Ultrastructural and cytochemical aspects of the female gonad of Geoplana burmeisteri (Platyhelminthes, Tricladida, Terricola)

The ultrastructure of the female gonad of the land planarian Geoplana burmeisteri was investigated by means of electron microscopy and cytochemical techniques. It consists of two small germaria located ventral to the intestine and of two irregular, lateral rows of vitelline follicles, both enveloped by a tunica composed of an extracellular lamina and an inner sheath of accessory cells. Accessory cell projections completely surround developing oocytes and vitellocytes. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of the rough endoplasmic reticulum (RER) and Golgi complexes. These organelles appear to be correlated with the production of egg inclusions of medium electron density, about 1.5-1.8 microm in diameter, which remain scattered in the ooplasm of mature oocytes. On the basis of cytochemical tests demonstrating their glycoprotein composition, these inclusions were interpreted as residual yolk globules. Vitellocytes are typical secretory cells with well-developed RER and Golgi complexes that are mainly involved in the production of yolk globules and eggshell globules, respectively. Eggshell globules appear to arise from repeated coalescence of small Golgi-derived vesicles and, at an intermediate stage of maturation, show a multigranular pattern. Later, after vesicle fusion, they reach a diameter of 1.3-1.6 microm when completely mature and show a meandering/concentric pattern, as is typical of the situation seen in most Proseriata and Tricladida. The content of yolk globules is completely digested by pronase, while the content of eggshell globules is unaffected. Mature vitellocytes contain, in addition, a large quantity of glycogen and lipid droplets as further reserve material. On the basis of the ultrastructural characteristics of the female gonad described above and in relation to the current literature, we conclude that G. burmeisteri appears to be more closely related to the freshwater triclads, in particular to members of the Dugesiidae, than to the marine triclads.     

An ultrastructural study of oogenesis in the planarian Schmidtea mediterranea (Platyhelminthe, Paludicola)

The ovary of the freshwater planarian Schmidtea mediterranea has been studied for the first time using both light and electron microscopy methods. The ultrastructure of the ovary revealed two types of cells: accessory cells and germinal cells at various stages of differentiation, distributed along a maturation axis. Initially, oogonia underwent cytoplasm growth due to the development of organelles, such as endoplasmic reticulum, Golgi complex, and mitochondria, which are all involved in the production of cytoplasmic inclusions or yolk globules. It is shown that the chromatoid body and fibrogranular aggregates may participate in the synthesis of vitelline inclusions. When completely mature, the oocytes have become larger, due to the accumulation of nutritive inclusions, which are round in shape and have a paracrystalline structure. These inclusions are interpreted as being yolk globules and may represent a kind of nutritive material for the developing embryo. These ultrastructural features of the ovary agree with the available phylogenetic tree, based on morphological and karyological characters that considers Schmidtea group as a genus and not a subgenus. The presence of sperm between the oocytes suggests that fertilization may occur within the ovary, representing an uncommon condition within the Triclads, in which fertilization usually takes places outside of the ovaries.     

Anatomy and ultrastructure of the female reproductive system of Pleioplana atomata (Platyhelminthes: Polycladida)

The ultrastructure of the female reproductive system of the polyclad flatworm Pleioplana atomata is described. Numerous ovaries are scattered throughout the entire body but are mainly concentrated on the dorsal side. Within an ovary, a germinative zone with oogonia and prefolicular cells is located in the dorsal part of the ovary. The remaining part of the gonad is filled with previtellogenic and early vitellogenic oocytes enwrapped by follicular cells. During previtellogenesis, oocytes produce numerous eggshell globules, which are distributed into the cortical area of the cell in later stages. Eventually, these globules release their contents into the space between the eggshell cover and oolemma. Similar types of globules are also found in others flatworms, and may represent useful phylogenetic characters. Entolecital, vitellogenic oocytes pass to paired uteri, where vitellogenesis is completed. The remainder of the female reproductive system consists of paired thin uterine ducts that join a vagina. The distal part of the long, curved vagina forms a large Lang's vesicle, while the proximal part is connected to a female atrium leading to a female gonopore. We hypothesize that Lang's vesicle functions in the digestion of excess sperm received. Two kinds of different shell (cement) glands that release their secretion into the vagina are identified. Both are unicellular glands and each gland cell connects to the lumen of the vagina via an individual canal. Similar glands in other acotylean polyclads have been implicated in the formation of eggshell covers. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

