Ultrastructure of the Ovary of the Hagfish Eptatretus stouti

Ultrastructure of the ovary of the Pacific hagfish was studied. Attention was paid to the mesovarium, small oocytes, oocytes of intermediate size, large oocytes, postovulatory follicles shortly after ovulation, postovulatory follicles considerably after ovulation, and preovulatory atretic follicles. The progressive changes of thecal and granulosa layers in the course of egg development and degeneration are described. No cells showing the ultrastructural characteristics associated with steroidogenesis were found.     

Ultrastructure of the Neurohypophysial Lobe of the Hagfish,Eptatretus stouti (Cyclostomata)

The neurohypophysial lobe is a thin-walled sac that, except for a few blood vessels, lacks any anatomical link with the adenohypophysis. Its wall consists of ependymal, fiber and palisade zones and is surrounded by blood vessels. The lobe is differentiated into distinct dorsal and ventral regions. The dorsal wall is doubly innervated by Gomori-positive axons arising in the anterior hypothalamus and by Gomori-negative fibers of unknown origin. Its surface is covered by an extensive vascular plexus. The ventral wall is innervated only by Gomori-negative fibers and is sparsely supplied with a few fine capillaries. All of the ependymal cells in both regions have the same ultrastructural appearance. The Gomori-positive or Type I axons are identified at the electron microscope level as fibers containing elementary granules with a diameter of 150–230 run. The Gomori-negative or Type II fibers contain dense-cored vesicles that vary from 80–125 nm in diameter. Both Type I and II fibers form synaptic-like complexes with the processes and end-feet of the ependymal cells. Type I axons also abut on the basal lamina bounding the perivascular spaces. It is suggested that the agranular reticulum of the ependymal cells may provide a transport pathway for neural products that are destined for release into the circulation. It is also possible that the ependyma itself is a target of neural activity.     

Gill ultrastructure of the Pacific hagfish Eptatretus stouti

At the gross anatomical level, hagfish gills show unusual features not seen in any other fish gills. Our study was undertaken to determine if peculiarities also characterize the microscopic anatomy and ultrastructure of hagfish gills. To the contrary, branchial respiratory lamellae of Pacific hagfish were found to resemble the lamellae of lampreys, elasmobranchs, and teleosts, often down to the finest subcellular details. As in other fish, hagfish lamellae are lined by epithelium containing pavement cells with organelles indicative of a secretory function, basal cells showing undifferentiated cell features, and branchial ionocytes. The ionocytes are identical to chloride cells of teleosts in cytostructure, distribution, and abundance. There are pillar and marginal capillaries in hagfish gill lamellae. Pillar cells contain bundles of 5-nm microfilaments, and they associate with collagen columns as in other fish. Hagfish pillar cells do exhibit odd features, however: They cluster (groups of up to nine were seen), and their extracellular collagen columns are rarer than in other fish gills (averaging only two columns per three pillar cells). Other special features of hagfish gills are the following: lipid droplets and smooth endoplasmic reticulum are well developed in all cell types; pavement cells secrete a lipomucous product (stains with periodic acid-Schiff, Alcian blue, and Sudan black B); and goblet cells are absent. The presence of "chloride cells" in hagfish is puzzling, as hagfish body fluids are iso-osmotic to seawater and there is no need to osmoregulate for sodium chloride; the ionocytes contain carbonic anhydrase, suggesting a function in acid/base regulation.     

Ovulation and Eggs of the Hagfish Eptatretus burgeri

Abstract Ripe females and males of Eptatretus burgeri were kept in cages both in the sea and in the aquaria for about 2 months of the normal spawning period around October. All the 9 ripe females in the sea ovulated all their eggs (200) and deposited 178 of them. Only 5 eggs were ovulated and deposited in the aquaria, although there were 17 ripe females and some of them were injected with various hormones in an effort to induce ovulation. No eggs were fertilized. A peculiar surface pattern of hexagonal ridges is described around the entrance of the micropylar canal on the bottom of the micropylar funnel in the ovulated eggs.     

