Norihiko Uto

Presence of Rhodopsin and Porphyropsin in the Eyes of 164 Fishes, Representing Marine, Diadromous, Coastal and Freshwater Species—A Qualitative and Comparative Study

There are two types of visual pigments in fish eyes; most marine fishes have rhodopsin, while most freshwater fishes have porphyropsin. The biochemical basis for this dichotomy is the nature of the chromophores, retinal (A1) and 3‐dehydroretinal (A2), each of which is bound by an opsin. In order to study the regional distribution of these visual pigments, we performed a new survey of the visual pigment chromophores in the eyes of many species of fish. Fish eyes from 164 species were used to examine their chromophores by high‐performance liquid chromatography—44 species of freshwater fish, 20 of peripheral freshwater fish (coastal species), 10 of diadromous fish and 90 of seawater fish (marine species) were studied. The eyes of freshwater fish, limb freshwater fish and diadromous fish had both A1 and A2 chromophores, whereas those of marine fish possessed only A1 chromophores. Our results are similar to those of previous studies; however, we made a new finding that fish which live in freshwater possessed A1 if living near the sea and A2 if living far from the sea if they possessed only one type of chromophore.

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.

The motility and fertility of golden hamster sperm cultured in BSA‐free medium

Summry— Before fertilization, capacitation and the acrosome reaction in mammalian spermatozoa must be completed. The motility and fertility of hamster sperm were examined in four kinds of modified Tyrodes solution with or without bovine serum albumin (BSA). Since the presence or absence of polyvinylalcohol (PVA) in the media was another variable, its effect on the sperm motility and fertility was also studied. Sperm were incubated in four different media for up to 6 h at 37.5°C. After 4 h of incubation in the media containing BSA alone or BSA and PVA, sperm were hyperactivated, showing a high sperm motility index (SMI) and were able to fertilize more than 80% of eggs. However, their fertility rapidly decreased during further incubation. In contrast, sperm in the medium containing PVA and no BSA showed low SMI scores after 4 h. However, during the following 2‐h period, the SMI progres‐sively increased and sperm were hyperactivated. Furthermore, the hyperactivated sperm in the PVA containing medium were able to effectively fertilize eggs. Our results indicate that hamster sperm can be capacitated in BSA‐free medium and that capacitation occurs much more slowly in such a medium. We suggest that PVA is a reasonable alternative to BSA in in vitro fertilization and that this slowly progressing system may be a good model for studying various steps in sperm activation.

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO3, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd3+), a Ca2+ channel blocker, on spicule formation. Gd3+ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd3+, of which levels are considered to be insufficient to block Ca2+ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd3+, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd3+ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd3+ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca2+ channels were not inhibited. These results suggest that Gd3+ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca2+ channels.