An ultrastructural study of oogenesis in a marine triclad

The ultrastructural features of oocyte differentiation were studied in the marine triclad Cercyra hastata. Oocytes at several stages of maturation, each surrounded by follicle cell projections, are present within each of the two ovaries. A pre-vitellogenic and a vitellogenic stage have been detected in the oogenesis of C. hastata. The pre-vitellogenic stage is mainly characterized by an increase in the nuclear and nucleolar volume and activity, and the appearance and development of cortical granule precursors which are elaborated by the Golgi complex. In early phases of the vitellogenic stage, intense delamination and blebbing of the nuclear envelope occurs which probably contributes to an increase in number of cytoplasmic membranes and to transfer of nuclear material to the cytoplasm. The rough endoplasmic reticulum is extensively developed and often assumes a ‘whorl’ array. Several areas of yolk precursor formation appear in the whorls. Numerous 2–5 μm protein yolk globules are subsequently formed which appear surrounded by a double membrane (cisternae of the smooth endoplasmic reticulum) and become randomly distributed throughout the cytoplasm of mature oocytes. The peripheral ooplasm is occupied by a monolayer of electron-dense cortical granules. Finally, the evolutionary significance of the autosynthetic mechanism of yolk production is discussed.     

Occurrence of Proteinaceous 10-nm Filaments throughout the Cytoplasm of Algae of the Order Dasycladales

Previously, whole-mount electron microscopy of nuclei extruded together with residual cytoplasm from the rhizoids of several algal species of the order Dasycladales has revealed the occurrence of an intra- and perinuclear network of 10-nm filaments morphologically indistinguishable from that of mammalian vimentin intermediate filaments. The present investigation demonstrates the existence of a filament system throughout the cytoplasm of the rhizoid, stalk, and apical tip of these giant cells. However, while the perinuclear 10-nm filaments interconnecting the nuclear surface with a perinuclear layer of large, electron-dense bodies filled with nucleoprotein material are of smooth appearance, those continuing within and beyond the perinuclear bodies are densely covered with differently sized, globular structures and, therefore, are of a very rough appearance. The filaments in the very apical tip of the cells are mainly of the smooth type. The transition from smooth to rough filaments seems to occur in the numerous perinuclear dense bodies surrounding the large nucleus. Digestion of the rough filaments with proteinase K removes the globules from the filament surface, revealing that throughout the nonvacuolar, intracellular space the filaments have the same basic 10-nm structure. On the other hand, gold-conjugated RNase A strongly binds to the filament-attached globules but not to the smooth, perinuclear, and the proteinase K-treated, rough filaments. In addition, an antibody raised against Xp54, a highly conserved protein which in Xenopus oocytes is an integral component of stored mRNP particles, decorates the rough but not the smooth 10-nm filaments. These results support the notion that the 10-nm filament system of Dasycladales cells plays a role in the transient storage of ribonucleoprotein particles in the cytoplasm and possibly fulfils a supportive function in the actomyosin-based transport of such material to various cytological destinations.     