Spermiogenesis in the Hagfish Eptatretus burgeri (Agnatha)

The fine structure of spermatid differentiation in a primitive vertebrate, the hagfish, whose spermatozoa bear acrosomes, was investigated. In early round spermatids, the acrosomal vesicles were spherical and located in a shallow nuclear indentation, flanked by the plasma and the nuclear membranes. The vesicle underwent a transition through lens-shaped and cap-shaped stages until it attained the shape of a bell in mature spermatozoa. Electron-dense acrosomal material that appeared as deposits in three portions of the vesicle finally joined in the center region at a late stage. Condensation of chromatin occurred in the anterior region of the nucleus. During transformation of the spermatids, many regularly spaced microtubules appeared beneath the plasma membrane except in the anteriormost region of the cell. The microtubules in a single alignment lay parallel to one another and encased the nucleus diagonally. During an early stage, the centrioles changed their orientation from perpendicular to longitudinal and rotated to become parallel to the long axis of the nucleus. Thus, the flagellum lay nearly straight along the cell axis. A cytoplasmic canal appeared transiently during the early stage. A droplet of cytoplasm was eliminated after descending along the flagella. The features of spermiogenesis in hagfish, which lies between invertebrates and vertebrates, are compared with those of other animals.     

Effects of Estradiol and Testosterone in the Hagfish Eptatretus burgeri

Effects of exogenous estradiol and testosterone on the gonad and pituitary were examined in the hagfish. Estradiol caused degeneration of the ovary. Testosterone had no effect on the ovary. Neither steroid had a significant effect on the testis. Neither estradiol nor testosterone provoked a conspicuous change in adenohypophysial cells. Therefore, a feedback mechanism between gonadal steroids and gonadotropin either does not exist or plays a very minor role. The degeneration of the ovary may be due to a pharmacological effect of estradiol.     

Morphometics and Gonadal Development of the Hagfish Eptatretus cirrhatus in New Zealand

Hagfishes have been the target of commercial fisheries in many areas of the world, with the catch processed for leather and for human consumption. A fishery has been operating in New Zealand waters for the last six years, harvesting the bearded hagfish, Eptatretus cirrhatus. The fishery has thus far been unregulated. Based on samples collected dockside over a two-year period, this report expands the morphometric database for this species, provides information on the size and weight of the harvested animals, determines the sizes at the onset of gonadal development and the minimum sizes at sexual maturation for males and females, and indicates that E. cirrhatus, like most other hagfish species, has no specific breeding season. Although females appear in the population at smaller sizes, the sex ratio for mature animals is 1:1 and the sizes of the largest males and females are comparable. The changes observed in sex ratio as a function of TL suggest differences in the timing and rates of gonadal development in females versus males rather than protogyny. Based on the size of the eggs, the number of eggs per female, the proportion of the population that contains large eggs, and the number of postovulatory females, it is clear that E. cirrhatus, like other hagfish species, are potentially vulnerable to overexploitation.     

Distinct Ig H chains in a primitive vertebrate, Eptatretus stouti

Serum Ig from the Pacific hagfish, Eptatretus stouti, was isolated by affinity chromatography using a specific mAb (H.45). Analysis of the approximately 210-kDa molecule by SDS-PAGE under reducing conditions revealed two H chains of approximately 77 kDa (H1) and approximately 70 kDa (H2) and L chains of approximately 30 kDa. H1 and H2 were shown to differ with respect to their peptide maps, amino-terminal amino acid sequences, and reactivity to the mAb H.45, suggesting that they represent discrete H chain isotypes. Two-dimensional nonreducing/reducing SDS-PAGE demonstrated that H and L chains were covalently linked predominantly as H-H-L and H-L configurations. Noncovalently bound L chains were also found. H-H-L complexes were shown to contain H1-H2 heterodimers of H chains in addition to H1-H1 homodimers.     

Cyclical Changes in the Testis of the Hagfish Eptatretus burgeri (Cyclostomata)

The development of the spermatogenetic stages in the testis of the hagfish Eptatretus burgeri was studied by means of histological techniques. These animals have an annual reproductive cycle with a spawning season in October. Spermatocytes begin to appear in January and reach a maximum in March. The development of spermatids of stage I and stage II starts in April and June, respectively. In August about 35% of the testicular follicles contain spermatids of stage II. The gonadosomatic index shows a more or less constant increase from November until August. During the spawning season, no animals with well developed testis could be trapped. About 10% of male hagfish bear undeveloped gonads.     

