For precise temporal activation of the egg during amphibian fertilization, the sperm must provide a signal for egg activation at the time of membrane binding or fusion between sperm and eggs. A fertilizing sperm causes a Ca² wave which is both necessary and sufficient for egg activation at amphibian fertilization. The Ca² wave seems to be mediated by IP3-receptors on the endoplasmic reticulum and by IP3 produced by hydrolysis of PLC activated by a Src-related protein tyrosine kinase (Xyk) in Xenopus eggs. We have proposed three different hypotheses for initiation of egg activation in amphibian eggs: the Ca² -influx model, the membrane receptor model, and the soluble factor model. The membrane receptor model and the soluble factor model seems to be applied to the monospermic Xenopus fertilization and the physiologically polyspermic Cynops fertilization, respectively. The Ca² wave at egg activation induces a positive fertilization potential which prevents entry of a second sperm in fertilization of monospermic species. In physiologically polyspermic urodele eggs, several sperm enter the egg at normal fertilization, but only one sperm nucleus with a centrosome participates in the embryonic development. The degeneration of accessory sperm nuclei is closely involved in differential distributions of both γ-tubulin and cyclin B in the egg cytoplasm, which causes developing a larger sperm aster and earlier entry into M phase in a zygote nucleus, respectively. We have discussed the molecular mechanisms of egg activation and polyspermy blocks in amphibians and make some comparisons with other vertebrates, such as fishes and mammals.

The G protein messages in Octopus vulgaris photoreceptor were isolated and characterized with molecular biological techniques. Four classes of G protein α subunit cDNA were isolated from an octopus eye cDNA library: OvGα_i, OvGα_o, OvGα_q and OvGα_s. Northern blot analysis of octopus tissues showed abundant expression of OvGα_q in the eye, and specific expression of OvGα_o in neural tissues. OvGα_i was expressed in all tissues studied. In situ hybridization revealed that OvGα_q message was expressed in almost all photoreceptor cells. Based on these results, a possible phototransduction pathway in the octopus visual cells is discussed.

L-Cysteine is a competitve inhibitor of the pyruvate kinase (PK) isozymes from the body wall and introvert of the sipunculan Phascolosoma arcuatum exposed to normoxia or anoxia. Firstly, the 1/V versus 1/[phosphoenolpyruvate] plot shows that the V_max values of PK isozymes from these body parts were unaffected by L-cysteine with exception of the body wall from the anoxic worm. Secondly, the Dixon plot shows that the percentage inhibition of PK activity by L-cysteine decreased with increasing concentrations of phosphoenolpyruvate. Kinetic properties of PK isozymes from the body wall and introvert of P. arcuatum could be altered by anoxic exposure. In anoxia, the modified PK isozymes from these body parts had lower affinity to phosphoenolpyruvate and L-cysteine. Despite the increase in K_i(L-cysteine) value upon anoxic exposure, the [I]_0.1, [I]_0.5 and [I]_0.9 values of L-cysteine for these PK isozymes were lower than those obtained for the normoxic worms. L-Cysteine was a more effective inhibitor than alanine for PK isozymes from the body wall and introvert. However, the effect of L-cysteine on PK was tissue specific and it had no effect on the isozyme obtained from the internal organs.

Visual features of the wing color, with special reference to the UV (ultraviolet) color, of the British subspecies of the cabbage butterfly, Pieris rapae rapae and its mating behavior were investigated. Both sexes of the British subspecies were found to lack UV color and differed only slightly in color in the visible color range, with female wings more yellowish. It follows that they show only slight sexual dimorphism in wing color. It was shown that the initial mate recognition was mediated visually, based on the wing color. Males discriminated between the sexes visually, but only marginally and were occasionally observed to approach other males mistakenly. The resting males approached by female-searching males displayed a flutter response, deterring the approaching males from attempting to copulate with them; i.e. it functioned as “mechanical isolation mechanism” against maladaptive copulatory attempts between males. The results are discussed in terms of the comparative ethology of the mating behavior with that of the Japanese subspecies. It is suggested that the British subspecies is ancestral to the Japanese subspecies.

A new reliable bioassay method has been developed for detection of female sex pheromone(s) of the helmet crab Telmessus cheiragonus using artificial sponges based on the grasping behavior of male crabs. The seawater, male urine, and pre- and postmolt female urine were tested for pheromonal activity which resulted in the detection of activity only in urine from pre- and postmolt females.

