Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.     

The relationships between corpora allata and fat body and haemolymph lipids in the adult female desert locust

Allatectomy of 1-day-old female desert locusts resulted in an accumulation of lipid in the fat body. This accumulation of lipid was due to the continuation of lipid deposition in the fat body after the period of somatic growth. Somatic growth and feeding activity were unaffected by allatectomy, and so could not be indirect causes of fat body lipid accumulation. Lipid accumulation in allatectomized locusts is more likely to be related directly to a lack of juvenile hormone. Implantation of active corpora allata into 1-day-old adult female locusts resulted in a premature development of oöcytes and a decrease in fat body lipid accumulation; somatic growth was not inhibited. Implantation of active corpora allata into old allatectomized locusts resulted in a decrease in the fat body lipid content and the onset of oöcyte development. The lipid synthetic activity of the fat body, measured by the incorporation of ¹⁴C-actate into total fat body lipid, was greatly increased in allatectomized locusts after the period of somatic growth. The protein synthetic activity of the fat body, measured by the incorporation of ³H-leucine into total fat body protein, remained low after the period of somatic growth in allatectomized insects. Juvenile hormone might thus have a dual effect on fat body metabolism, that is suppressing lipid synthesis and stimulating vitellogenic protein synthesis. Increased synthesis of lipid by the fat body would then account for the accumulation of lipid in the fat body after allatectomy. Inhibition of release of lipid from the fat body is unlikely to play a part in the accumulation as allatectomy had no effect on haemolymph lipid concentrations.     

Vitellogenesis by the American cockroach: Haemolymph and follicle protein patterns during vitellogenin synthesis

Radioactive ¹⁴C-leucine is removed from the blood within 4 hr of injection during the first 2 days of the vitellogenic cycle. Injections during the 3rd to 6th day result in leucine retention and a rise in labelled protein.Label appears in the follicle by day 3 with most of the protein being incorporated during day 5. Comparison of haemolymph and follicle proteins suggests that fat body synthesis, subsequent haemolymph transport and follicle uptake all occur primarily on days 4 and 5 of the cycle.In vivo follicle incubations reveal ¹⁴C-leucine uptake during the last 4 days of the cycle. During days 4, 5, and 6, leucine is incorporated into protein by the follicle. Injections of ¹⁴C-haemolymph proteins into 6 day females result in the incorporation of label into the terminal oöcytes.     

Incorporation of 14C leucine into the fat body of the cockroach Periplaneta americana during oöcyte development

The fat body of Periplaneta americana incorporates labelled leucine into the protein during the period of oöcyte formation. The protein isolated from the ovary shows radioactivity only in the 20 hr treated animals; in the 1 hr treated animals the ¹⁴C activity is below background levels. The protein contents of the fat body and ovary were measured during the reproductive stages. The measurements indicate that the fat body may not store the protein which it synthesizes during reproduction.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Absorption and transport of dietary lipid in Pieris brassicae

The digestion and absorption of dietary glycerol tri(1-¹⁴C)oleate and oleic acid-1-¹⁴C and subsequent transport of the label was followed during the fifth instar in Pieris brassicae. The rate of incorporation of the label in tissue lipid was similar in both diets. Triolein was hydrolysed to free fatty acids (FFA), diglycerides (DGL), and monoglycerides (MGL). DGL were rapidly absorbed. FFA were less readily absorbed and some were excreted. In the gut wall the label was found in phospholipids (PL), triglycerides (TGL), DGL, and FFA. In haemolymph most of the label was in DGL and PL, but later appeared also in TGL and sterol esters (SE). The results suggest that DGL are released from the gut wall and carried in haemolymph into the fat body. In the fat body lipid is stored mainly as TGL, and released as DGL, TGL, and SE. The turnover of oleate in haemolymph DGL is rapid in comparison to haemolymph SE or TGL. Synthesis of PL in gut lumen is apparent. Much of this PL is excreted but some may be absorbed.     

