Preliminary results on the reproduction of a deep-sea snailfish Careproctus rhodomelas around the active hydrothermal vent on the Hatoma Knoll, Okinawa, Japan.

Deep-sea snailfish Careproctus rhodomelas were collected from an active hydrothermal vent using a remotely operated vehicle (R.O.V. Hyper-dolphin) and a pressurized device (Deep-Aquarium). Careproctus rhodomelas exhibited a cystovarian-type ovary containing a small number of developing oocytes at different stages, suggesting that the fish is a batch-spawner that spawns large eggs (c. 6·0 mm) several times within its life span. In vitro culture of the oocytes in the presence of human chorionic gonadotropin showed that oestradiol-17ß production fluctuated with oocyte development, suggesting that the oocytes were at the vitellogenic stage.

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP, and acidification of yolk granules.

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.

Effects of cGMP and cAMP on light responses of the photosensory pineal neurons in the lamprey, Lampetra japonica.

We examined in this study how external cyclic nucleotides affect the light response mechanism of the pineal photoreceptors and explored the existence of parietal eye type of photoreceptor of which the internal cGMP concentration increased during the light response. Pineal organs of river lampreys, Lampetra japonica, were treated with 8-bromo guanosine 3',5'-cyclic monophosphate (8Br-cGMP) or 8-bromo adenosine 3',5'-cyclic monophosphate (8Br-cAMP) before light stimuli, and the light responses were recorded from the second order neurons, chromatic or achromatic-type neurons. Excitatory and inhibitory light responses of the chromatic-type neuron became obscure by 9 and 3 mM 8Br-cGMP without changing the spontaneous spike discharge in the dark. 8Br-cAMP (3 mM) increased the frequency of spontaneous spike discharge, though it did not inhibit the light responses themselves. The inhibitory light response of the achromatic-type neuron decreased after adding 3 mM 8Br-cGMP, and it was unchanged by 3 mM 8Br-cAMP. The spontaneous spike discharge of the neurons in the dark was not affected by the cyclic nucleotides. The mechanism of these results can be explained if cGMP is an intracellular second messenger of light responses in the pineal photoreceptors and the blocking effect on photoresponses by externally applied 8Br-cGMP is caused by compensating for the reduction in intracellular cGMP by light. However, it does not indicate that the parietal eye type of photoreceptor found in lizard participates in the chromatic and achromatic-type responses in the lamprey pineal organ.

Multiphotoreceptor and multioscillator system in avian circadian organization.

Photoperiodism and circadian rhythms have been studied intensively in birds because Aves are typical seasonal breeders and diurnal animals. Light is the most important environmental factor involved in entrainment of circadian rhythms and photoperiodism. The eyes and the extraocular photoreceptors, such as the pineal organ and hypothalamus, are reported to have an important function not only for photoreception but also for circadian organization in nonmammalian vertebrates, including birds. In this report, we review the roles of the eyes, pineal organ, and deep brain as the components of the multiphotoreceptor and multioscillator system in avian circadian organization.

Light- and temperature-dependence of the melatonin secretion rhythm in the pineal organ of the lamprey, Lampetra japonica.

To identify the characteristics of the oscillator located in the pineal organ, we examined the effects of temperature and light on melatonin secretion rhythm using pineal organs in cultures. At 20 degrees C, the melatonin rhythm was obvious: low secretion during the daytime and high during the nighttime. When the temperature was lowered from 20 to 10 degrees C, the melatonin rhythm disappeared. When the temperature was returned from 10 to 20 degrees C, the rhythm quickly reappeared. The plasma melatonin level was measured in living lampreys kept at 7 degrees C to establish the melatonin profile at low temperature in vivo: secretion was not significantly different between daytime and nighttime. Under continuous light conditions, the melatonin elevation normally seen during the subjective night became obscure after 72 h. When the LD cycle was shifted by 6 h (phase-advanced or phase-delayed), the melatonin rhythm shifted to remain in the same phase relation to the LD cycle. This re-synchronization took several LD cycles. The results indicate that, in cultures, the melatonin secretion rhythm in the pineal organ of the lamprey is both light- and temperature-sensitive, and that in vivo, the melatonin rhythm is not the critical factor maintaining the locomotor activity rhythm of the lamprey. The role of the pineal organ and melatonin in the circadian organization of the lamprey is discussed.

