Proteolytic processing of Blattella germanica vitellin during early embryo development.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 15:119-135 (1990) Proteolyt ic Processing of Mattella germanica Vitellin During Early Embryo Development John H. Nordin, Esther L. Beaudoin, and Xiaodong Liu Department of Biochemistry and Program in Molecular and Cellular Biology University of Massachusetts, Amkerst, Massachusetts In the eggs of the cockroach Blattella germanica, vitellin (Vt) utilization i s initiated 4 days postovulation by the proteolytic processing of its three subunits. These reactions yield a specific set of peptides that are consumed by the developing embryo. A yolk proteinase activity, believed central to this processing event, has been investigated. First expressed at day 3 postovulation, just prior to Vt’s processing, its specific activity with synthetic substrates increased fourfold to 18-fold through day 6. In addition, a mixing experiment showed that these proteinases(s) can also process Vt’s large subunits in vitro. A relationship between Vt processing and proteinase specific activity was also noted with two B. germanica translocation heterozygotes, which displayed differences in the extent of Vt processing. One group of eggs (group A) failed to process any Vt subunit. A second group (6)processed the M,102,000 subunit but not the M,95,000. A third group (C) processed their Vt normally. Proteinase specific activities in the yolk of translocant’s eggs at day 6 mirrored the extent of processing, being highest i n group C eggs and effectively absent from the yolk of group A eggs. Eggs defective in Vt processing also contained arrested embryos. It i s concluded that the yolk proteinase activity described here participates in Vt processing at day 4 postovulation. Microscopic exarnination of yolk obtained from eggs of wild type females showed that, as processing began in vivo (day 4), the yolk granules also underwent an abrupt decrease in size from diametersof15-30ym to3-10pm. Yolkgranulesof those translocant’s eggs that were defective in Vt processing did not undergo this size decrease, suggesting that granule reorganization and Vt proteolysis may be linked functionally. Key words: yolk proteinase, yolk granules, translocation heterozygote Acknowledgments: This research was supported by National Science Foundation grant no. DCB 88-19371. The authors thank Dr. C.-M. Yin for the statistical analysis and Drs. J.R Purcell, J.G. Stoffolano, and C.-M. Yin for their critical reading of the manuscript. Received May 7,1990; accepted July 5,1990. Address reprint requests to Dr. John H.Nordin, Department of Biochemistry, Lederle Graduate Research Center, University of Massachusetts, Amherst, M A 01003. 0 1990 Wiley-Liss, Inc. 120 Nordin et al. INTRODUCTION Insect Vg" is sequestered by the oocyte from the hemolymph by adsorptive endocytosis and, following transient localization in endosomes, segregates into storage vesicles called yolk spheres or granules as the egg-bound form known as vitellin [reviewedin 1-31. Although a general understanding of the mechanisms underlying these steps has emerged, much less is known about Vt utilization in insects. However, some aspects of the degradative process have been described. It is clear that Vt degradation in the egg is coordinated with embryo development. This has been demonstrated for Locusta migratoriu , Blattella germmica [5,6], Culliphora erythrocephala , Bombyx mori , and Xhodnius prolixus . Most important, however, Vt utilization is not a random degradation of the molecule but involves an ordered consumption of constituent polypeptides. This can be preceded by structural modifications of the subunits. For example, the Vts of Leucophaea maderae [lo], Periplanetu americana [ll],13. germunicu [5,6,12,13]and Caruusius morosus [141 are proteolytically processed prior to utilization, but the Vts of X.prolixus , Drosophilu melanogaster , and Phormiu regina (J.H. Nordin and A. Cecchettini, unpublished data) are not. Individual subunits of Vt can be degraded simultaneously, as reported for D. melanogaster and B. germanicu  or at different times, as in C. erythrocephala  and C. morosus . Proteolytic processing appears to be a characteristic that distinguishes the utilization of class I  Vts [6,10,11,13,14]from class I1 Vts, which are degraded without prior proteolytic modification [9,15]. This may reflect the fact that class I Vts contain large subunits (M, 120,000-180,000), whereas class I1 Vts do not. It is also clear that modifications of covalent structure such as oligosaccharide trimming  or dephosphorylation can accompany or precede proteolysis. The proteolytic processing of B. germmica Vt is an important aspect of its physiological life, occurring during biosynthesis, upon uptake into the oocyte and finally following formation of the ootheca [5,6,12]. This last event occurs at day 4 postovulation, just prior to the onset of the majority of the embryo's growth. The three subunits of Vt are processed, and a specific set of lower-molecularweight products accumulates . These are then consumed gradually during the remaining 14 days of development . It is evident that the entire