Transcriptional regulation of the gene for epidermal growth factor-like peptides in sea urchin embryos.

Exogastrula-inducing peptides (EGIPs), which are epidermal growth factor-related peptides of the sea urchin Anthocidaris crassispina, are substances that elicit abnormal gastrulation (exogastrulation) during embryogenesis of the sea urchin. In the present study we have examined the regulation of the expression of the EGIP precursor gene (EGIP) in sea urchin embryos. Whole mount in situ hybridization showed that EGIP is zygotically expressed afterthe onset of gastrulation in subdomains of the embryonic and larval ectoderm. The expression is confined in early gastrulae to small ectodermal regions adjoining the vegetal plate, which progressively expand to almost the entire ectoderm except the oral hood and postoral tips of the arms in later stages. In adults the expression is restricted to the ovary. Zygotic EGIP expression is sensitive to dissociation of embryonic cells, as well as to disruption of the extracellular matrix (ECM) with 5-cis-hydroxyproline, suggesting requirements for interaction with neighboring cells and/or with the ECM. The expression of reporter genes (chloramphenicol acetyl transferase and green fluorescent protein) under the regulation of the 4.6 kb upstream region of EGIP is temporally and spatially similar to that of the endogenous gene, showing that EGIP expression is regulated at the transcription level during embryogenesis by the cis-elements within the 4.6 kb upstream region.

Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos.

The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae.

Association of the sea urchin EGF-related peptide, EGIP-D, with fasciclin I-related ECM proteins from the sea urchin Anthocidaris crassispina.

Exogastrula-inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP-D (EGIP-D-binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP-alpha and EBP-beta) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell-binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC-32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP-D as does native EBP, suggesting the interaction between EGF-related proteins and fasciclin I-related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer.

Expression of a src-type protein tyrosine kinase gene, AcSrc1, in the sea urchin embryo.

By screening a cDNA library and 3'-rapid amplification of cDNA ends, the cDNA for a non-receptor type protein tyrosine kinase from the sea urchin Anthocidaris crassispina was analyzed. The deduced protein (AcSrc1) with the highest identity of about 60% to mammalian Src family kinases shows the characteristic features of the Src family. AcSrc1 mRNA is maternally expressed in unfertilized eggs, while zygotic expression is first detected in blastulae and continues through the pluteus stage. Zygotic mRNA expression, visualized by in situ hybridization, is detected specifically in archenteron at the gastrula stage, while it is restricted in plutei to the midgut and hindgut, suggesting specific roles for AcSrcl in the formation and/or functions of the digestive tract. Meanwhile, western blot analysis has shown that the AcSrc1 protein is constantly expressed throughout embryogenesis. By immunostaining, it was found that the protein (distributed evenly in the cytoplasm of unfertilized eggs) is translocated to the membrane after fertilization. All through the following development, AcSrcl was localized to the peripheries of different embryonic cells, although at a relatively low level of localization at the boundaries between adjacent cells.

Induction of metamorphosis in the sand dollar Peronella japonica by thyroid hormones.

The larva of the sand dollar Peronella japonica lacks a mouth and gut, and undergoes metamorphosis into a juvenile sand dollar without feeding. In the present study, it was found that thyroid hormones accelerate the metamorphosis of P. japonica larvae. The contents of thyroid hormones in larvae increased gradually during development. Thiourea and potassium perchlorate, inhibitors of thyroid hormone synthesis, delayed larval metamorphosis and simultaneously repressed an increase in the content of thyroxine in the larval body. These results suggest that the P. japonica larva has a system for synthesis of thyroid hormones that act as factors for inducing metamorphosis.

Purification of EGIP-D-binding protein from the embryos of the sea urchin Anthocidaris crassispina.

Exogastrula-inducing peptides (EGIPs) are intrinsic factors that are present in eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. In the present study, we isolated an EGIP-D-binding protein (EBP) from a homogenate of mesenchyme blastulae. EBP had an apparent molecular weight of 33,000. The N-terminal amino acid sequence of EBP had a sequence homology to HLC-32 and bep4 identified in other sea urchin embryos. In addition to its ability of binding to EGIP-D, EBP also inhibited exogastrulation induced by EGIP-D. These results suggest that EBP plays an essential role in EGIP-D-induced exogastrulation.

The protein tyrosine kinases of the sea urchin Anthocidaris crassispina.

