Acrosome reaction in spermatozoa of the hagfish Eptatretus burgeri (Agnatha)

Fertilization of the hagfish or myxiniformes, a member of the most primitive vertebrate group and an animal of phylogenic interest, is unknown. Here, induction of an acrosome reaction for spermatozoa in the hagfish, Eptatretus burgeri, was successfully achieved by treatment of mature spermatozoa with ionomycin and excess Ca2+. The spermatozoon produced an acrosomal process that elongated from the apex of the long sperm head. The reaction bears resemblance to that of invertebrate spermatozoa rather than that of vertebrate spermatozoa. The result provides insights into the phylogenetical changes that have occurred in this sperm reaction.     

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish,Eptatretus burgeri

Summary Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.We wish to express our appreciation to Professor Hideshi Kobayashi for the guidance of this study. It was aided by grants from the Ford Foundation and the Ministry of Education to Professor H. Kobayashi, and also by grants from the Population Council, New York and the Ministry of Education to Professor S. Ishii.     

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.     

Chromosome elimination in the Japanese hagfish, Eptatretus burgeri (Agnatha, Cyclostomata)

The modal number of chromosomes in Eptatretus burgeri was 36 in 297 (81.8%) of 363 somatic cells, 52 in 13 (30.2%) of 43 spermatogonia, and 25 (46.8%) or 26 (40.3%) in 162 of 186 first spermatocytes. The relative amount of DNA in a somatic cell to that in a spermatogonium averaged 79.2%. C-band-positive chromatin was observed along almost the entire length of the associated dumbbell-shaped bivalents and part of two other bivalents in the metaphases of first spermatocytes, but was rarely observed in somatic cells. These results indicate that, in E. burgeri, chromosome elimination takes place during the early stages of cleavage, except for the ancestral germ-line cells.     

FMRF -amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

Summary Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material.Antisera to ACTH, -MSH and pancreatic polypeptide gave no immunoreaction in hagfish brain or pituitary. D aspartic acid - F phenylalanine - L leucine - M methionine - R arginine; - W tryptophan - Y tyrosine     

Effects of dark-light step transitions on circadian locomotor activity rhythms of the hagfish,Eptatretus burgeri

Captive hagfish,Eptatretus burgeri, were subjected to step transitions from continuous dark (DD) to continuous light (LL) and their locomotor activity patterns recorded. Free-running activity rhythms occurred in both the DD and LL regimes. The timing of the transition influenced the circadian period (τ), the relationship between the period response (δτ, or τ{inLL}–τ{inDD}, change in the circadian period) and the former being represented by a cosine curve δτ became most positive and most negative when hagfish underwent a DD-LL transition shortly before the beginning of the duration of activity (α) and shortly after the end of such, respectively. The phase response (δ-phase, change in the relative timing of the activity phase) was characterized by a delay in the activity phase after the DD-LL transition, although its magnitude bore no relation to the timing of the transition.     

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.     

X-ray microanalysis on the thyroid follicle of the hagfish, Eptatretus burgeri and lamprey, Lampetra japonica.

Intracellular dense bodies, cytoplasmic matrices, and luminal colloids in the thyroid follicle of cyclostomes, hagfish and lamprey were examined to identify the distribution of iodine using an energy dispersive X-ray microanalyzer attached to a scanning transmission electron microscope. High level of iodine was detected only in the large dense body of the hagfishes, while it was too slight in quantity to detect by this method in the cytoplasmic matrix as well as in the luminal colloid. In the adult lamprey thyroid, an appreciable amount of iodine was detected in a few large dense bodies. In mice and rats, it is very hard to detect iodine in the luminal colloid, intracellular colloid droplet, and in the lysosomal dense granule by this method, though these structures have been well known to contain a fairly large amount of iodine which is combined with thyroglobulin. These facts mean that intracellular dense bodies in the thyroid follicular epithelium of the cytoclostome, especially of the hagfish have an extremely larger amount of iodine. These bodies are considered to be secondary lysosomes or residual bodies containing reabsorbed colloid materials which are highly condensed.     

Experimental studies on the light sense in the hagfish,Eptatretus burgeri andParamyxine atami (Cyclostomata)

Photo-reception and sensitivity to light were studied in two Japanese hagfishes,Eptatretus burgeri living in shallow water andParamyxine atami living in water of about 100m depth. Both species responded to general illumination by first moving the tail or head and then by swimming. Local illumination revealed that regions most sensitive to light were the skin of the tail in both species and a line of unpigmented skin running down the back ofE. burgeri. The light sensitivity of the lensless eyes, which are situated below the skin, was very weak in both species.P. atami showed shorter reaction time to light thanE. burgeri. No change in skin colour was induced either by almost complete hypophysectomy or by continuous illumination against a white background. Under-water observations with SCUBA revealed that free movingE. burgeri responded well to illumination uncovered during the night, but the ones buried in mud, with only the heads uncovered did not.     

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.     

