Amphibian Vitellogenin: Properties, Hormonal Regulation of Hepatic Synthesis and Ovarian Uptake, and Conversion to Yolk Proteins

Oogenesis, the early events of primary oocyte growth (meiotic arrest, synapsis, ribosomal gene duplication), and folliculogenesis can be seen to particular advantage in the germinal ridge of the syngnathan ovary. After budding off the germinal ridge (a compartment of the luminal epithelium), nascent follicles then enter into a linear array of developing follicles within which temporal and stage-specific events can be correlated with spatial distribution. Prominent features of the later phase of primary oocyte growth include intense transcriptional activity and the formation and subsequent dispersal of the Balbiani vitelline body (mitochondrial cloud) concomitant with an increase in cytoplasmic organelles and volume. Further oocyte growth is characterized by a period of cortical alveolus (in teleosts) or cortical granule (in anurans) formation, in which Golgi elements play a predominant role, and finally vitellogenesis. The latter process, which is responsible for the preponderance of oocyte growth, includes the hepatic synthesis and secretion of vitellogenin (VTG), the uptake of VTG from the bloodstream into the oocyte by receptor-mediated endocytosis, and the transport of VTG via endosomes and multivesicular bodies to forming yolk platelets. In the process, VTG is proteolytically cleaved into the yolk proteins, which assume either a monoclinic (in cyclostomes) or orthorhombic (in teleosts and amphibians) crystalline array. Other structures associated with the growing oocyte are also briefly discussed, including nuage, the vitelline envelope, intercellular junctions between the oocyte and overlying follicle cells, pigment, intramitochondrial crystals in ranidae, and annulate lamellae.

Vitellogenin (Vg) and vitellogenin‐derived yolk proteins of growing oocytes and ovulated eggs were purified and characterized to evaluate structural changes of yolk proteins during oocyte growth and maturation in barfin flounder, Verasper moseri, a marine teleost which lays pelagic eggs. Native circulating vitellogenin, estimated to be a 520 kDa protein using Superose 6 column chromatography, cleaved into the 410 kDa major yolk protein, lipovitellin (Lv), in vitellogenic oocytes. An additional minor yolk protein at 19 kDa in native form, which was immunoreactive to an antiserum raised against Vg, and a highly phosphorylated 38 kDa band of phosvitin (Pv) in SDS‐PAGE with reduction were also identified in vitellogenic oocytes. Analysis of the amino acid composition of the 19 kDa yolk protein showed it to be similar to the β′‐component of egg yolk from salmonid fish. The 410 kDa Lv in vitellogenic oocytes was proteolytically cleaved into an 170 kDa major yolk protein during final oocyte maturation. The close similarity of amino acid composition between the two proteins combined with the results of lipid analysis suggested that the 410 kDa Lv cleaved into two homologous 170 kDa monomeric Lv. Both 19 kDa yolk protein (β′‐component) and Pv became undetectable after oocyte maturation. We propose that the three classes of yolk proteins (Lv, Pv, and β′‐component) are products of Vg cleavage, and that all of them undergo proteolysis again during oocyte maturation. © 1995 Wiley‐Liss, Inc.

The in vivo uptake and processing of vitellogenin (VTG) into developing oocytes of rainbow trout were studied. Oestrogenized trout injected with 32P and 3H‐leucine incorporated these labels into newly synthesized vitellogenin. This dual‐labelled VTG was purified from the serum and injected intraperitoneally into sexually maturing female trout. The 32P. 3H‐VTG was readily taken up from the peritoneal cavity into the blood, without degradation, and thus became available for sequestration by developing oocytes. Ovarian uptake of 32P. 3H‐VTG was very rapid. Eight h after injection, approximately half of the labelled VTG was in the blood, but this figure had fallen to 12% after 48 h, and to 0.2% after 2 weeks. This fall in blood 32P. 32H‐VTG level coincided with increases in the level of both radiolabels in the ovary. Eight hours post‐injection, a significant amount of 32P. 32H‐VTG was found in the ovary homogenate. Most of this material was intact, primarily representing VTG bound to the follicle surface. However, small amounts were contained within the follicles and probably represented newly sequestered VTG which had not yet been degraded into the yolk proteins, lipovitellin and phosvitin. Twenty‐four hours post‐injection, approximately 25% of the injected 32P. 3H‐VTG had been sequestered by the ovary. Although a small proportion of the labels were associated with intact VTG, the majority of the 3H and 32P eluted with lipovitellin and phosvitin, respectively, the yolk proteins derived from VTG. After 2 weeks, nearly 70% of the 32P. 3H‐VTG initially injected had been incorporated into the ovaries, all of which was present as labelled yolk proteins. The rate of uptake of VTG was highest in the fish with the largest follicles, where it approached 120 ng.mm2 follicle surface h−1. The results demonstrate that both the uptake and processing of VTG by trout oocytes are rapid features that are reflected in the rate of ovary growth seen during exogenous vitellogenesis.

