Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called “lysin” to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm trypsin-like proteases, acrosin and spermosin, the latter of which is a novel sperm protease with thrombin-like substrate specificity, are essential for fertilization in H. roretzi. These molecules contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. In this review, I describe recent progress on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat.

Effects of application of glutamate and glutamatergic ligands were studied to characterize the receptors for glutamate present on the soma membrane of the dorsal unpaired median (DUM) neurons in the thoracic ganglia of the cockroach, Periplaneta americana, using the intracellular recording technique. Application of L-glutamate did not block the GABA-response, and application of β-guanidino-propionic acid, a competitive antagonist for GABA, failed to block the response to L-glutamate. These results indicate that most of L-glutamate action may not be mediated by a GABA-activated channel. To examine glutamate receptor types on the DUM neurons, glutamate receptor agonists were applied. The ionotropic glutamate receptor (iGluR) agonists evoked depolarizations with the following relative rank of order of potency: kainate>AMPA>quisqualate. Metabotropic glutamate receptor (mGluR) agonists also elicited membrane depolarizations or hyperpolarizations associated with an increase in membrane conductance. The mGluR agonists evoked depolarizations or hyperpolarizations with the following relative rank of order: L-CCGI>1S, 3R-ACPD>L-AP4. Depolarization of the same DUM neuron was detected following exposure of kainate and L-CCG-I, suggesting the coexistence of distinct iGluR and mGluR types. A membrane permeable cAMP analog, CPT-cAMP, could not mimic the effect of mGluR agonists. The mGluR selective antagonists, MCCG and MCPG, failed to antagonize the response to mGluR agonists. The involvement of cAMP in the mGluR response was not confirmed in DUM neurons. Although the functional roles of these receptors are unknown, it might be possible then that these extrasynaptic receptors have a modulatory effect on the excitability of the DUM neurons.

Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-toserosal flux of Na was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na₂HPO₄ seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na and the arsenate-sensitive Na flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO₄ symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.

The mechanical properties of smooth muscles in aorta and vas deferens were studied in mice with a mutated basic calponin locus to learn the physiological function of calponin. The intact smooth muscles were stimulated with high KCl and the force development was compared between calponin deficient (knockout, KO) mice and wild type (WT) ones. The isometric force induced by various concentrations of high KCl was lower in KO than in WT both in aorta and in vas deferens. The length-force relations were compared between KO and WT. The active isometric force in KO was significantly lower at most muscle lengths examined than in WT without the change in resting force both in aorta and in vas deferens. In vas deferens, the rate of force development after quick release in length at the peak force was significantly faster in KO than in WT. The above results show that the force development is lower and the rate of cross-bridge cycle is faster in KO mice than in WT ones, suggesting that calponin plays basic roles in the control of the contraction of smooth muscle.

Wing colors of the four species of Chrysozephyrus butterflies were analyzed by a spectrophotometer. As the dorsal wing surface of males showed a strong reflectance when the specimen was tilted, measurements were made by the tilting method. The dorsal wing surface of males which appears green to the human eye reflected UV (315–350 nm) as well as green light (530–550 nm). The reflectance rate of UV to visible green light varied among species with a higher rate for C. hisamatsusanus and C. ataxus, and a lower rate for C. smaragdinus and C. brillantinus. The peak wavelength and the peak height did not shift when the specimen was exposed to direct sunlight at least for 16 hr. Artificial removal of scales by scratching the wing surface decreased reflectance. Blue marks on the forewings of C. brillantinus, C. hisamatsusanus and C. ataxus females reflected UV to visible light of short wavelength, and orange marks on the dorsal surface of the forewing and the ventral surface of the hindwing of C. samaragdinus females showed a higher reflectance at longer wavelengths.

We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%−93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1–1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%−10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.

It is well characterized that melanophores in the tail fin of Xenopus laevis tadpoles are directly photosensitive. In order to better understand the mechanism underlying this direct photosensitivity, we performed a retinal analysis of the tail fins and eyes of Xenopus tadpoles at stages 51-56 using high performance liquid chromatography (HPLC). Following the extraction of retinoids by the formaldehyde method, a fraction containing retinal and/or 3,4-didehydroretinal isomers from the first HPLC analysis were collected. These isomers were then reduced by sodium borohydride to convert retinal and/or 3,4-didehydroretinal isomers into the corresponding retinol isomers to prepare for a second HPLC analysis. Peaks of 11-cis and all-trans 3,4-didehydroretinol were detected in the eyes and tail fins containing melanophores, but they were not detected in the tail fins without melanophores. The amounts of 11-cis and all-trans 3,4-didehydroretinol were 27.5 and 5.7 fmol/fin, respectively, and the total quantity of 3,4-didehydroretinal was calculated at approximately 5×10⁶ molecules/melanophore. These results strongly suggest the presence of 11-cis and all-trans 3,4-didehydroretinal in melanophores of the tadpole tail fin, which probably function as the chromophore of photoreceptive molecules.