Paramecium bursaria shows many kinds of circadian rhythms, including a mating reactivity rhythm. P. bursaria cells normally contain several hundred Chlorella in the cytoplasm as endosymbionts. We found an interesting mutant strain (Ok2) from nature. Chlorella-contain green cells (Ok2) showed a normal mating reactivity rhythm in a constant light condition (LL) or constant darkness (DD). However, Chlorella-free white cells (Ok2w) derived from Ok2 did not display a mating reactivity rhythm in LL. In DD, they showed a normal mating reactivity rhythm. When Ok2w cells were infected with Chlorella isolated from green cells that show normal mating reactivity rhythms, they exhibited a circadian mating rhythm in LL. The green Ok2 cells reverted to the non-reactive state in LL by treatment with the herbicide paraquat. Sugar components in the cytosol of Ok2 and Ok2w were analyzed by HPLC, and four kinds of sugars were identified in Ok2 cells of day time. When maltose and maltotriose were added to Ok2w cell culture, the mating reactivity rhythms appeared in white cells in LL. These results suggest that the photosynthetic products of symbiotic Chlorella are closely related to the expression of circadian rhythms in a mutant strain of P. bursaria.

Mottled mosaic strains in the silkworm, induced by X-ray irradiation, contain chromosomal fragments carrying the larval body marking genes that are lost occasionally during larval development. In one of the mosaic strains, mottled zebra (Ze^m), the somatic loss of the chromosomal fragment is presumed to cause the mosaic pattern, but the fragment has not yet been identified. Here, we showed that Ze^m individuals have an extra small chromosomal fragment (Ze fragment) using genetical and cytological methods. The rate of loss of the Ze fragment, calculated based on the data of segregation distortion, is higher than one from a different mottled strain, mottled striped (p^Sm). Fluorescent in situ hybridization with theBombyxtelomeric sequence (TTAGG)_n as a probe demonstrated that the Ze fragment has a telomeric repeats at only one chromosomal end, although the fragment of p^Sm (p^S fragment) has the repeats at both ends. These data show that the broken ends of chromosomal fragments generated due to X-ray irradiation could be basically healed by de novo addition of the telomeric repeats and the structural difference of telomere may be related to the stability of chromosomal fragments.

The avian hatching process is considered to comprise a series of complicated phases including the digestion of vitelline membrane and egg white and the breakdown of shell membrane and the eggshell. The present study focuses on the first phase, i.e., the digestion of vitelline membrane in Japanese quail Coturnix japonica. When embryos were subjected to immunocytochemical tests with an anti-Xenopus hatching enzyme antibody, staining was observed on day 0 of incubation in the outermost ectodermal cells of blastoderms, and later in the ectodermal cells of yolk sacs in the area vitellina. It was confirmed by electron microscopy that what had been specifically stained in these (two locations) were secretory granules in the cells. A 57 kDa protein was detected by immunoblot tests of the extracts of yolk sac material from the area vitellina. Ultrastructural disintegration of the vitelline membrane followed the advance of the yolk sac toward the vegetal pole. We propose that the step-by-step digestion of the vitelline membrane from the animal pole side toward the vegetal pole one is accomplished by an enzyme produced by the outermost ectodermal cells which show a temporary secretory activity.

Male members of the seaweed pipefish, Syngnathus schlegeli, incubate eggs in the brood pouch located on the tail. The eggs are directly spawned into the brood pouch by inverted copulation after intensive courtship, and the fertilization takes place in the brood pouch. During gestation, the brood pouch is filled with viscous fluid that seems to be of maternal origin, indicated by the absence of mucous secretion of the brood pouch epithelium. The spermatozoa are considered to swim in viscous ovarian fluid during fertilization. These findings indicate that the environment for fertilization is equivalent to that for internal fertilization. The pipefish spermatozoa had a bullet-shaped nucleus (3×0.6 μm), a spiral mitochondrion and an elongate flagellum (ca. 85 μm) with centrioles embedded in deep basal fossa. Based on the morphological features, the pipefish spermatozoon may be categorized in introsperm (internally fertilizing sperm). The spermatozoa swim straight by beating the entire length of the flagellum (84.1±43.8 μm/sec, ±SD, n = 3). The number of spermatozoa in the testis was extremely small (1–2 nuclei per whole transverse sectional area). The mode of fertilization is considered to enable the reduction of the spermatozoan density without deteriorating the success of fertilization. Apart from the typical spermatozoa, another type of spermatozoa with the head about 3 times as large as that of typical spermatozoa was observed. The atypical spermatozoa swim in circles (45 turn/min). Possible natures of the atypical spermatozoa are discussed.