We have recently shown that extensive proliferation of liver parenchymal cells takes place in adult male Xenopus frogs in response to estradiol-17 beta, which also induces synthesis and secretion of vitellogenin, the precursor of yolk proteins. We demonstrate here that liver parenchymal cells from adult male animals can be maintained for several weeks in a defined tissue culture medium containing added insulin, dexamethasone, and triiodothyronine. Under these conditions the cells do not divide, but can synthesize DNA. Maximum DNA synthesis occurs in cells that have achieved monolayer morphology under low plating densities. Estradiol-17 beta causes a dose-dependent increase in the number of cells synthesizing DNA, as well as inducing synthesis of vitellogenin. Estrogen-dependent, but not background, DNA synthesis is inhibited by the antiestrogen tamoxifen. These results imply that estradiol-17 beta acts directly on liver cells to initiate DNA replication, probably by interaction with a receptor protein and induction of specific gene transcription.

Yolk

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].

Glucocorticoids act together with estrogens and thyroid hormones in regulating the synthesis and secretion of Xenopus vitellogenin, serum albumin, and fibrinogen

The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with35S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.

The selectivity of protein sequestration by vitellogenic oocytes of the rainbow trout, Salmo gairdneri, was investigated by comparing the rates of uptake of radiolabelled vitellogenin (VTG) and bovine serum albumin (BSA). Fish were injected simultaneously with equal amounts of both 3H‐VTG and 14C‐BSA and sampled subsequently at varying time invervals to assess the rates of uptake of these proteins into the developing oocytes. Both labelled proteins were taken up rapidly into the blood after their injection into the peritoneal cavity. Once in the blood, they became available for sequestration by the vitellogenic oocytes. There was both rapid and selective uptake of 3H‐VTG; within 72 hours half of the 3H‐VTG injected was present within the ovary, whereas only 2.5% of the 14C‐BSA was similarly located. Once within the oocyte, 3H‐VTG was specifically and rapidly processed to yield its constituent yolk proteins, which included 3H‐lipovitellin. In contrast, after 72 hours 14C‐BSA was present in the oocyte as the native, intact protein. However, after 13 days, whereas the yolk proteins had undergone no further processing, remaining stable within the oocyte, much of the BSA within the ovary had been degraded to an array of smaller peptides. Furthermore, after 26 days, whereas all of the 3H‐VTG endocytosed remained within the oocytes as yolk protein, at least half of the BSA taken up initially had been exocytosed. Calculation suggested that 3H‐VTG was sequestered about 60 times more rapidly than 14C‐BSA. Other tissue did not incorporate 3H‐VTG to any significant degree. These results suggest that VTG was sequestered into vitellogenic oocytes by receptor‐mediated endocytosis, whereas BSA, along with other extraneous proteins, was taken up adventitiously.

Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes

Differention of the animal-vegetal axis in Xenopus laevis oocytes : I. Polarized intracellular translocation of platelets establishes the yolk gradient

Endocrine control of gametogenesis and spawning induction in the carp

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus

Parenchymal cells purified from Xenopus liver and maintained in primary culture synthesize vitellogenin in response to estradiol-17β and serum albumin in response to dexamethasone

Little is known about how metabolism changes during development. For most animal embryos, yolk protein is a principal source of nutrition, particularly of essential amino acids. Within eggs, yolk is stored inside large organelles called yolk platelets (YPs). We have gained insight into embryonic nutrition in the African clawed frog Xenopus laevis by studying YPs. Amphibians follow the ancestral pattern in which all embryonic cells inherit YPs from the egg cytoplasm. These YPs are consumed intracellularly at some point during embryogenesis, but it was not known when, where or how yolk consumption occurs. We have identified the novel yolk protein Seryp by biochemical and mass spectrometric analyses of purified YPs. Within individual YPs, Seryp is degraded to completion earlier than the major yolk proteins, thereby providing a molecular marker for YPs engaged in yolk proteolysis. We demonstrate that yolk proteolysis is a quantal process in which a subset of dormant YPs within embryonic cells are reincorporated into the endocytic system and become terminal degradative compartments. Yolk consumption is amongst the earliest aspects of differentiation. The rate of yolk consumption is also highly tissue specific, suggesting that nutrition in early amphibian embryos is tissue autonomous. But yolk consumption does not appear to be triggered by embryonic cells declining to a critically small size. Frog embryos offer a promising platform for the in vivo analysis of metabolism.