Immunocytochemical observations on pineal organ and retina of the Antarctic teleosts Pagothenia borchgrevinki and Trematomus bernacchii.

In spite of the unique conditions they have to operate under, the pineal organs of Antarctic fishes have not previously been examined. We determined immunohistochemically that in the end-vesicles and the pineal stalks of Pagothenia borchgrevinki (a species found directly beneath the sea-ice) as well as Trematomus bernacchii (a species preferring somewhat deeper water than the former) at least two populations of physiologically-different cells occurred that displayed reactions indicative of typical vertebrate photoreceptors. Comparisons with immunocytochemically treated retinal sections from the eyes of the same two species showed that anti-opsin reactivity, characteristic of rods, was particularly strong in the lumina of the pineal stalks of both species. Anti-visinin reactions stained cones in the retinal sections of both fishes and occurred throughout the pineal organs, but in particular in the end vesicles of the pineals of both species. The difference in preferred habitat depth between the two species appears to have had very little influence on both retinal and pineal immunocytochemistry. It is concluded that the pineal organs of both species, at least during the austral summer, exhibit signs of being directly photo-sensitive.

Internalization of urinary trypsin inhibitor in human uterine fibroblasts.

We have characterized the molecular species and internalization of urinary trypsin inhibitor (UTI) in human uterine fibroblasts. Link protein (LP) has previously been identified as one of the cell-associated UTI binding proteins. The truncated forms of UTI were readily detectable in the cells after incubating the cells with purified UTI. Immunoblotting analysis with a panel of domain-specific antibodies revealed that the UTI species lacked the amino-terminal domain of UTI, but contained the carboxyl-terminal domain. We have examined whether LP is involved in the UTI internalization in the cells. Internalization of 125I-labelled UTI was blocked by the intact UTI, but not by the carboxyl-terminal domain of UTI. Treatment with a polyclonal antibody to the UTI binding domain of LP partially inhibited UTI binding to the cells, but did not significantly prevent UTI internalization. In addition, preincubation of the cells with hyaluronidase reduced the UTI binding to the cells, but had no effect on the rate with which UTI was internalized. These data allow us to conclude that there are at least two different mechanisms for internalization of UTI. The major one is via unknown UTI receptors in a Ca2+, Mg2+-sensitive manner and another is via LP.

Identification and characterization of the cell-associated binding protein for urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20,642-20,647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH2-terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan-hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.

Three-dimensional reconstruction of serotonin-immunoreactive photoreceptors in the pineal organ of the river lamprey, Lampetra japonica.

Serotonin-immunoreactive (5-HT IR) photoreceptors are present in the pineal complex (pineal and parapineal organ) of the river lamprey, Lampetra japonica. They are so-called modified pineal photoreceptors and have been regarded as photoneuroendocrine cells which secrete melatonin. We reconstructed 5-HT IR cells with a computer to demonstrate their three-dimensional structures from optical sections taken by a confocal laser scanning microscope. The 5-HT IR cell possesses a basal process, and it appears that the process does not branch out. These processes contact each other at the basal region of the end vesicle, and a process extends to the soma of the neighboring 5-HT IR cell. These findings were obtained by three-dimensional analysis with a computer, which is a useful technique to demonstrate the interaction between cells. We suggest that the 5-HT IR photoreceptors interact with one another.

Melatonin excretion rhythms in the cultured pineal organ of the lamprey, Lampetra japonica.

Pineal organ of the lamprey, Lampetra japonica, is essential to keep the circadian locomotor activity rhythm as previously reported. In this paper, we tried to show that an endogenous oscillator is located and is working in the pineal organ. When the pineal organs were excised and cultured in a plastic tube with M199 medium at 20 degrees C, melatonin secretion rhythms were clearly observed under both light-dark and continuous dark conditions. The circadian secretion of melatonin continued for more than five cycles under the continuous dark condition. This indicates that the pineal organ has an endogenous oscillator and that the melatonin secretion rhythm is controlled by this oscillator. These findings suggest the possibility that the locomotor activity rhythm of the lamprey is under the control of the oscillator in the pineal organ.

Light-modulated subcellular localization of the alpha-subunit of GTP-binding protein Gq in crayfish photoreceptors.