population of Vt molecules in the yolk is modified within this brief 24 h period because after day 5 the M, 102,000 and M, 95,000 subunits are absent. Processing of the M, 50,000 subunit occurs more slowly and is not complete until day 6-7 .The major processing products with molecular weights of approximately 53,000,50,000,45,000, and 20,000 (2 peptides) were identified, and the relationship of each of these to the individual subunits of mature Vt in freshly ovulated eggs was established by using Western blots with subunit-specific antibodies . Our laboratory is conducting a number of studies aimed at understanding how Vt utilization is regulated in B. germmica. One of these is an analysis of yolk during the first 7 days postovulation for enzymes that might be important in the proteolytic processing described above. In the course of this work, we *Abbreviations used: AZA = azoalbumin; B C A = bis-cinchinonicacid; BSA = bovine serum pepstatin A = isovaleryl-L-valyl-L-valyl (35,4S) albumin; leupeptin = N-acetyl-leucyl-leucyl-arginal; 4-amino 3-hydroxy 6-methylheptanoyl L-alanyl (3S,4S) 4-amino 3-hydroxy 6-methyl heptanoic acid; PMSF = phenyl methyl sulfonyl fluoride; SDS = sodium dodecyl sulfate; Vg = vitellogenin; Vt = vitellin; Z-AMC = benzoyloxycarbonyl L-phenylalanyl L-arginyl 7-amido 4-methyl coumarylamide; Z-LNE = benzoyloxycarbonyl~-lysyl(4-nitro)phenyl ester. Proteolytic Processing of Vitellin 121 have utilized two B. germanica translocation heterozygotes bearing, respectively, translocations between chromosomes 3 and 12 (TH 3;12) and chromosomes 7 and 12 (TH 7;12). These mutants produce oothecae in which 40% to 50% of the embryos fail to mature, many being arrested early in development [17-191. Certain characteristics of the eggs of these translocants led us to consider the possibility that arrested eggs might be defective in processing their Vt and that they would provide information useful for the broader investigation. Results of preliminary experiments with one of these mutants  supported this premise. In this present work, we explored the relationship between yolk proteinase activity, processing of Vt at day 4 postovulation, yolk granule structure, and embryo development using both wild-type and translocant B . germanica. MATERIALS AND METHODS Materials BSA, 2-LNE, AZA, Z-AMC, leupeptin, PMSF, SDS-PAGE high-molecularweight markers, acrylamide, Coomassie brilliant blue, and Triton X-100 were obtained from Sigma Chemical Co., St. Louis, MO. Reagents for silver staining of gels were purchased from Bio-Rad, Rockville Centre, NY. Insects Developmentally synchronous B. germunica were reared in the laboratory at 30°C . In our laboratory, the first 24 h postovulation is defined as day zero. Two B. germanicu translocation heterozygotes-TH 3;12 and TH 7;12-were kindly provided by Dr. Mary Ross, Department of Entomology, Virginia Polytechnic and State University, Blacksburg, VA. Stocks were maintained by crossings as described by Ross and Cochran [17,18]. Isolation of Yolk Oothecae were carefully detached from females at various times after ovulation, secured on the stage of a dissecting microscope with a small piece of modeling clay, and their eggs were scored for color and texture of yolk as described previously [6). To isolate yolk from individual eggs in an ootheca, the egg was punctured with a minutien pin, nonadjacent eggs being chosen when possible to avoid contaminationby yolk from an adjoining compartment. A fine-tipped glass microcapillarywas used to draw yolk samples. When pooled yolk was prepared, multiple punctures were used, and the yolk was removed with the tip of an automatic pipettor. The yolk was suspended in tubes containing between 50 pl and 200 p1 of buffer, depending on the experiment. Unless noted otherwise, diluents used were buffer A; 110 mM sucrose, 5 mM HEPES, 150 mM NaCI, pH 7.0, or buffer B; 110 mM sucrose, 145 mM NaC1, 14 mM KC1,18.3 mM phosphorous acid, 4 mM CaC12,5.1 mM MgS04, pH 6.8 (modified from ). In certain experiments, individual eggs were also examined for the presence of an embryo dorsal midline to ascertain embryo viability [181. Electrophoresis SDS-PAGE was conducted in a Bio-Rad Mini-Protean I1 cell by a modification [22) of the Laemmli procedure . Gels were visualized with either Coomassie blue dye or with silver staining. 122 Nordin et al. Proteinase Assays Yolk samples were withdrawn from oothecae between day 0 and day 6 postovulation and suspended in buffer A and their proteinase activities measured at 22°C using the following assays : Z-LNE. Yolk, 20 to 150 pg of protein, Z-LNE, 0.06 pmol and sodium acetate buffer, pH 4.2,lOO pmol, were mixed in a final volume of 400 ~ 1The . increase in absorbance at 326 nm was monitored for 5 to 7 min. Product formation was linear for at least 12 min under these conditions. A reagent blank without yolk was included for each reaction. AZA. Yolk, 300 to 500 pg of protein, and sodium acetate buffer, pH 4.2,3.75 kmol, were added to a series of microcentrifuge tubes followed by 1.8 mg AZA (60 pl final volume). Reactions were terminated by adding 600 p1 of chilled 5% trichloroacetic acid. After standing on ice for 1h, samples were centrifuged