A novel RNA helicase-like protein during early embryonic development in silkworm Bombyx mori: molecular characterization and intracellular localization.

In order to understand the molecular mechanism of development during early embryogenesis in diapause and non-diapause of the silkworm, mRNA from diapause and non-diapause eggs was compared using the differential display technique. We cloned the full length of a cDNA encoding a novel RNA helicase-like (RHL) protein by the RACE method using a cDNA fragment which was one of the specific cDNAs in the non-diapause eggs. A BLAST search using the predicted amino acid sequence of RHL revealed a low homology (21-25% identity of its partial length) with that of the DEAD-box RNA helicase. Gene expression of the RHL gene of the diapause and non-diapause eggs was investigated by RT-PCR until 60h after oviposition. Amplified RHL cDNA was observed through all the stages in the non-diapause eggs, while in the diapause eggs, cDNA was found in eggs 0-12h after oviposition but disappeared 24-60h after oviposition. When the diapause eggs were activated by HCl treatment after chilling at 4 degrees C for 6 days from 48h after oviposition (artificial diapause termination), cDNA was observed from 12h after HCl treatment. We also investigated the immunohistochemical distribution and localization of RHL in non-diapause eggs using anti-recombinant His-tag RHL antiserum. RHL was distributed in blastoderm cells and yolk cells and was localized in the nucleus and the cytosol of yolk cells. These data suggest that RHL has an important role in the early embryo of the silkworm.

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.

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary.

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-L-Phenylalanyl-L-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.

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

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 Tyrode's 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 degrees 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 progressively 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.