Gq-type GTP-binding protein (Gq) plays an important role in invertebrate visual phototransduction. The subcellular localization of the alpha subunit of visual Gq in crayfish photoreceptor was investigated immunocytochemically and biochemically to demonstrate the details of the rhodopsin-Gq interaction. The localization of Gq(alpha) changed depending on the light condition. In the dark, Gq(alpha) was localized in the whole rhabdoms as the membrane-bound form. In the light, half of the Gq(alpha) was localized in the cytoplasm as the soluble form. The translocation of Gq(alpha) was reversible. The light-modulated translocation possibly controls the amount of Gq that can be activated by rhodopsin. In vitro hydroxylamine treatment of rhabdomeric membranes suggested that the translocation was regulated by the fatty-acid modification of Gq(alpha).

Norepinephrine-induced phosphorylation of the transcription factor CREB in isolated rat pinealocytes: an immunocytochemical study.

In the present study we investigated whether norepinephrine, which stimulates melatonin biosynthesis in the mammalian pineal organ, causes phosphorylation of the cyclic AMP responsive element binding protein (CREB) in rat pinealocytes. Cells isolated from the pineal organ of adult male rats and cultured on coated coverslips were treated with norepinephrine, beta- or alpha 1-adrenergic agonists for 12, 5, 10, 20, 30, 60 or 300 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (p-CREB). Treatment with norepinephrine or beta-adrenergic agonists resulted in a similar, time-dependent induction of p-CREB immunoreactivity, exclusively found in cell nuclei. The alpha 1-adrenergic agonist phenylephrine did not induce p-CREB immunoreactivity at low doses (0.1 microM) or when high doses (10 microM) were applied in combination with a beta-antagonist (propranolol, 0.1 microM). This indicates that induction of CREB phosphorylation is elicited by beta-adrenergic receptor stimulation. The response was first seen after 10 min and reached a maximum after 30 to 60 min when more than 90% of the cells displayed p-CREB immunoreactivity. The intensity of the p-CREB immunoreactivity showed marked cell-to-cell variation, but nearly all immunoreactive cells were identified as pinealocytes by double-labeling with an antibody against the S-antigen, a pinealocyte-specific marker. The results show that norepinephrine stimulation induces p-CREB immunoreactivity by acting upon beta-adrenergic receptors in virtually all rat pinealocytes. The findings support the notion that phosphorylation of CREB is a rather rapid and uniform response of pinealocytes to noradrenergic stimulation and thus is an important link between adrenoreceptor activation and subsequent gene expression in the rat pineal organ.

Dissociation of photoreceptor cells from the pineal organ of the lamprey, Lampetra japonica.

Photoreceptor cells, nerve cells and supporting cells were dissociated from the pineal organ of the river lamprey, Lampetra japonica, by the use of 10 U/ml papain solution at 28 degrees C for 20 min, followed by repeated trituration. With the aid of Nomarski interference-contrast optics, photoreceptor cells, nerve cells and supporting cells were readily identified. Electron-microscopic examination revealed that isolated photoreceptor cells display an outer segment endowed with a few lamellar disks and connected to the inner segment (ellipsoid) via a connecting cilium. The structural features of the dissociated photoreceptor and supporting cells strongly resemble the morphology of the respective cellular elements in situ. We succeeded in culturing dissociated cells for time periods up to 48 h when the procedure described in detail was applied.

Immunocytochemical localization of serotonin and photoreceptor-specific proteins (rod-opsin, S-antigen) in the pineal complex of the river lamprey, Lampetra japonica, with special reference to photoneuroendocrine cells.