The complete primary structures of α^D-2- and β-globin of hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, have been constructed by amino acid sequencing analysis in assistance with nucleotide sequencing analysis of PCR fragments amplified using degenerate oligo-nucleotide primers. Using computer-assisted sequence comparisons, the α^D-2-globin shared a 92.0% sequence identity versus α^D-globin of Geochelone carbonaria, a 75.2% versus α^D-globin of Aves (Rhea americana) and a 62.4% versus α^A-globin of Hb A expressed in adult red blood cells of Geochelone gigantea. Additionally, judging from their primary structures, an identical β-globin was common to the two hemoglobin components, Hb A and Hb D. The α^D-2- and β-globin genes contained the three-exon and twointron configurations and showed the characteristic of all functional vertebrate hemoglobin genes except an abnormal GC dinucleotide instead of the invariant GT at the 5′ end of the second intron sequence. The introns of α^D-2-globin gene were both small (224-bp/first intron, 227-bp/second intron) such that they were quite similar to those of adult α-type globins; the β-globin gene has one small intron (approximately 130-bp) and one large intron (approximately 1590-bp).

A phylogenetic tree constructed on primary structures of 7 α^D-globins from Reptilia (4 species of turtles, 2 species of squamates, and 1 species of sphenodontids) and two embryonic α-like globins from Aves (Gullus gullus) and Mammals (Homo sapiens) showed the following results: (1) α^D-globins except those of squamates were clustered, in which Sphenodon punctatus was a closer species to birds than turtles; (2) separation of the α^A- and α^D-globin genes occurred approximately 250 million years ago after the embryonic α-type globin-genes (π' and ζ) first split off from the ancestor of α-type globin gene family.

Nicotinic acetylcholine receptors (nAChRs) switch from the embryonic-type (α₂βγδ subunits) to the adult-type (α₂βεδ subunits), and disappear besides the neuromuscular junctions with the development of trunk and limb skeletal muscles. However, little is known about this process during the embryonic development of masseter muscle. To identify the time course of the nAChR transition from embryonic day (E) 11 to the newborn stage in mouse masseter muscle, we analyzed the expression level of δ, ε, and γ subunit mRNAs by competitive polymerase chain reaction in combination with reverse transcription as well as distribution of δ subunit protein by immunohistochemistry. The nAChR δ subunit mRNA was initially detected at E11, showed an approximately 25-fold increase (p<0.0001) between E11 and E17, and plateaued thereafter until the newborn stage. Immunostaining for δ subunit was observed in the whole portions of masseter myofibers at E17 and birth, suggesting that the nAChR elimination does not begin even at the newborn stage. The ε subunit mRNA initially appeared at E17, and increased in quantity by 144% (p<0.0001) up to the newborn stage. The quantity of γ subunit mRNA increased by approximately 240% (p<0.0001) between E11 and E17, and then decreased by 22% (p<0.05) from E17 value at the newborn stage. The beginning of the expression of the ε subunit mRNA was coincident with the beginning of the decrease in the quantity of the γ subunit mRNA, suggesting that the nAChR subunit switch begins at E17.

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin α subunit in the ovary was also investigated.

Inhibin α subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin α subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin α subunit on days 1 and 2. On the other hand, positive reactions for inhibin βA and βB subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin βB subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin α subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin.

These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin βA from βB subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin α subunit in interstitial cells of the cyclic hamster.

The role of photoperiod in adult testicular maintenance and body weight and juvenile development was assessed in male Mongolian gerbils (Meriones unguiculatus). Gerbils were raised on a 14L (14 hr of light) photoperiod. In the first study, adult gerbils with functional testes were transferred to thirteen different photoperiods (0L, 2L, 4L, 6L, 8L, 10L, 12L, 14L, 16L, 18L, 20L, 22L, or 24L) and body weights and testicular size were measured every week for 10 weeks. Body weights were similar in all groups. Testicular regression had occurred in animals housed on 0L, 2L, 4L, 6L, 8L, and 24L by week 10. In the second study, 14L-born prepubertal gerbils were transferred to thirteen different photoperiods as in the first study. Body weights and testicular development were examined for 10 weeks. At the end of 10 weeks the body weights of animals in all groups except 24L were similar to those of adults. Animals in 24L had a lower body weight gain. Exposure to 0L, 2L, and 24L inhibited testicular development and testes weights were significantly different from those of the other groups.

These results demonstrate that maintenance of body weight in adult gerbils appears to be independent of photoperiodic signal. Exposure to very long (24L) and short photoperiods (<10 hr) causes testicular regression in adult gerbils. Moreover, different photoperiods experienced in early life can influence prepubertal testis growth and body weight gain.

Using skeletochronology, we determined the age structure of adult Hynobius nebulosus from Kyoto in the breeding season of 1998. From previously marked individuals, the lines of arrested growth proved to be formed once per year, indicating the number of winters each salamander experienced. The age at first reproduction was estimated to be 2.8–2.9 yrs of age in males and 3.8–3.9 yrs in females. The oldest males and females were 9.8–9.9 and 5.8–5.9 yrs of age, respectively, and, therefore the longevity in this species was estimated to be more than 9 yrs for males and 5 yrs for females. The growth curve of male's body size estimated indicated that the growth rate much decreases after males attained sexual maturity. Because body sizes of adults greatly vary even within an age class, it is dangerous to estimate individual age from the size frequency data at least in adults. We discussed age properties in Hynobius by comparing lentic and lotic breeders.