at 6,5009 for 5 min and the absorbance of the supernatant solution monitored at 366 nm. Product formation was linear for at least 3 h under these conditions. Reagent blanks contained all components except yolk. Zero time samples were prepared by addition of acid immediatelyafter mixing the reaction components. Z-AMC. Yolk, 50 to 150 pg of protein, Z-AMC, 5 nmol, and 250 pmol of sodium acetate buffer, pH 4.2, containing 0.05% Brij 35, were mixed in a final volume of 1 ml and immediately transferred to a cuvette. The amount of hydrolysis product, N-methyl coumarin, was determined in an Aminco Bowman spectrofluorimeter using excitation and emission wavelengths of 370 nm and 460 nm, respectively. Fluorescence (uncorrected) was linear over the range of product concentrations encountered; the amount formed in the first 2 min was utilized in calculations. Reactions were linear for at least 5 min. Reagent blanks, omitting yolk, were run for each sample. Latency Experiments Two procedures were utilized as described below: Yolk samples obtained from wild-type oothecae at day 0 and day 6 postovulation were diluted in buffer A to a concentration of approximately 10 mg per ml. Each preparation was subjected to three freeze-thaw cycles at - 80°C and 22°C. Following this treatment, microscopic examination of an aliquot confirmed lysis of all yolk granules. A second aliquot was assayed for proteinase with 2-LNE as described above. Diluted samples not subjected to freezing and thawing were also included in the experiment as controls. Yolk specimens from day 0 and day 5 oothecae were diluted in buffer A at a concentration of 10 mg per ml and divided into two portions. One was diluted with an equal volume of buffer A containing 0.1% Triton-X 100, the other with an equal volume of buffer A. Microscopic analysis revealed complete lysis in the detergent-treated sample. Aliquots of each tube were then assayed for proteinase activity with Z-LNE as described above. Proteolytic Processing of Vt in Vitro Samples of yolk from eggs at day 0 and day 6 postovulation were drawn into duplicate sets of separate microcapillary tubes, and their ends were sealed with modeling clay. Additional tubes containing equal volumes of each of the above yolk specimens were also prepared. The tubes were then incubated at 30°C for 24 or 49 h, at which time their contents were discharged into 100 p1 of 150 mMNaC1. Prateolytic Processing of Vitellin 123 The diluted samples were assayed for protein concentration , and appropriate aliquots were then subjected to SDS-PAGE analysis as described above. Photomicroscopy Yolk was drawn from eggs at various times postovulation and mixed immediately with buffer B. Suspensions were placed either on a hanging drop microscope slide and photographed through a Nikon Diaphot microscope using a tungsten light source and Ektachrome film (ASA 160) or, when greater magnification was desired, on a printed depression slide with a well diameter of 3.5 mm (Cel-Line Associates, Inc. Newfield, NJ) and photographed through a Leitz Dialux microscope using Kodacolor film (ASA 200). Protein Determinations The concentrations of protein in the various yolk samples were measured by the procedures of Lowry et al. , Bradford , or Smith et al.  using a BSA standard. RESULTS Expression of Proteinase Activity in Wild-Type Eggs Correlates With Initiation of Vt Processing Yolk from wild-type eggs, at various times postovulation, was assayed for proteinases using three different substrates (Fig. 1).Two of these, Z-LNE and 2-AMC, detect certain cathepsins (L and B) ; AZA is widely employed to measure general proteolytic activity . The specific activities at each of the earlier sampling times were normalized with the day 6 value for that substrate set at 100% (Fig. 1, solid bar). Before day 3 postovulation, either no activity or a low baseline level of activity was found in yolk preparations when Z-LNE or AZA was employed as substrate. A somewhat higher activity (approximately 10% of that at day 6) was detected at day 2 with Z-AMC. Parallel increases in specific activity through day 6 were noted with the three substrates, suggesting that either one enzyme was being expressed or, if more than one, coordinate expression was occurring. Specific activity increments between days 3 and 6 were 4-,13-, and 18-fold for Z-AMC, AZA, and Z-LNE, respectively. Because 2-AMC and AZA contain amide (peptide) bonds, designation of the activity as proteolytic is confirmed. Most importantly, however, proteinase activation coincided temporally with the onset of Vt processing, suggesting it functions in this specific proteolytic event. To test whether proteinase deficiency in freshly ovulated eggs was caused by sequestration of enzyme(s) in membrane-bound organelles (making it inaccessible to substrates), yolk was subjected to two different treatments used to unmask cryptic enzyme activity. Yolk taken from eggs at days 0, 5, and 6 postovulation was exposed to cycles of freezing and thawing or to preincubation with Triton X-100 before assay for proteinase activity with Z-LNE. Neither treatment resulted in higher proteinase activities in any of the samples when compared with