No abstract

In eggs of the cockroach Blattella germanica, vitellin (Vt) utilization by the embryo is initiated at day 4 postovulation by the proteolytic processing of its three subunits to a specific set of peptides. A report from our laboratory (Nordin et al.: Archives of Insect Biochemistry and Physiology 15:119, 1990) described a yolk proteinase, activated at days 3-4, which processes the Vt. Further investigation of this event has focused o n the yolk granules. Granules from eggs 4-6 days postovulation contained a significant subpopulation which accumulated high concentrations of the dye acridine orange (AO), a fluorescent probe of vesicle acidification, while those from eggs 0-3 days postovulation did not. A 0 accumulation was caused by proton translocation and was not due to dye binding or a Donnan equilibrium. The temporal correlation of granule acidification with Vt processing suggests a role for this event in yolk proteinase activation in B. germanica. This hypothesis was supported by the finding that incubation of yolk from freshly ovulated eggs in vitro at p H of 5 and below resulted in Vt processsing. Yolk granules of the blowfly Phormia regina also became acidified but this occurred in the oocyte prior to egg deposition.

Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among t h e subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, t h e M, 160,000 subunit Vt is cleaved to products of M, 95,000 and M, 50,000. In association with an unprocessed M, 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 3OoC), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose-type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4-5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event.Although the predominant oligosaccharide of Vt is Man9GlcNAc2, the M, 50,000 subunit of egg-borne Vt contains a much higher proportion of Man6GlcNAc2 than t h e other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an ootheca permits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo-a-rnannosidase, exo-0-Nacetylgl ucosam i n idase, a-glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo-P-N-acetyl- glucosarninidase activities coincide with the day 4-5 proteolysis, while arnannosidase remains relatively constant throughout the first 6 days.Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.

No abstract

Proteolytic processing of the vitellin in Blattella germanica eggs occurs 4 days postovulation and is correlated with both the onset of its utilization and the major portion of the embryo's growth. Yolk phosphatase i s also expressed coincident with this event, and some aspects of its activation have been investigated. The enzyme is absent from the ooplasm (yolk) during the first 2 days following ovulation but increases approximately 20-fold in specific activity between days 3 and 4, when assayed at pH 3.9 or 4.8 and 9-fold at pH 6.5. No activation is observed for yolk-bound a-mannosidase, its specific activity remains elevated through the first 6 days following ovulation. This suggests that expression of the phosphatase is regulated independently of that of a-mannosidase in the yolk. Yolk with active phosphatase can dephosphorylate native vitellin, delipidated vitellin, and phosphocasein. Sucrose density gradient centrifugation of yolk obtained from eggs 4 days postovulation, revealed that phosphatase activity cosediments with material which reacts with antivitellin antibodies, while a-mannosidase and P-N-acetyl glucosaminidase are found near the top of the gradient. Oothecae derived from crossing certain translocational heterozygote males and wild-type females contain some eggs with severely depressed levels of yolk phosphatase in which embryos do not grow. Vitellin in these eggs fails to undergo proteolytic processing as late as day 5 postovulation and retains the subunit composition of freshly ovulated vitellin.