Oogenesis in pig ovaries during the prenatal period: ultrastructure and morphometry

Oogenesis in fetal pig ovaries comprises the successive changes from the primordial germ cells to the dictyotene oocytes in primordial ovarian follicles. In this study the observations were carried out with an electron microscope and stereological analysis was performed. At the ultrastructural level there are no differences between the primordial germ cells and oogonia, but oogonia are connected with the intercellular bridges. The onset of the dictyotene phase was accompanied by the changes in the cytoplasm of oocytes. Near the nucleus, the yolk nucleus is formed containing numerous Golgi bodies, endoplasmic reticulum (ER), mitochondria and granules. ER proliferates in contact with the external leaflet of the nuclear envelope forming the narrow ER cisterns. Between the nuclear envelope and ER cisterns, the vesicles with grey content are visible. The proliferating ER forms numerous concentric cisterns around the nucleus. Next, the most external cisterns fragment, detach, and then form the cup-like structures. These structures separate the distinct areas of cytoplasm-compartments, which contain mitochondria, ribosomes and lipid droplets. The cells of cortical sex cords of the ovary, which encloses the oocyte, form the follicles. The volume of oocytes in forming follicle increases due to the increase in the number of the cell inclusions: lipid droplets, vacuoles and yolk globules. In the oocytes of primordial ovarian follicles, the compartments are transformed into the yolk globules, which are encountered by a sheath of ER cisterns and the grey vesicles; they contain the mitochondria, lipid droplets and light vacuoles. The role of the compartments and yolk globules as metabolic units is discussed in comparison with similar structures of the mature eggs of pigs and other mammal species.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphonidae). I. Ovary structure and previtellogenic growth of oocytes

Glossiphonia heteroclita has paired ovaries whose shape and dimensions change as oogenesis proceeds: during early previtellogenesis they are small and club-shaped, whereas during vitellogenesis they broaden and elongate considerably. During early oogenesis (previtellogenesis), each ovary is composed of an outer envelope (ovisac) that surrounds the ovary cavity and is filled with hemocoelomic fluid, in which a single and very convoluted ovary cord is bathed. The ovary cord consists of germline cells, including nurse cells and young oocytes surrounded by a layer of elongated follicle cells. Additionally, follicle cells with long cytoplasmic projections occur inside the ovary cord, where they separate germ cells from each other. The ovary cord contains thousands of nurse cells. Each nurse cell has one intercellular bridge, connecting it to a central anucleate cytoplasmic mass, the cytophore (rachis); it in turn is connected by one intercellular bridge with each growing oocyte. Numerous mitochondria, RER cisternae, ribosomes, and Golgi complexes are transported from the nurse cells, via the intercellular bridge and cytophore, to the growing oocytes. Oogenesis in G. heteroclita is synchronous with all oocytes in the ovary in the same stage of oogenesis. The youngest observed oocytes are slightly larger than nurse cells, and usually occupy the periphery of the ovary cord. As previtellogenesis proceeds, the oocytes gather a vast amount of cell organelles and become more voluminous. As a result, in late previtellogenesis the oocytes gradually protrude into the ovary cavity. Simultaneously with oocyte growth, the follicle cells differentiate into two subpopulations. The morphology of the follicle cells surrounding the nurse cells and penetrating the ovary cord does not change, whereas those enveloping the growing oocytes become more voluminous. Their plasma membrane invaginates deeply, forming numerous broad vesicles that eventually seem to form channels or conducts through which the hemocoelomic fluid can easily access the growing oocytes.     

A complex cortical reaction leads to formation of the fertilization envelope in the lobster,Homarus

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron-dense granules and structures resembling “bottlebrushes.” At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in-vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring-shaped elements of the RV and the moderately electron-dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.     

Ultrastructural and cytochemical studies of the female gonad of Prorhynchus sp. (Platyhelminthes,Lecithoepitheliata)