Cyclical Changes in the Ovary of the Hagfish Eptatretus burgeri (Cyclostomata)

Abstract The development of eggs and the degeneration of ovulated follicles in Eptatretus burgeri was studied over a period of one year. It was found that small resting eggs with a diameter of about 1.5 mm continue to grow in November. By the spawning season in October of the following year, the follicles attain a length of about 24 mm. Only one fertilized egg containing an embryo of 7.5 mm length was obtained. The ratio of males to females was found to vary throughout the year. The annual increase of body length in correlation to ovarian development in the hagfish is calculated to be 4 to 5 cm.     

A Scanning Electron Microscopic Study on the Thyroid Follicle of the Hagfish Eptatretus burgeri

The external form of the follicles was mostly spherical or oval and the large oval follicles reached more than 300 times 180 μm in size. Cytoplasmic inclusions of various sizes were observed in the follicular epithelium. Follicles were composed of hexagonal epithelial cells, and on the apical surface of the cells many microvilli were found (380–500 microvilli per cell) with globular or amorphic cytoplasmic protrusions. In a few cells a central cilium was also observed. From these results a relationship between thyroid structure and function in the hagfish is discussed.     

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish,Eptatretus stouti (Myxinoidea)

Summary The distribution of FMRFamide-like immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish,Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral ganglionic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinet terminal arborization occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that FMRFamide may play a role in the central control of branchiomotor activity.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

Immunocytochemical localization of somatostatin and vasotocin in the brain of the Pacific hagfish,Eptatretus stouti

Summary The brain of the Pacific hagfish, Eptatretus stouti, was studied immunocytochemically using antisera against somatostatin (SRIH), arginine vasopressin (AVP), and adrenocorticotropic hormone (ACTH). SRIH-immunoreactive perikarya were distributed bilaterally in the postoptic nucleus and in the hypothalamic nucleus. Although several short, stained fibers were observed in the vicinity of the perikarya, SRIH-immunoreactivity was not found in the neurohypophysis, nor in other parts of the brain. On the other hand, presumed arginine vasotocin (AVT) perikarya were distributed in an arc-shaped region extending from the posterior part of the preoptic nucleus to the anterior-most end of the hypothalamic nucleus and projected their fibers to the neurohypophysis. Most presumptive AVT perikarya were located close to the paired prehypophysial arteries near the anterior end of the postoptic nucleus. In the neurohypophysis, abundant presumptive AVT-fibers terminated in the posterior dorsal wall, although some fibers terminated in the anterior dorsal wall and only a few fiber endings were found in the ventral wall. No ACTH-positive cells were detected in the hagfish brain or in the pituitary gland.Supported from a grant from the National Science Foundation PCM 8141393     

Blood and extracellular fluid volume in whole body and tissues of the Pacific hagfish, Eptatretus stouti