cDNA cloning and 5′-RACE experiments were conducted on β-amyloid precursor protein (APP) using porcine ovary mRNA. The isolated cDNA clone and the clones generated by 5′RACE spanned 3,051 bp containing the complete open reading frame of APP which consisted of 770 amino acid residues. The amino acid sequence of porcine APP was 97.8, 97.1, and 97.4% homologous to those of human, mouse, and guinea pig APPs, respectively. The expression of APP in several porcine tissues was examined by Northern blot analysis. The ovaries and adrenal glands showed a strong expression of the APP mRNA, as did the granulosa cells from small and large follicles of the porcine ovary. RT-PCR analyses using two primer sets revealed that the porcine ovary expressed at least four types of APP mRNAs. Western blot analysis was conducted using the extract of granulosa cells and the fluid of ovarian follicles, and the results indicated that the follicular fluid contained soluble APP in relatively high content. These results suggest that APP undergoes proteolytic processing and/or degradation within the follicles during follicular development.

Acetylcholine, released from cholinergic nerve terminals, innervated to adrenal chromaffin cells, evokes catecholamine release from the chromaffin cells. In the previous studies neostigmine, an acetylcholinesterase inhibitor, nicotine and oxotremorine, a muscarinic receptor agonist, hardly induce catecholamine release from adrenal medulla of 21-day-old rats. Not only adrenaline but also noradrenaline is released from the chromaffin cells by insulin-induced hypoglycemia in fasted 21-day-old rats, whereas preferential adrenaline release occurs in fasted 8-week-old rats. The purpose of the present study was to characterize the catecholamine output induced by hypoglycemia in 21-day-old rats. Hexamethonium, a nicotinic receptor antagonist, blocked the adrenaline and noradrenaline release from the chromaffin cells almost completely as judged from measuring the catecholamine content and observing the morphological changes of the chromaffin cells. Nicotine or oxotremorine, injected into the fasted animals, induced catecholamine release. In the multiple steps of the chromaffin granule exocytotic pathway one or several steps are probably inactive in infant rat and these steps become to be fully active by starvation. The nicotinic receptors of the chromaffin cells in infant rats mainly contribute the secretion induced by hypoglycemia. It has been postulated that the nicotinic receptors are primarily concentrated in the synaptic zones of the chromaffin cell membrane and are involved in the physiological stimulation, whereas the extrasynaptic regions contain a mixture of the nicotinic and muscarinic receptors, these are activated by injected secretogogues.

In Bombyx 5th instar larvae, haemolymph trehalose concentrations were maintained in a range of 8-14 mM during the feeding period and then decreased to approximately half the level concomitantly with the occurrence of a gut purge. The decrease occurred as well in larvae that were starved for 29 hr before the gut purge and those that were ligated around the neck. In contrast, ligation between the thorax and abdomen suppressed such a decrease in trehalose concentrations. These results indicated that thoracic factor(s) was involved in the decrease in the haemolymph trehalose concentration. Injections of ecdysteroids into the isolated abdomens of day 5 larvae resulted in a decrease in haemolymph trehalose concentrations. These findings suggest that an increase in the haemolymph ecdysteroid titer at the wondering stage brings about a decrease in the haemolymph trehalose concentrations. Ecdysteroids thus appear to play an important role in the changes in the carbohydrate metabolism at the larval-pupal transformation.

In mammals, the pituitary hormone prolactin is also produced in various extrapituitary tissues and may act in an auto/paracrine fashion. To explore the comparative aspects of extrapituitary prolactin, the distributions of prolactin transcripts were investigated in extrapituitary organs of the goldfish, African clawed frog and mouse by reverse transcription-polymerase chain reaction (RT-PCR). For comparison, the amount of the transcript in mouse tissues was also estimated using competitive RT-PCR. In the goldfish, the transcript was detected in the ovary, testis, liver, kidney, spleen, gill, muscle and brain in slightly lower abundance than in the pituitary, but not detected in the intestine. In the frog, the transcript was detected in the following organs with an order of abundance: pituitary >> brain > testis and ovary. In the mouse, the transcript was detected in the pituitary, brain, testis, and ovary, and its copy number per μg of total RNA was estimated at ~10¹⁰ in the male pituitary, ~10⁴ in the placenta, hypothalamus and testis, ~10³ in the thymus and experimentally induced deciduoma, and ~10² in the ovary. These results suggest that the origin of extrapituitary prolactin goes back to the common ancestor of fish and tetrapods, but that distinct evolution has occurred in each lineage. The significance of extrapituitary PRL in non-mammalian species is also suggested.