The female gonad of Prorhynchus is heterocellular (neoophoran organization) and consists of an unpaired, elongate germovitellarium enveloped by a finely granular extracellular lamina. It is composed of a posterior germinative area where early oocytes are randomly associated with differentiating vitellocytes and a growth area with follicular organization. In each follicle a single oocyte is surrounded by a layer of vitellocytes. By electron microscopy, the oocytes showed features typical of non-vitellogenic germ cells; they had chromatoid bodies, annulate lamellae, lipid droplets and R.E.R. and Golgi complexes producing small granules with a multilamellar pattern. Vitellocytes showed features typical of secretory cells with the R.E.R. and Golgi complex developed to a great extent and involved in the production of type A and type B globules, respectively. We speculate that type A globules are shell-globules and type B globules are yolk. The structure, composition and role of vitellocyte globules of Prorhynchus are compared with those of homologous inclusions from other Platyhelminthes.Abbreviations A type A globule - B type B globule - ECL extracellular lamina - GC Golgi complex - L lipid - RER rough endoplasmic reticulum - O oocyte - V vitellocyte     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

THE ORIGIN OF PROTEIN AND FATTY YOLK IN RANA PIPIENS : I. Phase Microscopy

Study of living frog oocytes with the phase microscope has shown that the early yolk appears in two forms. One of these, the protein yolk, consists of thin, dense, plate-like bodies which in face view are almost always regular hexagons. The other form, the fatty yolk, occurs as clusters of globules of varying sizes. The plate-like bodies occur both singly and in clusters. As the oocytes mature these plate-like bodies grow in size while retaining their hexagonal outline. Mitochondria have been observed to increase in length and numbers as the oocytes mature; they are rods or filaments at all stages of growth up to an oocyte diameter of 300 microns. The oocyte cytoplasm gradually becomes packed with long mitochondria, plate-like bodies, and clusters of globules.     

Morphological differences in nuclear materials released from hamster sperm heads at an early stage of incorporation into immature oocytes,mature oocytes,or fertilized eggs

To elucidate the effects of ooplasmic factors on the early morphological changes in hamster sperm heads within the ooplasm, immature ovarian oocytes at the germinal vesicle stage (GV oocytes), ovulated fully mature oocytes, and fertilized eggs at anaphase II or the pronuclear stage (PN eggs) were examined in detail 15–30 min after insemination or reinsemination. Thin-sectioning studies demonstrated distinct materials released from the sperm nucleus over the entire postacrosomal nuclear surface immediately after disappearance of the sperm nuclear envelope. The release occurred in all of the oocytes and eggs prior to or even in the absence of subsequent chromatin decondensation. Depending upon the stage of the penetrated oocyte or egg, however, the materials varied in morphology: several hemispherical projections of amorphous material within mature oocytes; a number of electron-dense globules within GV oocytes and PN eggs; and both forms within eggs at anaphase II-telophase II. These observations and the fact that only the release of the amorphous material was accompanied by sperm chromatin decondensation indicate that this release was the initial process of chromatin decondensation, whereas the release of the globules resulted from a deficiency or lack of ooplasmic factors affecting the sperm nucleus. Restriction of the release in both forms of material to the late meiotic phase suggests changes in the factors associated with progression of meiosis. To approach an understanding of the mechanism of successful decondensation of sperm chromatin, the ooplasmic factors considered responsible for the stage-dependent release of nuclear materials are discussed. © 1996 Wiley-Liss, Inc.     

Ultrastructural and cytochemical studies on the germarium of Dicrocoelium dendriticum (Plathelminthes,Digenea)

