Franco Giorgi

Vitellogenesis in Insects

Fertilized eggs are the crossroads of development. They represent both the origin and the biological goal of each individual organism. Eggs provide in two ways for the embryo they enclose: They contain developmental instructions given by the mother to direct the initial phases of embryogenesis (see Chapters 11–13), and they are usually provisioned with nutritive substances to support the embryo until it can obtain its own food. This chapter concentrates on a portion of the second aspect of oogenesis—how storage materials come to reside in insect eggs.

DIFFERENTIAL VITELLIN POLYPEPTIDE PROCESSING IN INSECT EMBRYOS

Abstract This review focuses on the current status of knowledge regarding the process of vitellogenin (Vg) uptake and vitellin (Vt) storage in insect oocytes. We also consider the overall morphology of the yolk sac as an embryonic organ allowing for both temporal and spatial differentiation of yolk degradation to provide the primary food supply for the embryo. In this context we describe the evidence that demonstrates the occurrence of Vt polypeptide processing and how it may be affected by maternally derived proteases stored in the yolk granules and by the ubiquitin–proteasome system in the cytosol. The extent of Vt polypeptide processing induced by these proteases will be correlated with the structural modifications affecting yolk granules and vitellophages in developing insect embryos. To accomplish these goals the ultrastructural and cytochemical composition of yolk granules during vitellogenesis and embryogenesis will be reviewed. Last but not the least, our current understanding of the role played by acidification of yolk granules in the activation of maternally derived proteases will also be examined.

In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster.

SummaryPinocytotic activity has been analyzed in Drosophila oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm.In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone.

Vitellin degradation in developing embryos of the stick insect Carausius morosus

Abstract Two vitellins of the stick insect Carausius morosus are utilized progressively during embryonic development. The relative titre of each vitellin was determined as a function of the developmental time using rocket immunoelectrophoresis with the specific anti-vitellin serum. The vitellins differ in the rate of utilization, one declining faster than the other. The pattern of vitellin in embryos differing in developmental time was demonstrated by polyacrylamide gel electrophoresis and subjected to immunoblotting using an anti-vitellin serum. Polypeptides E20, E9 and E5 that appeared de-novo during embryonic development were identified as fragments of the vitellins originally accumulated in newly laid eggs. Embryos differing in developmental stages were exposed to [35S]-methionine in vitro. Polypeptides labelled in vitro differed in molecular weight from those reactive to the anti-vitellin serum. Based on the above observations, it is concluded that vitellin degradation in C. morosus entails a stepwise degradation of vitellin polypeptides leading to the appearance of several derivatives of smaller molecular masses.

Intercellular bridges in ovarian follicle cells of Drosophila melanogaster

SummaryIntercellular bridges have been detected in ovarian follicle cells of Drosophila melanogaster. These bridges occur widely between follicle cells of previtellogenic chambers, while, in vitellogenic chambers, they become restricted to the columnar follicle cells. Usually, only one bridge is detectable between adjacent follicle cells, but a single cell may form two cytoplasmic continuities.The fine structure of the intercellular bridges is similar to that previously described in the development of Drosophila. The bridge wall consists of two layers of which the more external is more electron dense and thinner than the inner one.The role played by the intercellular bridges in the determination of a synchronous differentiation of the linked follicle cells is discussed in relation to the known behaviour of these cells in the secretion of the egg covering precursors.

Yolk granules are differentially acidified during embryo development in the stick insect Carausius morosus

Abstract. Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.

Cytochemistry of late ovarian chambers of Drosophila melanogaster

SummaryLate ovarian chambers of Drosophila melanogaster have been examined by ultrastructural cytochemistry in an attempt to characterize some of the transformations which precede the completion of oogenesis. From stage 11 onward peroxidase activity is present in the endoplasmic reticulum of both nurse cells and oocyte, as well as in the egg-covering precursors of the columnar follicle cells. Catalase activity is restricted to the very last stages of oogenesis (stage 13–14) and appears to be located in membrane-bound organelles of the ooplasm which are continuous with the endoplasmic reticulum. Because of the presence of catalase as well as by their structural appearance, these organelles are to be identified as microperoxisomes. Catalase activity becomes cytochemically detectable in the ooplasm somehow in coincidence with the formation of glycogen. Furthermore, glycogen is first formed in intimate association with alpha-1 yolk platelets. On the basis of these findings it is suggested that glycogen synthesis occurs by a process of gluconeogenesis.