Whole-body and 20 individual-tissue (51)Cr-RBC (red cell space; RCS) and (99)Tc-diethylenetriaminepentaacetic acid (extracellular space; ECS) spaces were measured in seven unanesthetized Pacific hagfish (Eptatretus stouti). Volume indicators were administered via a dorsal aortic cannula implanted the previous day. Blood samples were collected at 6, 12, 18, and 24 h after injection. Tissues were removed at 24 h and radioactivity was measured; tissue water content (percent of wet weight) was determined by desiccation at 95 degrees C for 48 h. Mixing rates of both indicators were identical and were essentially complete by 12 h, indicating that blood convection is the rate-limiting process. At 24 h, the whole-body RCS was 19.3+/-2.1 mL kg(-1) body weight, and the ECS was 338.5+/-15.2 mL kg(-1) body weight. Blood volume estimated from the 24-h RCS and the mean central hematocrit (14%) was 137.9 mL kg(-1) body weight. Liver RCS (118.6+/-30.5 microL g(-1) tissue weight) was twice that of any other tissue and was also the most variable, ranging from 59 to 263 microL g(-1), whereas liver ECS (406.0+/-34.3 microL g(-1)) was in the range of other tissues, and water content (66.9%+/-3.5%) was low. Gill RCS (55.9+/-5.7 microL g(-1)), ECS (415.3+/-37.7 microL g(-1)), and percent water (83.1%+/-0.8%) were higher than most other tissues. RCS, ECS, and percent water were consistently lowest in ovum (1.1+/-0.02 microL g(-1), 111.1+/-4.3 microL g(-1), 51.3%+/-3.5%, respectively). Tongue, notocord, and myotome had generally lower RCS (2.1+/-0.4, 2.2+/-0.5, 7.1+/-0.1 microL g(-1), respectively) and ECS (121.2+/-7.0, 246.3+/-17.4, 185.3+/-16.7 microL g(-1), respectively), although their water content was in the midrange (74.7+/-0.5, 81.2+/-1.6, 74.4%+/-0.6%, respectively). Skin had a low RCS (6.8+/-1.1) and midrange ECS (387.5+/-28.0) but very low water content (61.2%+/-2.1%). These findings confirm that hagfish blood volume is at least twice as large as other fish, whereas our estimate of extracellular fluid volume is larger than previously reported and more in line with the predicted interstitial volume. RCS, ECS, and water content vary, often independently, between tissues, which may perhaps be indicative of specific tissue needs or functions. A distinct spleen is lacking in hagfish, and the liver appears to serve this function by sequestering red cells. To our knowledge, this is the first report of tissue ECS in Myxiniformes.     

Ependymal Absorption of Peroxidase into the Third Ventricle of the Hagfish Eptatretus burgeri (Girard)

Abstract The ependymal cells of both dorsal and ventral walls lining the lumen of the neurohypophysis of the hagfish, Eptatretus burgeri, absorb peroxidase injected into the third ventricle. Peroxidase diffuses rapidly into the connective tissue separating the neurohypophysis from the adenohypophysis, and also into the connective tissue located between the cell nests of the adenohypophysis.     

Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti)

Summary The hagfishes (cyclostomes) are known to secrete copious amounts of mucus mainly by the holocrine mode from the slime glands. Stressed animals release two types of cells (gland thread cells, GTCs; gland mucous cells, GMCs) which rupture on contact with water and rapidly form a mass of viscous mucus. Herein we report some key sequential events of this process and document a novel role for cytoskeletal polymers. After electrostimulation of Pacific hagfish (Eptatretus stouti), the exudate was collected in a stabilization buffer and GTCs segregated from GMC vesicles. Water was added progressively to mixtures of known quantities of these entities. The changing mucous composition and properties were monitored by light- and electron microscopy, viscometry and immunogold assay. Sequentially, the threads uncoil from GTCs, aggregate with the vesicles, the vesicles rupture and release mucin-like substances, at least some of which adhere to the thread. It was found that the intermediate filament (IF)-rich threads markedly facilitate hydration and modulate the viscoelastic and cohesive properties of the resultant mucus. It was speculated that the thread abets localization of mucus in an aqueous environment and promotes adhesion of mucus to surfaces such as the fish integument. As judged by immunostaining in situ, GTCs, as well as several cell-types in the epidermis, contain keratin-like components. The role of biopolymers on the properties of teleost and mammalian mucus is discussed.     

An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish (Eptatretus stouti). This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4-13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K(m) values of 10-45 microM. The antiviral nucleoside drugs 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and 2',3'-dideoxyinosine were also transported. Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent K50 value for Na+ activation was >100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7-13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.     

Sex Difference in the Cloacal Gland in the Hagfish,Eptatretus burgeri,and its Possible Significance in Reproduction

The cloacal gland of the hagfish, Eptatretus burgeri, was studied histologically in four seasons. Before the breeding season (spring and summer) the male cloacal gland was larger than the female cloacal gland. The gland was largest in the males with a more mature testis. After the breeding season (autumn and winter) there was no sex difference in the size of the cloacal gland. The cloacal gland consists of mucus and thread cells, as do the lateral slime glands. The sperm may be entangled within slime in the cloacal gland and be shed outside as a slimy sperm mass. Such a slimy sperm mass may play an important role in reproduction of the hagfish.