Norio Yoshizaki

Multiple Vitellogenins (Vgs) in Mosquitofish (Gambusia affinis): Identification and Characterization of Three Functional Vg Genes and Their Circulating and Yolk Protein Products

Abstract The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17β (E2) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa β′-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E2 treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.

Participation of oviducal pars recta secretions in inducing the acrosome reaction and release of vitelline coat lysin in fertilizing toad sperm

Jellyless coelomic eggs of the toad, Bufo bufo japonicus, are not fertilizable when inseminated in jelly components that assure fertilization of dejellied uterine eggs. However, when coelomic eggs were pretreated with an extract from the uppermost portion of oviduct (pars recta) and inseminated in jelly, a high frequency of fertilization was obtained. This activity was observed only in the pars recta extract (PRE) prepared from pituitary-injected, ovulating females. Treatment of coelomic eggs with the PRE followed by addition of sperm without jelly resulted in lysis of the vitelline coat (VC). Likewise, the supernatant of the PRE preincubated with sperm had a strong VC lytic activity. The Triton X-100 extract of sperm preincubated with the PRE exhibited less than 20% of the proteolytic activity in extracts of control, untreated sperm. Electron microscopy of sperm revealed that the PRE treatment induces a specific loss of the acrosomal cap. Electron microscopically, distinct filament bundles of the VC in coelomic eggs became less conspicuous, after eggs passed through the pars recta or were treated with the PRE. Epithelial cells in the pars recta contained unique nonciliated cells that possess electron-dense granules distinct from those in jelly-secreting cells in lower oviducal portions. The number of these granules in each cell increased significantly upon injection of pituitary hormone into females. Immunohistochemical observations proved that the specific antigen(s) localized in nonciliated cells in the pars recta is not detected in the VC of coelomic eggs, but is deposited in the VC during passage of eggs through the pars recta. It is concluded that the substance(s) secreted from the pars recta cells is deposited in the VC, and induces the acrosome reaction and release of VC lysin in sperm to ensure that they make a successful fusion with the egg plasma membrane.

Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion

There are two hatching enzyme homologues in the zebrafish genome: zebrafish hatching enzyme ZHE1 and ZHE2. Northern blot and RT‐PCR analysis revealed that ZHE1 was mainly expressed in pre‐hatching embryos, whereas ZHE2 was rarely expressed. This was consistent with the results obtained in an experiment conducted at the protein level, which demonstrated that one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid. Therefore, the hatching of zebrafish embryo is performed by a single enzyme, different from the finding that the medaka hatching enzyme is an enzyme system composed of two enzymes, medaka high choriolytic enzyme (MHCE) and medaka low choriolytic enzyme (MLCE), which cooperatively digest the egg envelope. The six ZHE1‐cleaving sites were located in the N‐terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. The digestion manner of ZHE1 appears to be highly analogous to that of MHCE, which partially digests the egg envelope and swells the envelope. The cross‐species digestion using enzymes and substrates of zebrafish and medaka revealed that both ZHE1 and MHCE cleaved the same sites of the egg envelope proteins of two species, suggesting that the substrate specificity of ZHE1 is quite similar to that of MHCE. However, MLCE did not show such similarity. Because HCE and LCE are the result of gene duplication in the evolutionary pathway of Teleostei, the present study suggests that ZHE1 and MHCE maintain the character of an ancestral hatching enzyme, and that MLCE acquires a new function, such as promoting the complete digestion of the egg envelope swollen by MHCE.

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica, comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA‐CrA‐Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron‐dense granules distinct from those in the jelly‐secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.

Fine structure of oviducal epithelium of Xenopus laevis in relation to its role in secreting egg envelopes

During passage through the oviduct, the egg of Xenopus laevis acquires the following substances or envelopes: electron‐dense particles in the vitelline coat, the prefertilization layer in and around the vitelline coat, and four layers of jelly. Both gross anatomical observation and light and scanning or transmission electron microscopic observations of various levels of oviduct revealed that the first two are produced in the pars recta 1 and 2 portions, and that jelly layers 1–4 are added in the pars convoluta 1–4 respectively. The relative lengths of these portions were 0–0.044, 0.044–0,055, 0.055–0.52, 0.52–0.70, 0.70–0.87 and 0.87–1.00 from anterior to posterior. Along the whole length of the oviduct, the epithelium was comprised of ciliated cells and various types of secretory cells. Except for the pars recta 1 portion, there were two or three types of secretory cells in each portion of the oviduct, indicating that the structure of each stratum of the egg envelope is formed by an interaction of products from the specific types of secretory cells in each portion of the oviduct.

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis

An ovarian extract of Xenopus laevis exhibited in SDS‐PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin‐sensitive and inhibited by monospecific anti‐rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.

Two‐step consumption of yolk granules during the development of quail embryos

The mechanism of yolk consumption was studied morphologically and biochemically in Japanese quail Coturnix japonica. The amount of yolk granules in the yolk (or ‘yolk cell’) decreased in two steps during embryonic development. In the first step, during days 0–4 of incubation, the yolk‐granule weight decreased at a rate of 13 mg/day. This decrease was due to segregation by endodermal cells that were newly formed in the developing yolk sac. In the second step after day 6, the decrease was drastic at a rate of 29.8 mg/day during days 6–12 and very slow thereafter. The decrease at the second step was due to the enzymatic digestion of yolk granules by cathepsin D that coexisted in yolk spheres. This digesting reaction was triggered by the solubilization of the granules with high concentrations of salts that were supplied after disruption of the limiting membrane of yolk spheres. The ‘yolk cell’ seemed to die around day 5 of incubation. Thus the digestion products might be taken up together with yolk lipids by endocytosis into the endodermal cells and transported to blood vessels.

