Egg Case Protein 3: A Constituent of Black Widow Spider Tubuliform Silk.

Spider silk has outstanding mechanical properties, rivaling some of the best materials on the planet. Biochemical analyses of tubuliform silk have led to the identification of TuSp1, egg case protein 1, and egg case protein 2. TuSp1 belongs to the spidroin superfamily, containing a non-repetitive N- and C-terminal domain and internal block repeats. ECP1 and ECP2, which lack internal block repeats and sequence similarities to the highly conserved N- and C-terminal domains of spidroins, have cysteine-rich N-terminal domains. In this study, we performed an in-depth proteomic analysis of tubuliform glands, spinning dope, and egg sacs, which led to the identification of a novel molecular constituent of black widow tubuliform silk, referred to as egg case protein 3 or ECP3. Analysis of the translated ECP3 cDNA predicts a low molecular weight protein of 11.8 kDa. Real-time reverse transcription-quantitative PCR analysis performed with different silk-producing glands revealed ECP3 mRNA is predominantly expressed within tubuliform glands of spiders. Taken together, these findings reveal a novel protein that is secreted into black widow spider tubuliform silk.

From Somatic Cells to Oocytes: A Novel Yolk Protein Produced by Ovarian Somatic Cells in a Stony Coral, Euphyllia ancora.

To gain a better understanding of how corals form their eggs at both the molecular and cellular levels, we performed a differential screen (suppression subtractive hybridization) to identify genes related to oocyte development in a stony coral, Euphyllia ancora. Through the course of screening, a novel gene that contains three alternate repeats of fibronectin domain 2 and epidermal growth factor (EGF)-like domains, as well as an additional calcium-binding EGF-like domain (EGF-CA), was identified and tentatively named euphy after the scientific name of the coral, E. ancora. Quantitative RT-PCR revealed that expression levels of euphy increased in female colonies as the coral approached reproductive season. Tissue distribution analysis followed by mRNA in situ hybridization revealed that euphy is highly expressed in the ovarian (mesenterial) somatic cells in the body of E. ancora. Staining of tissue sections with an antibody against euphy protein (Euphy) revealed Euphy immunoreactivity in both ovarian somatic cells and oocytes. Subsequent Western blotting demonstrated the presence of abundant Euphy in unfertilized mature eggs. These results indicate that Euphy produced in the ovarian somatic cells is transported to and accumulates within oocytes as a yolk protein during oogenesis. We previously showed that two major yolk proteins, vitellogenin and egg protein, are similarly produced by ovarian somatic cells. Hence, the present study uncovered the third ovarian somatic-derived yolk protein in corals. Our data provide new information that contributes to a more comprehensive understanding of coral egg formation.

The impact of fertilization on the chicken egg yolk plasma and granule proteome 24 hours post-lay at room temperature: capitalizing on high-pH/low-pH reverse phase chromatography in conjunction with tandem mass tag (TMT) technology.

Chicken egg yolk is a rich source of nutrients providing high quality proteins, vitamins, minerals, carotenoids and antioxidants. Chicken egg yolk, recovered from whole egg within 24 hours post-lay has been utilized as a starting material in the preparation of a dietary supplement that has been demonstrated to lead to gains in muscle mass in a human clinical study. Further, an oil derived from chicken egg yolk has been utilized as a topical agent to treat third degree burn injury. The molecular changes that take place in fertilized, chicken egg yolk during the first 24 hours post-lay are not well understood. By studying how the protein composition of egg yolk varies with fertility status, one can utilize this knowledge to develop egg yolk-based products that have been optimized for specific applications. In this study, a direct quantitative comparison was made between the proteome of fertilized chicken egg yolk and the proteome of unfertilized chicken egg yolk, both maintained at 20 °C and analyzed within 24 hours post-lay. Egg yolk proteins from each fertility state were digested with trypsin, labeled with distinct chemical labels (tandem mass tag reagents) and then combined in a 1 : 1 ratio. A TMT-labeled tryptic digest derived from chicken egg yolk proteins (fertilized and unfertilized) was separated using high-pH/low-pH reverse-phase chromatography and analyzed using mass spectrometry. 225 protein identifications were made from this TMT-labeled tryptic digest based on a minimum of 2 unique peptides observed per protein. 9 proteins increased in abundance in fertilized egg yolk relative to unfertilized egg yolk and 9 proteins decreased in abundance in fertilized egg yolk relative to unfertilized egg yolk. Some proteins that increased in abundance in fertilized egg yolk play an important role in angiogenesis (pleiotrophin, histidine rich glycoprotein) and defense against pathogens (mannose-binding lectin, ß-defensin 11, serum amyloid P-component, ovostatin). Based on this study, fertilized chicken egg yolk may be more useful as a starting material relative to unfertilized chicken egg yolk for the purpose of enriching or isolating proteins with pro-angiogenic and anti-microbial properties.

Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.

Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

Upregulation of cornichon transcripts in the dorsolateral prefrontal cortex in schizophrenia.

Schizophrenia has been proposed to be associated with abnormal glutamatergic neurotransmission. The AMPA subtype of glutamate receptors (AMPARs) mediates fast excitatory synaptic transmission in the brain, and their trafficking and function is regulated in part by AMPAR auxiliary proteins including the cornichons (CNIH) and transmembrane AMPAR-regulatory proteins. Abnormal regulation of AMPARs through altered expression of these auxiliary proteins could induce changes in glutamatergic neurotransmission and thus the pathophysiology of schizophrenia. In this study, transcript expression of cornichon homologs 1-4 was measured in the dorsolateral prefrontal cortex from schizophrenia (N=25) and comparison (N=25) patient groups by comparative quantitative real-time PCR. Significant upregulation of CNIH-1, CNIH-2, and CNIH-3 mRNA expression was found in schizophrenia, with no change in CNIH-4 expression. To determine the effect of antipsychotic treatment on the expression of these genes, cornichon mRNA expression was assayed in the frontal cortex of rats treated chronically with haloperidol decanoate and no changes in any of the cornichon transcripts were found. Abnormal expression of the CNIH family of genes is consistent with cornichon-mediated AMPAR trafficking abnormalities in schizophrenia, and suggests a new mechanism contributing toward the pathophysiology of this illness.

Normal ventral telencephalic expression of Pax6 is required for normal development of thalamocortical axons in embryonic mice.

BACKGROUND: In addition to its well-known expression in dorsal telencephalic progenitor cells, where it regulates cell proliferation and identity, the transcription factor Pax6 is expressed in some ventral telencephalic cells, including many postmitotic neurons. Its functions in these cells are unknown. RESULTS: We generated a new floxed allele of Pax6 and tested the consequences of a highly specific ventral telencephalic depletion of Pax6. We used the Six3A1A2-Cre allele that drives production of Cre recombinase in a specific region of Pax6-expression close to the internal capsule, through which thalamic axons navigate to cerebral cortex. Depletion in this region caused many thalamic axons to take aberrant routes, either failing to turn normally into ventral telencephalon to form the internal capsule or exiting the developing internal capsule ventrally. We tested whether these defects might have resulted from abnormalities of two structural features proposed to guide thalamic axons into and through the developing internal capsule. First, we looked for the early pioneer axons that project from the region of the future internal capsule to the thalamus and are thought to guide thalamocortical axons to the internal capsule: we found that they are present in conditional mutants. Second, we examined the development of the corridor of Islet1-expressing cells that guides thalamic axons through ventral telencephalon and found that it was broader and less dense than normal in conditional mutants. We also examined corticofugal axons that are thought to interact with ascending thalamocortical axons, resulting in each set providing guidance to the other, and found that some are misrouted to lateral telencephalon. CONCLUSION: These findings indicate that ventral telencephalic Pax6 is important for formation of the Islet1-expressing corridor and the thalamic and cortical axons that grow through it. We suggest that Pax6 might affect thalamic axonal growth indirectly via its effect on the corridor.

The primary structure of a novel riboflavin-binding protein of emu (Dromaius novaehollandiae).