The pineal complex of the river lamprey, Lampetra japonica, was examined by means of immunocytochemistry with antisera against serotonin, the precursor of melatonin, and two photoreceptor proteins, rod-opsin (the apoprotein of the photopigment rhodopsin) and S-antigen. Serotonin-immunoreactive cells were observed in both the pineal and the parapineal organ. The proximal portion of the pineal organ (atrium) comprised numerous serotonin-immunoreactive cells displaying spherical somata. In the distal end-vesicle of the pineal organ, the serotonin-immunoreactive elements resembled photoreceptors in their size and shape. These cells projecting into the pineal lumen and toward the basal lamina were especially conspicuous in the ventral portion of the end-vesicle. In addition, single serotonin-immunoreactive nerve cells were found in this location. Retinal photoreceptors were never seen to contain immunoreactive serotonin; amacrine cells were the only retinal elements exhibiting serotonin immunoreaction. Strong S-antigen immunoreactivity was found in numerous photoreceptors located in the pineal end-vesicle. In contrast, the S-antigen immunoreactivity was weak in the spherical cells of the atrium. Thus, the pattern of S-antigen immunoreactivity was roughly opposite to that of serotonin. Similar findings were obtained in the parapineal organ. The rod-opsin immunoreaction was restricted to the outer segments of photoreceptors in the pineal end-vesicle and parapineal organ. No rod-opsin++ immunoreactive outer segments occurred in the proximal portion of the atrium. Double immunostaining was employed to investigate whether immunoreactive opsin and serotonin are colocalized in one and the same cell. This approach revealed that (i) most of the rod-opsin-immunoreactive outer segments in the end-vesicle belonged to serotonin-immunonegative photoreceptors; (ii) nearly all serotonin-immunoreactive cells in the end-vesicle bore short rod-opsin-immunoreactive outer segments protruding into the pineal lumen; and (iii) the spherical serotonin-immunoreactive cells in the pineal stalk lacked rod-opsin immunoreaction and an outer segment. These results support the concept that multiple cell lines of the photoreceptor type exist in the pineal complex at an early evolutionary stage.

Response patterns and neuronal networks of photosensory pineal organs.

Pineal organs of poikilotherm animals have photoreceptor cells with cone type structures of disk membranes of the outer segment. In the lamprey, Lampetra japonica, photoreceptors responded to light at 6 X 10(-4) lux as a threshold with hyperpolarization, which modulated the spike discharge of the ganglion cells. Electrophysiological data and high performance liquid chromatography analysis of visual pigments of the pineal suggested the presence of several types of photoreceptor cells. Actually, two different immuno-reactivities were observed in photoreceptors by using antibodies against rod specific protein and cone specific protein. Light information of the environment perceived by the pineal organ is transmitted to the central nervous system and contributes to the control of the locomotor activity rhythm of the lamprey.

Presence of retina-specific proteins in the lamprey pineal complex.

The pineal complex of river lamprey reacted with the antisera raised against retina specific proteins including bovine opsin, chick visinin and frog light-sensitive cyclic GMP phosphodiesterase (PDE). Immunoreactive materials stained with anti-opsin were evenly located at the outer segment of photoreceptor cells in the pineal organ and also found in the parapineal organ. Although anti-visinin stained the pineal and parapineal photoreceptor cells, the immunopositive photoreceptor cells were observed only at the lateral portion and not at the medial portion of the pineal organ. No immunoreactive materials were found in the pineal complex by the anti-PDE, whereas the anti-PDE reacted with photoreceptor cells of the retinal tissue. The data suggest that the pineal and parapineal retinas of lamprey contain opsin- and visinin-like proteins with different distribution in their photoreceptor cell layer as found in the lamprey retinal tissue.

Photoreception in pineal organs of larval and adult lampreys, Lampetra japonica.

A comparative study of the larval and adult pineal organs, which are sensitive to incident light, was carried out in the river lamprey Lampetra japonica, using intracellular recording from the pineal photoreceptors. The tissue overlying the larval pineal organ is transparent, whereas that over the adult pineal is translucent. The optical density of this oval pineal window in the adult lamprey was 1.2. In order to elucidate the early development of the larval pineal, the ratio r of the diameter (micron) of the pineal to the body-length (cm) was measured. The value of r was 62.5 in a small larva of 2.8 cm, 29.7 in a larger one of 14.3 cm, and 9.3 in an adult of 54 cm body-length. The intracellular response to light of the larval pineal was a hyperpolarization, showing fundamentally the same pattern as that of the adult pineal. It was possible to record a typical response even from the pineal of the smallest larva, 2.8 cm in body length, used in this study. The intensity-amplitude relationship was analysed after Naka-Rushton's hyperbolic equation. The value of sigma of isolated larval pineals was 0.88 log unit higher than that of adults. The value of n was larger in larvae, suggesting a sensitive reaction to changing photic stimulus. The spectral sensitivity was compared. The peak was at 505 nm in the larva, but 525 nm in the adult. A change of visual pigment in the pineal during metamorphosis is suggested.