controls (data not shown). Therefore, the low levels prior to day 3 postovulation are not due simply to vesiculation of the proteinase(s). We previously reported that two other yolk hydrolases, acid phosphatase and p-hexosaminidase, also display this same delay in expression; while yolk or-mannosidase had a rather constant activity from the time of oothecal extru- Nordin et al. 120 Z-LNE AZA Z-AMC DAY6( 0 1 2 3 4 5 6 Days Postovulation Fig. 1. Developmental profile of yolk proteinase activity. At various times postovulation, yolk from 10 to 15 eggs was pooled in buffer A, and the samples were assayed with each of the three substrates as described in Materials and Methods, Specific activitiesare normalized with the day 6 value for each substrate set at 100. sion through day 6 postmlation .Consequently, expression of these enzymes must be regulated independently. The ProteinaseM in Day 6 Yolk Can Process Vt Subunits in Vitro Additional evidence that the yolk proteinase(s) participates in processing of Vt subunits was obtained in an experiment where yolk from wild-type females at day 0 and day 6 postovulation were incubated for various times in vitro, individually or as mixtures, and their polypeptide patterns were examined by SDS-PAGE. Panel A of Figure 2 shows the peptides of the day 0 yolk sample incubated for 45 min (lane l),25 h (lane 2), and 65 h (lane 3). As expected, the M, 102,000, M, 95,000, and M, 50,000 subunit compositions were retained, and no expressiodactivation of proteinase occurred. The minor band at M, 85,100 is normally present in yolk but is not a Vt subunit. Panel B shows the polypeptide composition of the day 6 yolk sample incubated for the same periods in vitro; its principal bands are at M,s 50,000-53,000, M, 45,700, and M,s 20,000-22,000. However, the M, 102,000 and M, 95,000 Vt subunits of day 0 yolk (lane 1)are absent, and prolonged incubation did not change this gel pattern (compare lanes 2,3, and 4). Thus, there was no proteolysis of its constituent polypeptides. Proteolytic Processing of Vitellin 125 TH 3;12 Vt(kd1 0 A B C 6 - C 6 - 102\ 95/50- TH 7;12 Vt(kd1 0 A B 102\ 95/- 50 - Fig. 2. Yolk polypeptides in individual eggs of TH 3;12 and TH 7;12 at day 6 postovulation. Samples of yolk were removed from individual eggs using a glass microcapillary as described in Materials and Methods and diluted in buffer A. Aliquots were assayed for their protein content, and approximately 5 bg of protein were applied in each sample well for SDS-PAGE. Gels were visualized with silver stain. Representative gel lanes showing the polypeptide pattern typical of each group (A-C) are presented. Yolk samples at days 0 (lane 0) and 6 (lane 6) were from eggs of wild-type females. Because the gel containing the day 6 yolk from wild-type eggs was electrophoresed longer than the other samples, i t s components migrated further into the gel. Therefore, the location of the M,50,000 products is indicated by an asterisk. 126 Nordln et al. Panel C, lanes 2 and 3, shows the SDS-PAGE analysis of a separate sample of day 6 yolk. Because the Vt polypeptides in this egg had not been completely converted to the typical day 6 products when the yolk was isolated, certain aspects of the processing event can be seen. Yolk in lane 2 (24 h incubation in vitro) still contains some M, 95,000 subunit, but it had been processed by 49 h (lane 3). However, some M, 45,700 and M,s 20,000-22,000 peptides, typical of day 6 yolk, have formed. In lane 3, two transient processing intermediates of M, 88,000-90,000 (band b) and M, 82,OOGareevident. These products, formed by proteinase(s) which had been activated in vivo prior to yolk removal, have been observed also in samples of yolk drawn at day 5 postovulation [13 and unreported data]. The diminished staining of bands at M, 45,700 and M,s 20,000-22,000 in lane 3 was due to less protein in the sample analyzed. When the day 0 and day 6 yolk samples, shown in panels A and B, were mixed and then incubated, they produced banding patterns shown in panel D, lanes 2, 3, and 4. The content of the M, 102,000 subunit of Vt in the day 0 yolk component decreased within 45 min after mixing with the day 6 yolk (lane 2), as evidence by its lower staining intensity relative to that of the unincubated day 0 sample (lane 1).Proteolysis of this subunit continued through the 25 h time point (lane 3) and by 65 h of incubation was absent from the mixture (lane 4). The M, 95,000 subunit was also degraded, albeit more slowly. The pattern in lane 4 is essentially a combination of bands comprising those of panel B, lane 4, and panel C, lane 3. Transient intermediates of M, 88,000-90,000 (band b) and M, 82,000 are present (lane 4). Note also that these intermediates are not present in the individual day 0 or day 6 samples. Panel E shows another gel of the same samples illustrated in panel D when electrophoresed for 75 rnin (rather than 25 min as in panel D) to improve the separation of the slower moving bands. Lane 5 contains a day 0 yolk control. The pattern in lane 2 shows that the content of M, 95,000 subunit decreased within 45 min and that a product of M, 92,000-93,000, band a, which is not resolved in lane 2 of panel