Progesterone Is a Sperm-Releasing Factor from the Sperm-Storage Tubules in Birds

Because of the presence of sperm-storage tubules (SST) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm have entered the female reproductive tract, they can survive for a prolonged time in domestic birds, although the specific mechanisms involved in the sperm uptake into, maintenance within, and controlled release from the SST remain to be elucidated. In this report, we provide evidence that progesterone triggers the release of the resident sperm from the SST in the UVJ. The ultrastructural observation of the SST indicated that the resident sperm are released from the SST around 20 h after oviposition. When laying birds were injected with progesterone, most of the sperm were released from the SST within 1 h of injection. In situ hybridization analyses demonstrated the presence of the transcripts of membrane progestin receptor α in the UVJ, and the translated proteins were detected in the UVJ extracts by Western blotting. Moreover, the number of secretory granules in the SST epithelial cells fluctuates during the ovulatory cycle, and the progesterone administration mimics this phenomena. A binding assay using [(3)H]-progesterone indicated the presence of a high affinity, limited capacity, saturable and single binding site for [(3)H]-progesterone in the membrane fraction of the UVJ, and this receptor did not interact with the synthetic antiprogestin RU486. These results demonstrated for the first time that the progesterone stimulates the release of the resident sperm from the SST and that the release of the sperm might occur via membrane progestin receptor α-mediating signal transduction.

Purification and gene cloning of Fundulus heteroclitus hatching enzyme A hatching enzyme system composed of high choriolytic enzyme and low choriolytic enzyme is conserved between two different teleosts, Fundulus heteroclitus and medaka Oryzias latipes

Two cDNA homologues of medaka hatching enzyme − high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE) – were cloned from Fundulus heteroclitus embryos. Amino acid sequences of the mature forms of Fundulus HCE (FHCE) and LCE (FLCE) were 77.9% and 63.3% identical to those of medaka HCE and LCE, respectively. In addition, phylogenetic analysis clearly showed that FHCE and FLCE belonged to the clades of HCE and LCE, respectively. Exon–intron structures of FHCE and FLCE genes were similar to those of medaka HCE (intronless) and LCE (8‐exon‐7‐intron) genes, respectively. Northern blotting and whole‐mount in situ hybridization showed that both genes were concurrently expressed in hatching gland cells. Their spatio‐temporal expression pattern was basically similar to that of medaka hatching enzyme genes. We separately purified two isoforms of FHCE, FHCE1 and FHCE2, from hatching liquid through gel filtration and cation exchange column chromatography in the HPLC system. The two isoforms, slightly different in molecular weight and in MCA‐peptide‐cleaving activity, swelled the inner layer of chorion by their limited proteolysis, like the medaka HCE isoforms. In addition, we identified FLCE by TOF‐MS. Similar to the medaka LCE, FLCE hardly digested intact chorion. FHCE and FLCE together, when incubated with chorion, rapidly and completely digested the chorion, suggesting their synergistic effect in chorion digestion. Such a cooperative digestion was confirmed by electron microscopic observation. The results suggest that a hatching enzyme system composed of HCE and LCE is conserved between two different teleosts Fundulus and medaka.

Absorption, transportation and digestion of egg white in quail embryos

The present study was done to reveal how egg white is taken up by embryonic tissues, the pathway through which egg white is transported, and the location where it is digested during the development of the quail Coturnix japonica. Antiserum against quail ovalbumin was raised in rabbit and used as a probe. By immunoelectron microscopy, the uptake of ovalbumin on a small scale by receptor‐mediated endocytosis was observed in the ectodermal cells of the yolk sac on days four to seven of incubation. The uptake of egg white on a large scale by fluid‐phase endocytosis took place in the cells generally referred to collectively as the ‘albumen sac’. The ovalbumin was transported through the albumen sac into the extraembryonic cavity during days eight to 10, and then into the amniotic cavity through the amnion approximately on day 10. Ovalbumin was present in the intestinal lumen on days 11 and 14, but it was not digested in the intestinal epithelial cells. The ovalbumin was detected in the yolk of embryos after day 10. Immunoblot testing, as well as a fluoroimmunoassay, revealed that the location where the amount of ovalbumin was highest changed chronologically from the extraembryonic cavity on day 10 to the amniotic cavity on day 11, the intestinal lumen on day 12 and then to the yolk on day 13. Several low molecular proteins which cross‐reacted with the antiserum were observed in the extracts of the yolk. The reaction producing these proteins depended on low pH (approximately 3.0) and was inhibited by pepstatin A. The ovotransferrin was similarly digested. These results indicate that egg white is, for the most part, transported through the albumen sac to the yolk via the extraembryonic cavity, the amniotic cavity, and the intestinal lumen, and is digested in the yolk by aspartic proteinases.