Summary The unpaired germarium of Dicrocoelium dendriticum contains many female germ cells at different stages of maturation and is enveloped by a fibrous basal lamina-like structure and a multilayered cytoplasmic sheath whose origins and functions are discussed. The maturation process of primary oocytes occurs completely within the prophase of the first meiotic division. It has been divided into three stages, as previously suggested for monogeneans. Stage I corresponds to oogonia and early oocytes which are located in the distal germinative area of the gonad. These cells are characterized by a high nucleo/cytoplasmic ratio and a poorly differentiated cytoplasm. Stage II corresponds to maturing oocytes grouped in the central area of the gonad and exhibiting long synaptonemal complexes and a prominent nucleolus. The main feature of cytoplasmic differentiation is the increase in the number of RER and Golgi complex which are involved in the production of small electron-dense granules. Stage III corresponds to mature oocytes located in the proximal area of the germarium near the origin of the oviduct. In this stage, the granules become regularly distributed in a monolayer in the peripheral ooplasm and make contact with the oolemma. They show a distinctive complex structure, are composed of proteins and glycoproteins and do not contain polyphenols. Their possible role as cortical granules is discussed in relation to chemical composition and previous studies on other Plathelminthes. Neither yolk globules nor glycogen are present in the oocytes.Abbreviations I oogonium and early oocyte - II growing oocyte - III mature oocyte - cg cortical granule - cs cytoplasmic sheath - db dense body - ecm extra cellular matrix - ER endoplasmic reticulum - fl fibrous extracellular layer - gc Golgi complex - m mitochondria - N nucleus - nu nucleolus - RER rough endoplasmic reticulum - sc synaptonemal complex     

Ultrastructure of somatic tissues in the ovaries of a chaetognath (Ferosagitta hispida)

Transmission electron microscopy reveals that the ovaries of Ferosagitta hispida contain four somatic tissues. A myoepithelial ovary wall, continuous with a thin layer of peritoneocytes lining the coelomic cavity, encloses a fluid-filled ovarian space in which oocytes develop. Lamellar extensions of a “follicular reticulum” branch throughout the ovarian space and ensheath developing oocytes. This tissue has been overlooked in most previous studies of chaetognath ovaries. A bipartite oviductal complex extends the length of each ovary just within the lateral ovary wall. It consists of a flattened, blindly ending cellular tube, herein referred to as the cellular sheath, and an enclosed syncytium. Sheath cells secrete an electron-dense product into the ovarian space. Those sheath cells directly bordering the syncytium are contractile and are joined to the to the syncytium by gap junctions and microvillar interdigitations. The syncytium contains a complex of membrane-bounded lumina. The latter sometimes enclose sperm received during mating or ovulated eggs. Thus the syncytium serves both as a seminal receptacle and as a duct for passage of eggs to the outside. Contrary to several classical reports, the cellular sheath and syncytium of the oviductal complex do not separate at ovulation to form a temporary oviductal lumen.     

Histochemical observations on lipid changes during oogenesis in the poultry nematode,Ascaridia galli

During oogenesis lipids continue to increase in Ascaridia galli leading to the formation of massive accumulations in larger oocytes. In addition to neutral lipids (triacylglycerols), small amounts of cholesterol and granules containing phospholipids and lipoproteins are seen in the ooplasm of growing and mature oocytes. Lipid inclusions of oogonia which are in the form of coarse granules show a complex structure in young oocytes and form droplets and globules in growing and mature oocytes. Changes in the morphology of the oocytes are accompanied by changes in the distribution of lipids within the oocytes. Localization of lipids in rachis and ovarian epithelium along the length of the ovary is also described.     

Sarcocystis neurona (Protozoa: Apicomplexa): description of oocysts, sporocysts, sporozoites, excystation, and early development

Equine protozoal myeloencephalitis is a major cause of neurological disease in horses from the Americas. Horses are considered accidental intermediate hosts. The structure of sporocysts of the causative agent, Sarcocystis neurona, has never been described. Sporocysts of S. neurona were obtained from the intestines of a laboratory-raised opossum fed skeletal muscles from a raccoon that had been fed sporocysts. Sporocysts were 11.3 by 8.2 microm and contained 4 sporozoites. The appearance of the sporocyst residuum was variable. The residuum of some sporocysts was composed of many dispersed granules, whereas some had granules mixed with larger globules. Excystation was by collapse of the sporocyst along plates. The sporocysts wall was composed of 3 layers: a thin electron-dense outer layer, a thin electron-lucent middle layer, and a thick electron-dense inner layer. The sporocyst wall was thickened at the junctions of the plates. Sporozoites were weakly motile and contained a centrally or posteriorly located nucleus. No retractile or crystalloid body was present, but lipidlike globules about 1 microm in diameter were usually present in the conoidal end of sporozoites. Sporozoites contained 2-4 electron-dense rhoptries and other organelles typical of coccidian zoites. Sporozoites entered host cells in culture and underwent schizogony within 3 days.     