A, has formed. This band then decreases in staining intensity relative to the product of M, 88,000-90,000, band b, which increases in intensity by 65 h (lane 4). The fastest-moving component in the three middle lanes of this gel is the M, 82,000 processing intermediate. It should be noted that complete conversion of intermediates to the typical day 6 products was not attained, even though the 65 h incubation period was in excess of that required in vivo (24 to 36 h), and the two yolk samples were only diluted with each other. Whether this was due to some effect of yolk removal or to enzyme inactivation remains to be determined. Vt Processing at Days 4 to 5 Postovulation is Accompanied by a Size Decrease in Yolk Granules Between days 0 and 3 postovulation, eggs of B . germmica normally have a light color and viscous, turbid ooplasm. At this time, the yolk granules generally range in size from 10 to 30 p m in diameter (Fig. 3, panels G and H), but Fig. 3. Proteolytic processingof yolk polypeptides in vitro. Yolk was obtained from the oothecae of females at day 0 and day 6 postovulation, and each sample was incubated alone or after mixing as described in Materials and Methods. Samples were then diluted in 0.15 M NaCI, assayed for protein, and their polypeptides separated by SDS-PAGE. Approximately 10 pg of protein were applied in each sample well. Developed gels were stained with Coomassie blue. Proteolytic Processingof Vitellin A B D 127 C E The molecular masses, in kilodaltons (kd), of the major components were determined using appropriate standards (not shown). The letters a and b mark the positions of the transient Vt processing products with molecular weights of approximately92,OOO-93,000and 88,000-90,000, respectively. A: Day 0 yolk; B: Day 6 yolk. Samples in lanes 1 , 2 , and 3 of panel A and those in lanes 2 , 3 , and 4 of panel B were incubated 45 min, 25 h, and 65 h, respectively.C: A sample of day 6 yolk from a different ootheca incubated for 24 h (lane 2) and 49 h (lane 3 ) . Lane 1 of panels B and C contains a sample of day 0 yolk which was not incubated. D, E: Polypeptide compositions of a mixture of the same samples of day 0 and day 6 yolk illustrated in panels A and B, following incubation. In each panel, the samples in lanes 2 , 3 , and 4 were incubated 45 min, 25 h, and 65 h, respectively. Lane 1 of panel D and lanes 1 and 5 of panel E contain samples of day 0 yolk that were not incubated. Time of electrophoresis: A, 25 min; E, 75 rnin. 128 Nordin et al. diameters of 60 pm are not unusual. At days 4 to 5, the eggs assume a darker, mottled appearance, which reflects changes occurring in yolk organization [ 18,281. Because this change coincides temporally with proteolytic processing of Vt, yolk granules were monitored for change, microscopically, during the first week postovulation. Panel A shows the granule size distribution of wildtype yolk at day 4. At this time, when Vt processing is normally initiated, they began to decrease in size. Between days 4 and 5 postovulation, when yolk undergoes the transition described above, the granules continued their sharp decline in size. By day 5 the yolk was populated with a mixture of larger and smaller granules (panel B), and by day 6 (panel C) the larger ones were virtually absent, being replaced by vesicles approximately 3 to 10 pm in diameter (panel I). This size change phenomenon was not affected by use of different buffers of equivalent osmolality. In their studies of B. germmica translocation heterozygotes, Ross and Cochran  noted that yolk utilization was heterogeneous among eggs of the translocation heterozygotes TH 3;12 and TH 7;12. The yolk of some eggs underwent transitions in color and appearance at day 4 to day 5, as described above for eggs of wild-type females, whereas the yolks of other eggs in the same ootheca retained the appearance of freshly ovulated eggs. They proposed that this transition is a marker for embryo viability in the egg at that point in development. Our examination of yolk from eggs of TH females suggests that the lack of change in appearance at day 4 to day 5 postovulation probably reflects the fact that the yolk granules in these eggs do not decrease in size. Panels D-F show suspensions of yolk from the eggs of TH 7;12 females at days 4,5, and 6, respectively. In contrast to what occurs in wild-type eggs at day 4 to 5 postovulation, some TH 7;12 eggs retain both the number and larger average size granules typical of freshly ovulated eggs (panels E and F). This retention of large granules was also noted in yolk of some of the eggs of TH 3;12 females (data not shown). Certain Eggs in the Oothecae of TH 3;12 and TH 7;12 Females are Defective in Processing Their Vt In an earlier study of yolk hydrolases, we noted that some eggs of the translocant TH 7;12 were defective in Vt processing in vivo , but because only i? few eggs were analyzed, the scope of the study was expanded. SDSPAGE was conducted on the yolk from more than 100 individual TH 3;12 and TH 7;12 eggs at day 5 to day 6 postovulation. The results, summarized in Figure 4, illustrate the polypeptide patterns which typified the samples. In contrast to eggs from wild-type females, in