Emu riboflavin-binding protein (RBP) was purified from egg white and yolk, and its N-terminal amino acid sequence was determined. The molecular mass of emu RBP was estimated at approximately 48 and 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., 10 kDa larger than chicken RBP. The molecular mass of deglycosylated RBPs indicated that the content of oligosaccharide chain in emu RBP was approximately 3 times greater than that in chicken RBP. The gene encoding the RBP precursor was cloned from emu oviduct cDNA by PCR and found also in the liver and ovary cDNAs as well as oviduct cDNA. The complete cDNA consisted of an open reading frame of 714 bp encoding a protein of 238 amino acids. The amino acid sequence deduced from the cDNA sequence revealed that many essential structural features were conserved in emu RBP including 18 cysteine residues, 2 N-glycosylation sites, a clustered phosphorylation region, and riboflavin-binding sites. Two additional potential N-glycosylation sites were found in the amino acid sequences of RBPs from the emu and other sources such as the turtle and frog, which might in part account for the greater content of oligosaccharide chain of emu RBP as compared to chicken RBP.

Functional activity of human ZP3 primary sperm receptor resides toward its C-terminus.

Zona pellucida glycoprotein 3 (ZP3) has been ascribed as a putative primary sperm receptor during fertilization in humans. Herein, attempts have been made to delineate the functional domain of human ZP3. ZP3 has been cloned and expressed in a baculovirus expression system as N-terminal fragments (amino acid [aa] residues 1-175 [pAc-ZP3(1-175 aa)] and 23-175 [pBg-ZP3(23-175 aa)]) and as C-terminal fragments (aa residues 214-305 [pBg-ZP3(214-305 aa)] and 214-348 [pBg-ZP3(214-348 aa)]). ZP3 encompassing both N- and C-terminal fragments corresponding to aa residues 1-370 (pAc-ZP3([1-370 aa])) has also been expressed. Lectin-binding analysis with these recombinant proteins revealed the presence of N- and O-linked glycosylation. Significant induction of acrosomal exocytosis was observed when capacitated sperm were incubated with pBg-ZP3(214-348 aa), pBg-ZP3(214-305 aa), and pAc-ZP3(1-370 aa) (P < 0.05), whereas incubation with pAc-ZP3(1-175 aa) and pBg-ZP3(23-175 aa) failed to do so under similar experimental conditions. However, N- and C-terminal fragments labeled with fluorescein isothiocyanate revealed binding to the anterior head of capacitated human spermatozoa. Escherichia coli-expressed ZP3 C-terminal fragments and chemically deglycosylated pBg-ZP3(214-348 aa) failed to induce a significant (P > 0.05) increase in acrosomal exocytosis, suggesting the relevance of glycosylation in imparting functional activity to ZP3 C-terminal fragments. pBg-ZP3(214-348 aa)-mediated induction of acrosomal exocytosis is regulated by G(i) protein, extracellular calcium, GABA(A) [gamma aminobutyric acid (A)] receptor-mediated Cl(-) channel, and T-type voltage-operated calcium channels. Taken together, the results of these studies suggest that the functional activity of human ZP3 resides in its C-terminal domain.

Vitellocyte-specific expression of phospholipid hydroperoxide glutathione peroxidases in Clonorchis sinensis.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and may protect against lipid hydroperoxidation in biomembranes. We isolated full-length cDNA sequences encoding four different PHGPxs from a causative agent of cholangiocarcinoma, Clonorchis sinensis (CsGPx1, CsGPx2, CsGPx3 and CsGPx4). These sequences contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in their 3'-untranslated regions. The open reading frames were composed of six exons in the chromosomal segments of CsGPx1 (7705bp), CsGPx2 (5871bp) and CsGPx3 (3867bp) and five exons in CsGPx4 (5655bp). The positions of these introns were tightly conserved between the trematode and vertebrate PHGPx genes. Oxidative stimulation of viable worms with H(2)O(2) or paraquat resulted in 1.5- to 2-fold induction of the GPx activity. The CsGPx proteins were specifically localised in vitellocytes within vitelline follicles and premature eggs in the proximal uterus. In the eggs, glutathione, an electron donor for GPx, was co-localised with the CsGPx proteins, while thioredoxin, which is preferred by peroxiredoxin, was principally detected in the extracellular space between the embryonic cell mass and an eggshell. Our data may suggest a concerted or a specialised function between a thioredoxin-dependent enzyme(s) and GPx in protecting against H(2)O(2)-derived damage during maturation of the embryo and formation of the eggshell, in these catalase-lacking trematode parasites. The uniquely conserved genomic organisation and Sec-dependency amongst trematode and vertebrate PHGPx homologues will also provide insight into the evolutionary episode and functional/biochemical diversification of GPx proteins.

