pollution in marine mussels.

Role of DNA hypomethylation in the development of the resistance to doxorubicin in human MCF-7 breast adenocarcinoma cells.

The resistance of cancer cells to chemotherapeutic agents is a major clinical problem and an important cause of treatment failure in cancer. Mechanisms that have developed to guard cancer cells against anti-cancer drugs are major barriers to successful anti-cancer therapy. Therefore, the identification of novel mechanisms of cellular resistance holds the promise of leading to better treatments for cancer patients. In the present study, we used human MCF-7 breast adenocarcinoma cell line and its doxorubicin-resistant variant MCF-7/R to determine the role of alterations of DNA methylation of chemoresitance-related genes, such as multidrug resistance 1 (MDR1), glutathione-S-transferase (GSTpi), O(6)-methylguanine DNA methyltransferase (MGMT), and urokinase (Upa), in the development of drug resistance. The promoter regions of MDR1, GSTpi, MGMT, and Upa genes were highly methylated in MCF-7 cell line but not in its MCF-7/R drug resistant variant. The hypomethylated status of MDR1 gene was associated with overexpression of P-glycoprotein. We hypothesize that acquirement of doxorubicin resistance of MCF-7 cells is associated with DNA hypomethylation of the promoter regions of the MDR1, GSTpi, MGMT, and Upa genes.

The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells.

The CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways.

Selective resorption in nutritive phagocytes of the sea urchin Anthocidaris crassispina.

Phagocytic resorption during spermatogenesis was studied in the sea urchin Anthocidaris crassispina. Nutritive phagocytes in gonad absorbed both waste sperm cells and residual bodies discarded from maturing spermatids, and these materials were subsequently compartmented in heterophagosomes. Based on 180 heterophagosomes examined by transmission electron microscopy, over 99% of heterophagosomes contained either residual bodies or sperm cells only. Simultaneous resorption of sperm cells and residual bodies in a heterophagosome was uncommon, with only approximately 0.56% occurrence, suggesting that heterophagosomes have a selective resorption ability in nutritive phagocytes.

Sublethal effects of phenol on spermatogenesis in sea urchins (Anthocidaris crassispina).

Adult sea urchins, Anthocidaris crassispina, were exposed to 0.1 and 10 mgL(-1) phenol for 4 weeks. Abnormal sperm development was clearly evident in phenol-treated sea urchins, although no mortality was found throughout the exposure period. Occurrences of multinucleate sperm cells (including spermatocytes to spermatozoa) showed a significant increase from 0.07% in the control to 10.7% and 43.3% in the 0.1- and 10-mgL(-1) treatments, respectively (P<0.01). Likewise, sperm with electron dense, dark tails increased significantly from 8% in the control to 36.6% and 43.4% in the 0.1- and 10-mgL(-1) phenol-treated sea urchins, respectively (P<0.01). In addition, disruption of cytoplasmic membranous structures such as loss of mitochondrial cristae and distortion of mitochondrial membranes and nucleus envelope were commonly found in phenol-treated spermatogonia and spermatocytes. Previous studies have clearly demonstrated motility impairment and a concomitant reduction of fertilization capability in sea urchin sperm with dark tails and/or distorted mitochondria. Our current findings therefore suggest that chronic exposure to phenol at 0.1 mgL(-1) could lower the quality of sperm and reproductive success in sea urchins, which may threaten the survival of these ecologically important species. The observed impairment of spermatogenesis by phenol might also occur in other invertebrate species.