which Vt is always processed by days 5 to 6, certain eggs of both TH 3;12 and TH 7;12 individuals were clearly defective in processing of the large Vt subunits to smaller polypeptides. The extent of Vt processing in an individual translocant egg placed it in one of three categories. In group A eggs, neither large subunit of the Vt was degraded proteolytically; the Vt retained the subunit composition normally present between day 0 and day 3 postovulation in both wild-type and TH eggs (Fig. 4, lane 0).Gels of this group’s yolk suggest that little degradation of the M, 50,000 subunit is occurring, because the M, 20,000 polypeptide, known to be derived from the M, 50,000 subunit [ 6 ] ,is present only in minor amounts. Proteolytic Processingof Vitellin 129 Fig. 4. Yolk granules from wild-type and PH 7;12 eggs during early development. At various times postovulation, yolk was withdrawn from eggs as described in Materials and Methods and diluted in buffer 6. A-F: Suspensions were placed on a hanging drop slide and photographed through a Nikon Diaphot microscope. A-C: Wild-type; 0-F:TH 7;12. A, D: Day4; 6, E: Day 5; C, F: Day 6. Bar length, 50 pm. C-I: Yolk from wild eggs was placed in awe11with a 3.5 mm diameter printed microscope slide and photographed through a Leitz Dialux microscope. C-I: Days 3 , 0 , and 6, respectively. Bar length-20km. 130 Nordin et al. The yolk of group B eggs, like that from group A, is characterized by retention, at day 6, of its M, 95,000 subunit. However, yolk from eggs in this group lacked the M, 102,000 subunit that characterized the polypeptide pattern of group A eggs. The gel patterns of yolk from eggs in group B also contained bands, principally those with molecular weights of approximately50,000-53,000 and 20,000-22,000, an indicator of Vt processing. In view of the fact that proteolysis of the M, 102,000 subunit was observed routinely in the yolks of group B eggs and, because the polypeptide composition of freshly ovulated eggs of both translocants always contained both large Vt subunits, it seems probable that these defects in group A and B eggs must involve a failure of Vt degradation rather than synthesis. We have observed that polypeptide patterns characteristic of yolk of both group A and B eggs occur as late as day 13 postovulation, indicating that this defect in Vt processing is not due simply to delayed proteolysis of these two subunits (data not shown). The fate of the M, 50,000 subunit in group B eggs could not be determined because another peptide of approximately M, 20,000, derived from the M, 102,000 subunit , occurs in group B eggs. Group C eggs show a Vt processing pattern at day 6 essentially like that of wild-type eggs; both the M, 102,000 and M, 95,000 subunits are absent. Because both large subunits were degraded in eggs of this group, it is presumed that the band present at M, 50,000 to M, 53,000 in yolk of this group contains the normal proteolytic product(s) detected by Western blots of peptides separated from wild-type eggs [131. Eggs in this group also underwent the transition in yolk color and texture and their embryos developed normally (see below). Included in Figure 4 are panels showing the polypeptide patterns of yolk of wild-type females at day 6 (lane 6 ) .These samples were electrophoresed longer than the others in the panel; therefore, the mobility of a M, 50,000 standard run with that particular gel is indicated by the asterisk to the right of the lane. TH Eggs Defective in Vt Processing Are Also Deficient in Yolk Proteinase Activity Assayed in Vitro The correlation between expression of proteinase activity (assayed in vitro) in yolks of wild-type eggs and the onset of Vt processing in vivo suggests that the enzyme(s) responsible for Vt processing is that assayed in vitro with synthetic substrates. Furthermore, the fact that the Vt of group A eggs possesses the normal complement of subunits, but is not processed, also suggests that this proteinase activity normally catalyzes this step in Vt utilization. However, it was critical to establish what relationship, if any, exists between yolk proteinase activity demonstrable in vitro and the extent of proteolytic processing in vivo in the same egg. To examine this issue, we made use of variations in processing of Vt's polypeptides that occur in yolk of TH 3;12 and TH 7;12 eggs. Oothecae of both mutant and wild-type females were removed at various times after ovulation, and the yolks of individual eggs were assayed for proteinase activity with Z-LNE; the extent of in vivo processing of the same egg's Vt was also determined by SDS-PAGE. The results, summarized in Table 1, are organized on the basis of the SDSPAGE results: group A (unprocessed Vt large subunits), group B (partiallyprocessed Vt), and group C (processed Vt). The mean proteinase specific activities Proteolytic Processingof Vitellin 131 TABLE 1. Vitellin Polypeptide Processing and Yolk Proteinase Specific Activities of Individual B. germanica Epes Days Postovulation TH 3;12 6 9 12 TH 7;12 Wild-type 6 8 13 6 8 10 A Unprocessed 0.040b ? O.O23l(3)’ 0.000 2 0.000’ (3) Groupa B Partially Processed * 0.235 0.0701 (11) 0.005 1 0.003’ (3) 0.311 0.0174l(5) -d 