A putative protein structurally related to zygote arrest 1 (Zar1), Zar1-like, is encoded by a novel gene conserved in the vertebrate lineage.

Identification and characterization of a bovine cDNA and the corresponding gene coding for a novel protein structurally related to Zar1, therefore called Zar1-like, are here reported for the first time. Structure of Zar1-like is similar to Zar1 gene, nevertheless they are located on distinct chromosomes. We demonstrated that the new gene as well as its genomic context are conserved along the whole vertebrate lineage. Analysis of the deduced protein primary structure showed a high conservation, among vertebrates, of the C-terminal region, where the putative presence of both zinc finger motifs and classical nuclear localization signals is also shared with Zar1. Bovine Zar1-like and the only two other available mRNA leader sequences (human and chicken) exhibit a number of upstream AUGs, suggesting that they are likely to be regulated at translational level. Expression patterns of the cattle transcripts show that Zar1-like is absent in early stages of embryo development, whereas Zar1 is expressed in matured oocytes and in in vitro produced pre-implantation embryos. In adult tissues Zar1-like transcript expression appears to be less restricted than Zar1, nevertheless, at least in bovine, both mRNAs are co-expressed in gonads, raising the question of a possible functional link.

Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR.

The expression levels of cytochrome P450 1A, p53 and vitellogenin were investigated in three different tissues of male medaka fish after exposure to diclofenac that is one of the main concerns among pharmaceuticals frequently found in sewage treatment plant (STP) effluents. The results showed that cytochrome P450 1A, p53 and vitellogenin were highly expressed in tissue-specific gene expression patterns after exposure to 8 mg/l and 1 microg/l of diclofenac. These elevated expression levels of three biomarkers suggested that diclofenac has potential to cause cellular toxicity, p53-related genotoxicity and estrogenic effects. It is also noteworthy that diclofenac has the potential to cause these effects even at an environmentally relevant concentration of diclofenac, 1 microg/l.

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins.

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Opisthorchis viverrini: identification of a glycine-tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis.

A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite's excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82-0.48) and specificity (0.97-0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present.

Evaluation of the contraceptive potential of brZPCp vaccine in BALB/c mice: their safety and efficacy.

In this study we have examined the potential ability of Microtus branditi partial ZPC (brZPCp) cDNA sequence (436-1150 nt) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in BALB/c mice using recombinant construction pCR3.1-brZPC(p). ELISA outcome indicated that antibodies could be generated by immunized mice, and IgG titer was increased compared to the control. Immunohistochemistry outcome indicated that antibodies could recognize native ZP in vivo, which in turn, prevented the binding of sperm to ovulated eggs. Antibodies could also recognize recombinant protein expressed by BL21 in vitro, which was confirmed by SDS-PAGE and Western blot. Fertility rate was reduced by 45% compared to the control immunized with pCR3.1. Meanwhile, there was no incidence of significant ovarian pathology in treated mice. This experiment indicates that this vaccine can elicit the specific antibody which binds exactly to the corresponding ZPC. This construction is proved to be immunogenic, and can reduce fertility without obvious oophoritis. The result in this study suggests a potentially important method for controlling population for its safety and easy production.

A vitellogenin chain containing a superoxide dismutase-like domain is the major component of yolk proteins in cladoceran crustacean Daphnia magna.