Vitellogenin fluctuations in haemolymph and fat body and dynamics of uptake into oöcytes during the reproductive cycle of Diploptera punctata

Haemolymph and fat body soluble protein titres have been examined during the reproductive cycle of Diploptera punctata, with particular emphasis on the occurrence of vitellogenin and its uptake into the developing oöcytes. Vitellogenin was first detected in the haemolymph of mated females 2 days after adult eclosion at about the same time that vitellin deposition in basal oöcytes began. Peak haemolymph titres of vitellogenin occurred on day 6, correlated with the completion of yolk uptake. Thereafter vitellogenin levels declined and were generally undetectable throughout most of gestation, rising again shortly before parturition in association with the second gonotrophic cycle. Total haemolymph protein levels were not correlated with vitellogenesis.Soluble fat body vitellogenin titres of mated females remained low during the first oöcyte growth period but then rose several-fold at its completion and remained high throughout pregnancy and the second gonotrophic cycle. Total fat body soluble proteins decline after adult eclosion in association with oöcyte growth.Vitellin accumulation in basal oöcytes was related linearly to increase in volume until the onset of chorion formation. Thus no post-vitellogenic growth period was detected.     

Yolk deposition in Douglas-fir beetle oöcytes: Possible rôle of RNA synthesis in the follicular epithelium

RNA synthesis and morphological changes in the follicular epithelial cells of oöcytes of Douglas-fir beetle, Dendroctonus pseudotsugae were studied during the reproductive phase. Inhibition of synthesis of DNA dependent RNA by actinomycin D injections blocked yolk deposition in the oöcytes as well as oviposition within the normal period. A mixture of radioactively labelled haemolymph and ovarial proteins was deposited as yolk proteins in the oöcytes of normal beetles. Such proteins were not deposited in the oöcytes of females injected with actinomycin D; the blockage of yolk deposition persisted even when such females were treated extraneously with juvenile hormone.     

Corpus allatum activity during overlapping cycles of oöcyte growth in adult female Melanoplus sanguinipes

Cycles of oögenesis in Melanoplus sanguinipes overlap to the extent that there are always 2 and occasionally 3 sets of vitellogenic oöcytes in the ovarioles at any one time. Three phases of vitellogenic oöcyte development can be distinguished: (1) An initial 24-hour phase of slow development (1.0–1.2 mm, 0.05–0.10 mm³). (2) A phase of rapid oöcyte growth (1.2–3.5 mm, 0.1–1.3 mm³). The duration of this phase is 2 days in the first cycle and 3 days in subsequent cycles. (3) A final phase of rapid oöcyte growth and maturation (3.5–4.5 mm, 1.3–2.8 mm³). Including the time taken for oviposition the duration of this latter phase is 3 days. Phases 1, 2 and 3 of cycles n + 2, n + 1 and n, respectively, overlap entirely. Activity of the corpora allata was measured using a radio-biosynthetic technique. A period of increased corpus allatum activity coincides with the initial part of phase 2 in each cycle. Each set of oöcytes is, thus, subject to 2 and occasionally 3 peaks of corpus allatum activity during development. Using these data a model of the control of oöcyte development has been devised     

Synthesis and deposition of yolk protein in adult Drosophila melanogaster

Newly eclosed Drosophila melanogaster females contain only previtellogenic stage oöcytes and no immunologically detectable female specific haemolymph protein. During the subsequent 48 hr the concentration of female specific protein in the haemolymph rises to a plateau value of 21 μg/μl; at this time yolk protein represents about one third of the total haemolymph protein in adult females. The first mature (stage 14) oöcytes are observed at 48 hr post eclosion. The female specific haemolymph protein and the major protein from mature oöcytes are electophoretically and immunologically the same or very similar. Injection of alpha amanitin into newly eclosed females inhibits the development of mature oöcytes and the degree of inhibition depends on the age of the female at the time of injection. Phenocopies of non-vitellogenic mutants result when alpha amanitin is injected into newly eclosed females; after 36 hr post eclosion no visible inhibition of vitellogenesis (as observed morphologically at 72 hr post eclosion) can be produced by alpha amanitin.     