0.053 & 0.0151(12) 0.191 & 0.056’ (16) 0.005 (2) 0.035 (1) 0.005 -c 0.002’ (8) 0.360 (2) 0.00 (1) - * - C Processed 0.994k 0.098’(10) 1.01 r 0.1602(3) 0.928 k 0.147‘(5) 0.855 -t 0.095’(21) 0.954 2 0.443(4) 0.508k 0.286’(6) 0.633 & 0.047(6) 0.811 0.181 (7) 0.923 i0.159(9) * “Determined by SDS-PAGE analysis of yolk Vt polypeptides. bMean proteinase specific activity (Z-LNE); A326min-’ mg protein k SEM. Means with the same superscript are not significantly different from each other (P 0.05). ‘Number of eggs. dNosample observed in this group. and the standard errors of the means are presented for each group. At day 6 postovulation, the average specific activities (U per mg protein) of yolk from group C eggs of TH 3;12 females (0.99) and TH 7;12 (0.86) approximated the value of 0.63 observed for the day 6 wild-type eggs examined. There was no significant difference between the mean specific activitiesof these three groups. Thus, the group C eggs of both translocants were considered to be wild-type eggs destined for normal development. Statisticalanalysis of the mean specific activities between older group C eggs was not made because of differences in their developmental age. However, group C eggs of TH 3;12, TH 7;12, as well as those of wild-type females, maintained the highest enzyme activity of all the groups throughout the experimental period. The very low specific activities of group A eggs of both translocants reflected the lack of Vt processing (which is always complete by day 6 postovulation in wild-type eggs). The specific activity of TH 3;12 group A eggs averaged some 25-fold lower than group C eggs, through the first 6 to 9 days’ postovulation. Day 9 eggs with no apparent Vt processing were also devoid of detectable proteinase activity. A parallel result was obtained at days 6 to 8 postovulation with the TH 7;12 translocant (a 17-fold reduction in activity among TH 7;12 group A eggs compared with group C eggs). The absence of processing of the Vt large subunits in any of the group A yolk samples, coupled with extremely low levels of activity with Z-LNE, clearly indicates the proteinase(s) required for the day 4 processing event, which occurs normally in group C eggs of both translocants and in wild-type eggs. At day 8 postovulation, TH 7;12 group A eggs had only about 0.5% of the specific activity of their group C eggs. Among the oothecae examined in this experiment, no egg with unprocessed Vt was found at days 12 to 13 in either translocants’ oothecae, but in another study of day 11eggs from TH 7;12 females, seven of the 21 eggs contained unprocessed Vt (data not shown). The proteinase specific activities noted from group B eggs of both translocants 132 Nordin et al. were generally elevated relative to their group A eggs, but not statistically different (Table I). One significant but puzzling aspect of the group B data is that one, but not both, of the large subunits is proteolytically processed. This could be explained if a second, quantitatively minor, proteinase participates in processing the M, 95,000 subunit but is missing in group B eggs. Another, but less likely, possibility is that the M, 95,000 subunit in the Vt of these eggs lacks a critical structural feature required for proteinase-catalyzed peptide bond cleavage. Interpreting the results with the group B eggs was also complicated by the fact that although practically all had proteinase specific activities higher than the average for group A, a few eggs placed in group B because of their SDS-PAGE pattern lacked detectable proteinase activity with Z-LNE. Due to the limited amount of yolk that can be obtained from a single egg, and because it is impossible to predict in advance if a defective egg belongs to group A or group B, assays with alternative substrates were not conducted with these eggs. In a few eggs of groups A and B, specificactivities were obviously much higher (or lower) than expected, based on the SDS-PAGE results. In these cases, it is possible that the total yolk sample was too small to obtain an accurate protein value. The fact that the proteinase specific activities of group A and B eggs of the TH 7;12 females are not statistically different at day 13 postovulation may be due to this cause. This single anomoly in the results is undoubtedly due to the wide range of values encountered with the wild-type females. Proteolytic Processing of Vt Correlates With the Development of TH 7;12 Embryos To ascertain the relationship between processing of Vt in an egg and the presence of a viable embryo, 33 eggs from the oothecae of seven TH 7;12 females were examined b r evidence of embryo development at days 12-13 postovulation (stage IX of Tanaka [18,28]) as described in Materials and Methods. The polypeptide contents of the yolk from these same eggs were also determined by SDS-PAGE. The results of this study showed that in each of 22 eggs, judged to have a developing embryo, its Vt large subunits were processed. Furthermore, 11eggs that lacked developing embryos all showed a polypeptide complement characteristic of yolk of freshly ovulated eggs. DISCUSSION The results of this investigation support the conclusion that the yolk proteinase detected in vitro is the same activity that processes Vt at day 4 in vivo. 1) The expression of, and large increase in, its specific activity coincide temporally with the proteolytic cleavages that occur in vivo. 2) Proteinase activity of day 6 yolk splits the M, 102,000 and M, 95,000 subunits of the Vt polypeptides of freshly ovulated eggs in vitro. 