A cDNA encoding vitellogenin (VTG), a precursor of a major yolk protein, vitellin (VTN), was isolated from cladoceran crustacean Daphnia magna. The deduced amino acid sequence of DmagVTG1, the polypeptide encoded by the cDNA, contained a possible signal peptide sequence of 16 amino acid (aa) residues. The possible mature form of DmagVTG1 consists of 1985 aa residues with a calculated molecular mass of 223,070 Da. The large lipid transfer (LLT) module and a part of the von Willebrand factor D (VWD) module found in the aa sequences of VTGs of many other organisms are well conserved in DmagVTG1. Phylogenetic analysis suggested that the LLT module of DmagVTG1 is more closely related to those of insect VTGs than those of decapodan crustaceans. A unique feature of DmagVTG1 is that it has a superoxide dismutase (SOD)-like domain at its NH(2)-terminus. Antisera against the SOD-like domain, the NH(2)-terminal part of the VTG domain and the COOH-terminal part of the VTG domain, respectively, were prepared and used for analysis of D. magna yolk proteins. Six species (I to VI) of major protein complexes were found in D. magna parthenogenetic eggs isolated immediately after ovulation. Complexes IV and V were the most abundant. DmagVTG1 was a component of Complexes III, IV and V, and the most abundant polypeptide in D. magna eggs. The protein complexes underwent gradual proteolysis during development. One of the primary sites of cleavage was between the two successive Arg residues located at the 1454th and 1455th positions of DmagVTG1.

A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction.

Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We have identified two glycoproteins, ZP1/gp97 and ZPC/gp42, as the major components of the chicken egg envelope. In the present study, another 42 kDa protein, designated ZPD, has been found as a new major component of the chicken egg envelope. ZPD was specifically released from the egg envelope by ultrasonication treatment without urea. ZPD cDNA was cloned using a chicken granulosa cell cDNA pool. The deduced amino acid sequence showed that preproprotein of ZPD is composed of 418 amino acid residues with four potential N-glycosylation sites and includes a ZP domain, common in vertebrate ZP glycoproteins, and a transmembrane domain. ZPD belongs phylogenetically to a distinct group from known ZP glycoprotein subfamilies, ZPA, ZPB, and ZPC. In two-dimensional gel electrophoresis ZPD proteins were identified to be several isoforms with different pI values between 5 and 7. ZP1, ZPC and the newly identified ZPD were confirmed to be the major components of chicken egg envelope by MS of proteolytic digests of whole egg envelope. The in vitro incubation of chicken sperm with calcium ionophore A23187 induced sperm activation, resulting in the fragmentation and release of a 41 kDa PNA (peanut agglutinin)-positive glycoprotein and the decrease or loss of sperm PNA-stainability. The incubation with ZPD and dimeric ZP1, but not ZPC and monomeric ZP1, also induced the decrease or loss of sperm PNA-stainability, suggesting the in vitro sperm activation by these ZP components. Collectively, ZPD might bind loosely to egg envelope matrix and play a key role in the sperm activation on avian sperm-egg interaction.

cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs.

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.

Identification of genes encoding mouse oocyte secretory and transmembrane proteins by a signal sequence trap.

The oocyte plays a key role in follicular development. At all stages of follicular development, oocytes interact with surrounding granulosa cells and promote their differentiation into the types of cells that support further oocyte growth and developmental competence. These interactions suggest the existence of an oocyte-granulosa cell regulatory loop that includes both secreted proteins and cell surface receptors on both cell types. Factors involved in the regulatory loop will therefore contain a signal sequence, which can be used to identify them through a signal sequence trap (SST). A screen of an oocyte SST library identified three classes of oocyte-expressed sequences: known mouse genes, sequences homologous to known mammalian genes, and novel sequences of unknown function. Many of the recovered genes may have roles in the oocyte-granulosa cell regulatory loop. For several of the known mouse genes, new roles in follicular development are implied by identification of their expression, for the first time, in the oocyte. The future characterization of novel sequences may lead to the identification of novel proteins participating in the regulatory loop.

The major yolk protein in sea urchins is a transferrin-like, iron binding protein.

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.