In this paper we report the effect of gonadectomy and/or long-term sex steroid (17β-estradiol and testosterone) treatment on binding activity of 17β-estradiol and testosterone binding proteins (EBP and TBP, respectively) in the plasma of the female of the green frog Rana esculenta. Experiments were carried out during different periods of the reproductive cycle when circulating levels of 17β-estradiol and androgens were : 1) low, 2) medium; 3) medium-high; 4) high. This study shows that EBP, but not TBP activity were affected by 17β-estradiol and testosterone treatment. The effect of the hormonal treatment changed according to the period of the reproductive cycle when it was carried out. Both 17β-estradiol and testosterone were ineffective when the circulating levels of 17β-estradiol and androgens were medium-high and high. On the contrary, the maximum effect was registered when circulating 17β-estradiol and androgens were at their minimum levels. Thus, our data indicate that binding activity of EBP apparent changes in response to 17β-estradiol and testosterone treatment varied according to the period of the reproductive cycle, an indication that studies on sex steroid binding proteins regulation should take into consideration the internal endocrine condition before drawing any final conclusion especially in species with a seasonal mode of reproduction.

Pituitary hormones regulate various physiological functions during spawning migration in salmonid. Cytological features of pituitary cells were therefore immunocytochemically examined by use of antisera against homologous hormones in pre-spawning chum salmon (Oncorhynchus keta) caught from Ishikari Bay and the Chitose River in October, 1996. Immunoreactivity and sizes of pituitary cells were determined by a computer-aided image analyzing technique. Immunoreactivity in growth hormone (GH) cells was stronger with enlarged cell sizes in freshwater (FW) fish than in seawater (SW) ones of both sexes. Majority of prolactin (PRL) cells also had significantly stronger immunoreactivity with enlarged cell sizes in FW fish than in SW animals of both sexes. Immunoreactivity in somatolactin (SL) cells was markedly stronger with enlarged cell sizes in FW fish than in SW ones of both males and females. In addition, greater portions of SL cells were strongly stained in FW animals than in SW ones. A greater portion of gonadotropin (GTH) I cells had stronger immunoreactivity with reduced cell sizes in FW fish of both sexes, when compared with SW fish. Conversely, GTH II cells had significantly stronger immunoreactivity with enlarged cell sizes in FW ones of both sexes. In proopiomelanocortin (POMC)-derived hormone producing cells, adrenocorticotropin (ACTH) cells had stronger immunoreactivity in FW animals of both sexes, while cell sizes did not change. In melanotropes, the cells immunoreactive to α-melanophore stimulating hormone (α-MSH) antiserum had stronger immunoreactivity with reduced cell sizes only in FW males, while the cells immunoreactive to β-endorphin antiserum had stronger immunoreactivity with reduced cell sizes in FW fish than in SW animals of both sexes. Implication of these results was discussed along with previous reports on gene expression of pituitary hormone precursors.

A new oribatid mite belonging to the family Ceratozetidae is described from the province of Biscay in the Basque Country, Northern Spain, being the second species for this genus in Palearctic Region. Edwardzetes ubali is proposed for this new species and main differences among the remaining Edwardzetes are presented in this paper.

The reef-building scleractinian coral Oulastrea crispata (Lamarck) has a conspicuous black skeleton. Skeletal pigments of O. crispata from 10 sites in Japan were measured quantitatively to compare geographic variation in skeletal color of the species. The level of pigment concentration varied from place to place, but showed no consistent pattern with the geographic distribution. Cross transplantation experiment between two of the sites showed that the skeletal color is changeable in both directions rather than being a genetically fixed trait at each site. Although environmental factor(s) controlling skeletal pigmentation has not been specified, it is assumed that light and sea water temperature are not involved in skeletal pigmentation.

A notodelphyid species inhabits in the colonial ascidian, Diplosoma virens that contains photosynthetic symbionts Prochloron sp. in the cloacal canal. The gut contents of the notodelphyids were examined by light and electron microscopy to estimate their diet. Since the gut contents are almost homogeneous, the notodelphyids are probably monophagous. The histochemical stainability of the gut contents suggests that they mainly feed on the host tunic. Although the host faeces and the fragments of the algal symbionts are sometimes found in the gut contents, they would be accidentally ingested by the notodelphyids.