3) The proteinase(s)of day 6 yolk, although active against synthetic substrates and Vt subunits of day 0 yolk in vitro, does not cleave further the peptides present in day 6 yolk, a property consistent with the presence of these peptides in yolk of older embryos [5,6].4)Translocant eggs which are defective in processing their Vt are also deficient in proteinase activity in vitro. We recently determined that proteinase activity, with either Z-LNE or AZA Proteolytic Processing of Vitellin 133 as substrates, is abolished by preincubation of yolk with leupeptin, but that it is insensitive to aprotinin (X. Liu and J.H. Nordin, unreported data). This observation and the fact that proteinase activity increments with the three substrates are essentially the same suggest that the B. germmica proteinase may be one cathepsin B-like  enzyme. Medina et al. described a cathepsin B in D. melanogaster yolk that degrades Vt [29,30]. However, in contrast to the protease(s) reported here and the trypsin-like enzyme of B. mori eggs , the D . rnelanoguster enzyme does not produce intermediate weight-processing products; therefore, their roles appear to be different. We are now attempting to define the B. germanica activity more precisely. As limited proteolysis is initiated at day 4, the yolk granules undergo a size decrease, suggesting that these events are linked functionally in B. germmica. Larger granules (30 to 60 p m diameter), typical of freshly ovulated eggs, diminish in number rather abruptly during days 4 to 5, being replaced by, or converted to, a population of vesicles 3 to 10 pm in diameter not evident earlier in development (Fig. 3). The possibility that proteolysis and granule reorganization are linked directly is strengthened by the fact that translocant eggs arrested in Vt processing also do not undergo a change in granule size (Fig. 3). In this regard, we have compared the peptide compositions of washed granules of both size populations in wild-type yolk. Eggs at days 0 to 3 contained only the M, 102,000, M, 95,000, and M, 50,000 subunits. The granules prepared from day 6 yolk contained less of the large subunits but did have peptides of sizes typical of yolk at day 6 postovulation (E. Beaudoin and J. Nordin, unreported data). Because Vt processing appears to take place within the granule, a precursor form of the processing proteinase(s) may be packaged with Vt, perhaps by fusion of granules with another vesicle. Although nothing is known about the biochemicalregulation of granule function during embryogenesis, ultrastructural information on a variety of species is available, and some studies suggest that granule restructuring is linked to Vt utilization. Granule sizes vary greatly among insects, ranging from 1 to 4 pm in diameter in D. melanoguster [2,33] to more than 60 pm in B. germmica (this work) and L. migratoriu . These structures are usuaIly distributed randomly throughout the ooplasm, but in the B. mori egg a portion of the granules appears to be organized in clusters . Although an abrupt decline occurs in the size of B. germanica granules during early embryo development, those of B. mori  and P. regina (A. Cecchettini and J. Nordin, unreported data) appear to decrease in number gradually during embryo development without changing size. Rather comprehensive ultrastructural studies describing changes which occur in granules during Vt degradation in the eggs of D.melanogaster and the brine shrimp Avternia salinu have also been presented recently [34,37]. What controls expression of proteinase and initiation of Vt utilization in the B. germanicu egg? Evidence from two in vitro studies currently being done in our laboratory suggests that acidification of the yolk granule may have an important role. We recently reported  that granules from yolk at days 4 to 6 accumulated the fluorescent weak base acridine orange, a widely used probe of vesicle acidification [39,40]. This accumulation (which did not occur in granules from freshly ovulated eggs) was prevented by preincubating granules with m-chloro cyanide phenylhydrazone, ammonium chloride, or monensin, 134 Nordin et al. reagents that dissipate proton gradients . We have also noted that incubation of day 0 yolk at pH 4.0 caused proteolytic processing of the Vt large subunits (J. Nordin and X. Liu, unreported data). The fate of the peptides that accumulate at day 6 postovulation also remains a subject of speculation. Although their primary role is undoubtedly nutritional, whether these, or smaller ones, function directly in the embryo is unknown. Inasmuch as Vt may transport other molecules into the insect oocyte for a specific purpose , it is not unreasonable to expect that certain processing intermediates of B. germanica might serve as more than just a source of amino acids. 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