Membrane-bound mRNAs are recruited from preinitiated ribonucleoprotein particles in injected Xenopus oocytes

Messenger RNA injected Xenopus oocytes exhibit a differential capacity for translation. mRNAs translated in the free cytoplasm are translated efficiently whereas mRNAs translated on the rough endoplasmic reticulum (RER) membrane are translated inefficiently. If mRNA injected oocytes are injected additionally with proteins isolated from the RER, enhanced translation of RER-bound mRNAs is observed. When examined by sucrose gradient centrifugation and RNA dot blots, most of the injected RER-bound mRNA sediments less than or equal to the 80 S monosome. The RER proteins recruit these preinitiated mRNAs onto polysomes as evidenced by a shift in sedimentation to the polysome region of a sucrose gradient. When examined by immunoblotting, the RER proteins are shown to contain a protein which reacts specifically with an antibody directed against docking protein (SRP-receptor protein). However, this putative docking protein does not appear to be the protein which actually recruits the preinitiated mRNAs onto polysomes.     

Oogenesis in Centropages typicus (Copepoda, Calanoida)

We carried out a complete study of oogenesis in Centropages typicus using structural, ultrastructural and cytochemical data. The usual stages of oogenesis, i.e. germinative phase, premeiosis, primary and secondary vitellogenesis, were found. The latter two stages were the most typical. Primary vitellogenesis consisted of endogenous yolk accumulations; these substances, probably of lipoprotein or lipoglycoprotein nature, were produced at the granular endoplasmic reticulum level and then stocked in the reticulum cavities. During secondary vitellogenesis, endogenous yolk production continued, but we mainly observed the development of exogenous yolk accumulation (lipid droplets and protein globules) in the ooplasm. These accumulations resulted from the fusion of very numerous pinocytotic vesicles arising from the oolemma and containing substances probably brought to the oocytes by the hemolymph. The effect of various proteases on the vitellus globules caused a more or less marked digestion of their contents, tending to prove their protein nature. The end of vitellogenesis was marked by the appearance of vacuolar formations with dense lamellae which could correspond to cortical granules.     

Secretory cells in the clitellar epithelium of Eisenia foetida (Annelida,Oligochaeta): A histochemical and ultrastructural study

Eight secretory cell types are identified in the clitellar epithelium of Eisenia foetida, of which five have been described in detail previously (i.e., the large granular, fine granular, metachromatic, orthochromatic, and small granular proteinacecus cells). The remaining three secretory cell types are mucus-producing cells specific to the clitellar epithelium (type 3), cells associated with the chaetal follicles (type 4), and cells that occur exclusively in the tubercula pubertatis (type 5). Type 3 cells secrete a mucus containing neutral and acid mucosubstances. Ultrastructurally, type 3 cells are characterized by membrane-bound globules 0.4 to 3.7 μm in diameter. The contents of the globules have a finely reticulate appearance. The secretion of type 4 cells contains a collagenlike protein and neutral and sulfated acid mucosubstances. Type 4 cell secretory granules are membrane bound and range in diameter from 0.8 to 1.6 μm. They contain large, electron-dense, spheroid cores which are surrounded by parallel orientated microfibrils 14 nm in diameter. Type 5 cells give variable responses to the histochemical techniques used in the present study. An elastinlike protein is detected in about half of the type 5 cells and acid and neutral mucosubstances in the remainder. At the ultrastructural level the secretory granules vary in shape from spheroid to polygonal. Their finely, electron-dense contents exhibit progressive swelling which results in the eventual rupture of the limiting membranes of the granules. The necks of types 3, 4, and 5 cells contain a peripheral ring of microtubles (20 ± 1 nm in diameter).