Cloning and characterization of Tc1 family-derived PPTN related transposons from ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri)

The Tc1 transposable element is the most widespread family among animal transposon and these elements consist of an inverted repeat (IR) sequence flanking a transposase gene that belongs to Class II type transposon, which is highly conserved in the genome of the nematode C. elegans. In order to characterize Tc1-like transposable elements from several fishes, PPTN (Tc1-like transposon was isolated from Pleuronectes platessa, marine flatfish species) IR primer-specific amplified elements were cloned from the genomic DNA of several fishes. Transposable elements were found in ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri) and named as PCTN and EBTN, respectively. Amino acid sequence alignment and phylogenetic analysis confirmed that the PPTN-like transposons belonged to the Tc1 superfamily of transposons, but they comprised a unique clade of Tc1-like transposons. The IR-PCR analysis using MMTS-IR and PPTN-IR specific primers from Paralichthys olivaceus (Paralichthyidae), Paraplagusia japonica (Cynoglossidae), P. yokohamae (Pleuronectidae) and Pagurus cornutus (Pleuronectidae) (within the same order, Pleuronectiformes but different families) exhibited mutually exclusive distribution of Tc1 family-derived PPTN and MMTS-like transposons in these fish genomes. These results indicate that Tc1 family-derived PPTN and MMTS related Tc1-like transposable elements have uniquely evolved in piscine genome, and can be used as phylogenetic markers for the distribution of subfamilies of Tc1-like transposon and the involvement of horizontal and vertical transmission in the evolution of fish genome.     

Fine structure of micropylar region during late oogenesis in eggs of the hagfish Eptatretus burgeri (Agnatha)

Using the hagfish, Eptatretus burgeri, the fine structure of formation of the micropylar region in hagfish eggs during the late stages of oogenesis was investigated for the first time, focusing on the bottom region of the micropyle and the egg surface. During these stages, many cells penetrated through the chorion and reached a pit of the egg surface, forming a shovel-like structure in two-dimensional sections. The cells, which we called micropylar cells, were separated from the chorion by a wall of amorphous material. In the pit, another fibrous layer filled the space between the egg surface and the anterior portion of the shovel-like structure. Microvilli coming from the egg surface were embedded in this layer. In later stages, the stack of micropylar cells loosened, and a space appeared between the anterior region of the shovel-like structure and the layer on the egg surface. Microvilli decreased in length and number. The pit region appeared likely to have a role in fertilization. The structures associated with the forming micropyle were markedly different from those observed in the same region of teleost fishes. A hypothesis that hagfish might show transitional structures in gametes from protochordates to teleosts is suggested.     

Distribution of 5-HT (serotonin) immunoreactivity in the central nervous system of the inshore hagfish,Eptatretus burgeri

Summary Immunohistochemical techniques were used to study the distribution of serotonin in the central nervous system of the hagfish,Eptatretus burgeri, in order to produce a detailed map of serotonin-containing structures. In the hypothalamus, many serotonin-containing neurons contacted the cerebrospinal fluid. Most of the serotonin-containing cell bodies were located in the raphe region, where they were compactly distributed at the level of the nucleus motorius tegmenti pars anterior but more diffusely distributed at the level of the nucleus motorius tegmenti pars posterior. Serotonin-containing cell bodies and varicose fibers were widely distributed throughout the brain and upper spinal cord segments, but the distribution density was not even. On the basis of its abundance, serotonin can be judged to have an important function in the control of the hagfish central nervous system. From a phylogenetic point of view, serotonin-containing neurons in the raphe region appear to be a common property of all classes of vertebrates studied except the lampreys, whereas serotonin-containing cerebrospinal fluid-contacting neurons may be considered to be a primitive condition in all nonmammalian vertebrates.     

Iodine distribution in the thyroid follicles of the hagfish,Eptatretus burgeri and lamprey,Lampetra japonica: Electron-probe X-ray microanalysis

Summary In the thyroid follicles of species of cyclostomes, a hagfish and a lamprey, the distribution of stable iodine was examined by electron-probe X-ray microanalysis. A high concentration of stable iodine, heterogeneously distributed, was observed in the follicular cells of hagfish thyroid follicles. In the lamprey a low concentration of iodine was seen in the follicular lumina. The relative values for stable iodine determined in this way corresponded to values obtained by a chemical analytical method.     

Construction of a bacterial artificial chromosome library from the inshore hagfish, Eptatretus burgeri: A resource for the analysis of the agnathan genome

The jawless fish occupy an important phylogenetic position for understanding the evolution of body plans, the origin of adaptive immunity and genome evolution in chordates. We describe here the construction of a large-insert bacterial artificial chromosome (BAC) library from the inshore hagfish, Eptatretus burgeri. The BAC library contains 93,978 clones with an average insert size of 100 kb and is estimated to represent threefold genome-equivalent coverage. The library was organized in three-dimensional pools to facilitate screening by PCR. We have screened this library by PCR and isolated several BAC clones; the average number of positive clones was compatible with the estimated genome coverage of the library. This BAC library, constructed for the first time from the jawless fish, should serve as a useful resource for the scientific community.     

Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly

The phylogenetic position of cyclostomes, i.e., the relationships between hagfishes, lampreys, and jawed vertebrates is an unresolved problem. Anatomical data support the paraphyly of cyclostomes, whereas nuclear genes data support monophyly of cyclostomes. Previous results obtained using mitochondrial DNA are ambiguous, presumably due to a lack of informative sequences. By adding the complete mtDNA of a hagfish, Eptatretus burgeri, we have generated a novel data set for sequences of hagfishes and of lampreys. The addition of this mtDNA sequence to the 12 taxa we have already used becomes sufficient to obtain unambiguous results. This data set, which includes sequences of mtDNA of animals closely related to the lamprey/hagfish node, was used in a phylogenetic analysis with two independent statistical approaches and unequivocally supported the monophyly of cyclostomes. Thus molecular data, i.e., our results and those obtained using nuclear genes, conclude that hagfishes and lampreys form a clade.     

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.