Determinants of oöcyte degeneration in Drosophila melanogaster

During normal oögenesis in many insects some of the oöcytes fail to mature; instead they degenerate and are resorbed. In this work oöctte degeneration was investigated in Drosophila melanogaster females and found to be limited to early vitellogenic stages (stages 8–10). Even when retained for up to 18 days by females, mature (stage 14) oöcytes showed unaltered protein patterns after separation by SDS polyacrylamide electrophoresis, indicating that protein breakdown, which is characteristic of degeneration, does not occur in chorionated oöcytes.A number of environmental parameters were shown to influence the percentage of degenerating oöcytes in females. Strong responses as reflected by increased stage-8 and 9 oöcyte degeneration were found in females subjected to suboptimal (but not starvation) medium, virgin females, females mechanically unable to oviposit, and females unable to locate suitable oviposition sites. Little or no response was seen in females subjected to crowding, however, since all of these environmental parameters except adult crowding have been shown to decrease fecundity, and therefore the rate of oöcyte production, it is suggested that oöcyte degeneration is a strategy for decreasing the rate of oöcyte production in Drosophila.     

Protein synthesis in the fat body of Leucophaea maderae during vitellogenesis

During the early stages of vitellogenesis in Leucophaea, vitellogenin accounted for most if not all of the secreted protein synthesized by the fat body. Synthesis began about 5 days after mating and continued until 24 hr or so before the formation of the oötheca. Ligation resulted in the degeneration of the oöcytes, the first evidence of which was seen within 24 hr. Ligation also curtailed the synthesis of vitellogenin at about the same time. Isolated abdomens treated with an analog of juvenile hormone commenced vitellogenesis within 12 to 24 hr and measureable oöcyte growth occurred after 5 days. Despite continued synthesis of vitellogenin, the oöcytes in isolated abdomens always degenerated.     

Cytochemical studies on vitellogenesis in Aspongopus obscurus (Hemiptera: Pentatomidae)

By employing cytochemical techniques it is possible to conclude that the oöcytes of Aspongopus obscurus develop fatty and compound yolks. The fatty yolk originates in close proximity to the Golgi bodies and is composed of neutral fat. Based on the source of origin, the compound yolk has been grouped into two categories: CY₁ and CY₂. While the CY₁ develops de novo in the central oöplasm, the CY₂ originates from precursors infiltrating into the oöcyte through the follicular epithelium. Both CY₁ and CY₂ are composed of carbohydrate (1:2 glycol group and glycogen), protein (tyrosine, histidine, NH₂, SH and SS groups) and RNA.     

Monoclonal antibodies as probes for processing of yolk protein in the mosquito; production and characterization

We have produced a library of monoclonal antibodies against yolk proteins of the mosquito Aedes aegypti. After the initial screening, 45 hybridoma cell lines were selected and cloned. Immunoblot analysis revealed three groups of monoclonal antibodies. One group recognized a 200-kDa polypeptide, the second a 68-kDa, and the third both of these polypeptides. While the affinity of binding by different antibodies varied widely, all monoclonal antibodies recognized these polypeptides only in extracts from vitellogenic fat bodies and ovaries. The antibodies were further characterized by video-enhanced immunofluorescence, which also showed that both yolk polypeptides originated in the fat body and accumulated in the oöcytes. The immunolocalization in trophocytes of the fat body suggested that monoclonal antibodies may recognize different stages of the secretory pathway of yolk polypeptides. Similar analysis of oöcytes indicated that our panel of antibodies recognizes different steps of processing of both 200-kDa and 68-kDa polypeptides, beginning with internalization by the oöcyte and ending with the final crystalline form in mature yolk bodies.     

Hormonal aspects of oögenesis in the flies Phormia regina and Sarcophaga bullata

The corpora allata (CA) and median neurosecretory cells (MNC) of Phormia regina and Sarcophaga bullata become active with increasing age of the fly, on a diet of sugar alone. To prevent or retard oögenesis the CA or MNCs must be removed shortly after emergence, with subsequent protein meals. Topical JH application partially compensates for CA or MNC removal. This shows that the MNC activate the CA, and not vice versa. The trauma of either operation slightly depresses egg development.Injection of ecdysone into both species in the stage of initial yolk deposition causes the primary oöcytes to degenerate. This leads to development of the penultimate oöcytes. Older and younger egg stages are not sensitive to ecdysone. In P. regina the application of JH to females with developing primary oöcytes stimulates yolk deposition in the penultimate oöcytes.     