Larval salivary gland proteins of the sheep nasal bot fly, (Oestrus ovis L.), are major immunogens in infested sheep

Tissue extracts from larval instars of the sheep nasal bot, Oestrus ovis, were resolved by gel electrophoresis under both native and denaturing conditions. Polypeptides resolved under these conditions were tested by immunoblotting against sera of infested sheep. Of all tissues examined in this study, salivary glands proved to be major immunogens in infested sheep. Salivary gland polypeptides were also detected in the washing solution as larval secretory products (LSP). To a minor extent, a few polypeptides from the larval cuticle were also found to be immunogenic, but they did not contribute to LSP. These results were further corroborated by nasal infestation of rabbits that also developed specific antibodies against larval salivary gland polypeptides from Oestrus ovis.

The Thyroid Disruptor 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl)-Ethane Appears to Be an Uncompetitive Inverse Agonist for the Thyrotropin Receptor

In this study, we aimed at establishing whether two previously identified thyroid disruptors, the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and Aroclor 1254 (a complex mixture of polychlorinated water), may inhibit thyrotropin (TSH) receptor (TSHr) activity. DDT and Aroclor 1254 were shown to inhibit both the basal and bovine TSH (bTSH)-stimulated accumulation of cAMP in Chinese hamster ovary (CHO)-K1 cells stably transfected with the TSHr. Furthermore, both DDT and Aroclor 1254 did indeed prevent cAMP accumulation, as induced by the constitutive activity of a point mutant TSHr(I486M) transiently transfected in African green monkey kidney fibroblast (COS)-7 cells. Neither trypsin digestion of the extracellular domain (ECD) nor deletion of the ECD in a mutant TSHr trunk transiently transfected in COS-7 cells counteracted the inhibitory activity of DDT and Aroclor 1254. DDT exerted a weak inhibitory activity against forskolin in both CHO-K1 and COS-7 cells, whereas it was nil against the agonists dopamine and 5′-(N-ethyl-carboxamido)-adenosine (NECA) in CHO cells stably transfected with the dopamine D1 receptor and in COS-7 cells transiently transfected with the adenosine type 2a receptor (A2a) receptor. Furthermore, DDT was inactive against the stimulation by isoproterenol of the endogenously expressed β2 adrenergic receptor in COS-7 cells. Conversely, Aroclor 1254 inhibited completely forskolin activity in CHO-K1 cells but not in COS-7 cells. Furthermore, it did not prevent accumulation of cAMP as induced by NECA in A2a transfected cells. The analog of DDT, diphenylethylene, was inactive against bTSH-induced increase in cAMP in CHO-K1 cells stably transfected with the TSHr. We interpreted these results as indicating that DDT and possibly Aroclor 1254 may have an uncompetitive inverse agonist activity for the TSHr.

The vitellin-processing protease of Blattella germanica is derived from a pro-protease of maternal origin

Proteolytic processing of vitellin in Blattella germanica embryos is accomplished by activation of a yolk-borne cysteine protease (Mr 29 000) derived from a pro-protease precursor of Mr 40 000 (Liu et al., 1997). In the present study, fat body, ovaries and embryos of different developmental stages were examined immuno-cytochemically with purified murine anti-proprotease antibodies (Liu, 1995) to determine the intracellular location of the pro-protease. Proenzyme was detected in discrete secretory granules of the fat body and in large lysosome-like vesicles of both the follicle cell cytoplasm and the cortical ooplasm of previtellogenic ovarian follicles. In vitellogenic oocytes, coated pits and vesicles are scantily labelled for proprotease and no clear gold pattern could be discerned over the yolk granules. During embryonic development, pro-protease is associated with some, but not all, yolk granules. In newlyovulated eggs (day 0), pro-protease is either distributed over the entire granule or confined to some internal vesicles. As development proceeds, it becomes associated with almost every yolk granule and restricted to the superficial layer. By day 6, pro-protease is evident over all yolk granules but the intensity of reaction has greatly diminished, due probably to conversion of the pro-protease to the mature enzyme. Yolk granules are flanked along their margin by vesicles that are stained after zinc-osmium fixation. This observation suggests that the pro-protease may be transferred between yolk granules via vesicular shuttling. B. germanica embryos of different developmental stages were also exposed to [(3)H]-DAMP. Data show that autoradiographic grains are not evenly distributed among closely adjacent yolk granules within vitellophagic cells, a result consistent with the known slight temporal asynchrony of the acidification event.