Actions of the juvenile hormone, 20-hydroxy-ecdysone,and the oöstatic hormone during oögenesis in the flies Phormia regina and Sarcophaga bullata

Application of juvenile hormone (JH) to sugar-fed Phormia flies leads to full ovarian development, i.e. the flies become autogenous. Application of JH to sugar + liver-fed Phormia leads to the simultaneous development of primary, secondary and tertiary oöcytes, suggesting the role of the oöstatic hormone is to shut off the action of the JH. This is consistent with the notion that the oöstatic hormone may act on the corpus allatum either directly or through the neurosecretory system. Application of 20-hydroxy-ecdysone to Phormia or Sarcophaga, when primary oöcytes have started to develop (stage 4A), causes the primary oöcytes to degenerate, with concomitant development of the secondary oöcytes.     

Oöcyte resorption during ovarian development in the blowfly Lucilia cuprina

Nulliparous females of a normal anautogenous strain of Lucilia cuprina mature all of their primary oöcytes after feeding ad lib on sheep's liver. Females fed measured inadequate amounts of protein-rich materia either fail to mature any oöcytes or mature less than their full complement. These mature oöcytes are smaller than in ad lib fed females. In females maturing no oöcytes, ovarian development ceases with all oöcytes in a pre-vitellogenic or early vitellogenic stage. When females mature only some of their oöcytes, the remainder are resorbed in early vitellogenesis. Few females mature less than 100 oöcytes, if they mature any at all.     

Juvenile hormone-induced vitellogenesis in Apterous4, a non-vitellogenic mutant in Drosophila melanogaster

D. melanogaster females homozygous for the ap⁴ mutant synthesize yolk protein and circulate this protein in the haemolymph at concentrations not different from concentrations found in normal females. However, ap⁴ females deposit little or no yolk protein into developing oöcytes. Topical application of a juvenile hormone analogue (JHA), ZR-515, stimulated sequestration of yolk protein by developing oöcytes of ap⁴ females. JHA had no detectable effects on haemolymph concentrations of yolk protein in either normal or ap⁴ females nor on the protein profiles obtained from electrophoresis of haemolymph samples.     

Evidence for haemoglobin uptake by oöcytes of Chironomus thummi

Developing oöcytes of a haemoglobin-containing fly, Chironomus thummi, have been investigated using the 3,3′-diaminobenzidine method for their endogeneous peroxidase activity. In the previtellogenic oöcytes the reaction product, which is thought due to the peroxidatic activity of the haemoglobins, is not observed within the oöcytes. Vitellogenic oöcytes appear active in the uptake and incorporation of the externally derived peroxidese-active material into the yolk. The reaction product which is first visualized in the extracellular spaces within the follicle, then the pinosomes and multivesicular bodies of the oöcyte, is later seen in the mature yolk granules. These observations are discussed in terms of their relation to the accumulation of haemoglobin as a part of the yolk.     

Synthesis and intercellular transport of ribosomal RNA in the ovary of the milkweed bug Oncopeltus fasciatus

Analysis of cellular and subcellular fractions obtained by dissection and subsequent purification of the main components of the Oncopeltus fasciatus ovary reveals that the uptake and incorporation of tritiated uridine into high molecular weight RNA differs markedly between the trophic syncytium and the oöcytes. The pattern of labelling indicates that the trophic syncytium is actively engaged in the synthesis of ribosomal RNA, but that the oöcytes do not synthesize detectable amounts of ribosomes. The ribosomal RNA which eventually appears in the cytoplasm of the oöcytes is postulated to come from the trophic syncytium via the nutritive cords. There is no other fraction of non-4 S RNA detectable in the oöcytes by these methods.