In order to know the function of protein tyrosine kinases (PTKs) in the development of sea urchin embryos, we performed reverse transcription-polymerase chain reaction (RT-PCR) to obtain partial cDNA fragments for PTK genes using primers to highly conserved regions of the PTK family. A total of seven PTK sequences were identified, two of which represented receptor PTK (RTK1 and RTK2), and five of which were non-receptor PTKs (NRTK1-5). RTK1 was highly similar to FGF receptor and Ret kinase, while RTK2 showed features of the insulin receptor family. NRTK1 and 2 belonged to the Src family and could be involved in egg activation at fertilization. NRTK3 showed the features of the Btk family kinases, while NRTK4 seemed to be a member of the Syk/ZAP70 family. NRTK5 is the Csk-type kinase of the sea urchin, which is known to negatively regulate the Src family kinases. RTK1 was not detected in unfertilized eggs and was activated after blastula stage. All the other PTK genes were expressed both maternally in unfertilized eggs and zygotically after fertilization, though each gene showed distinct temporal patterns.

The content and distribution of glycosaminoglycans in the ejaculates of normal and vasectomized men.

The glycosaminoglycans (GAGs) of sperm and seminal plasma of normal men and seminal plasma of vasectomized individuals have been identified and quantified by two dimensional (2D) electrophoresis. The sperm contains predominantly CSC and HS as well as significant amounts of DS which achieves a high level in the sperm of the youngest man, while HA and LSC are either undetectable or present in small quantities. In normal seminal plasma, characteristically, DS is essentially lacking whereas CSC is the major GAG and HA and LSC account for relatively high percentages. Interestingly, in the ejaculates of vasectomized men the DS content is relatively prominent and the HA concentration varies widely. The oversulfated chondroitin sulfates CSD/CSE were detected in 7 of the 37 specimens. Their presence in a normal human body fluid is reported for the first time and the previous observation of the youthful DS/CSC switch is expanded to this study.

Molecular cloning of a cDNA that encodes the precursor to several exogastrula-inducing peptides, epidermal-growth-factor-related polypeptides of the sea urchin Anthocidaris crassispina.

Complementary DNA clones for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are related to epidermal growth factor (EGF), were obtained from a cDNA library of late gastrula embryos using, as probe, the partial cDNA for one of the EGIP (EGIP-D) obtained by the reverse-transcription PCR method. The longest cDNA was composed of 1662 bp, and encoded a protein of approximately 36 kDa with a region that resembled a signal sequence. The deduced protein contains the sequences of EGIP-C, EGIP-D, and EGIP-A in that order, followed by the sequence for an unidentified EGIP-like polypeptide. When expressed in Escherichia coli as a fusion protein with beta-galactosidase, the product for the cDNA was specifically recognized by a rabbit antibody raised against EGIP-D that had been purified from embryos. Characteristic amino acid residues were found around the N-terminus and the C-terminus of each EGIP sequence, suggesting a specific processing mechanism for the generation of the individual EGIPs from the precursor. RNA-blot analysis revealed the presence of EGIP mRNA in unfertilized eggs. The level of this mRNA decreased gradually after fertilization, began to increase dramatically after the onset of gastrulation, and continued to increase through the pluteus stage. Genomic Southern-blot analysis suggested that this gene is present as a single copy. A homology search showed that the EGIP cDNA has a similarity to the cDNA for SpEGF2 which was cloned as a gastrula-specific gene in another sea urchin, Strongylocentrotus purpuratus.

Formation of the adult rudiment of sea urchins is influenced by thyroid hormones.

After the sea urchin embryo has developed to a pluteus larva, the adult rudiment (sea urchin rudiment) is formed, with other adult structures, on the left side of the larva and finally the juvenile sea urchin is formed after metamorphosis. We report here that thyroid hormones (THs) are involved in the formation of the adult rudiment and the adult-type skeleton and the resorption of larval tissues of the sea urchin. The contents of THs in the larval body were determined by radioimmunoassay after the separation of individual THs by HPLC. We confirmed the presence of THs in the larval body and in algae on which the larvae feed. The THs accumulate gradually following the development of the larva and reach maximum levels at the eight-armed stage when the adult rudiment is completed. These results suggest that the development of the larva is influenced by THs accumulated in the larval body. However, inhibitors for the synthesis of THs do not affect the development of the larval body, suggesting a supply of THs from algae.

Presence of a 16 kd protein in human seminal plasma counteracts the effects of the anti-fertility agent, gossypol.

Gossypol inhibited lactate dehydrogenase (LDH) noncompetitively in human spermatozoa. The inhibitory effect of gossypol on LDH was cancelled by the addition of human serum albumin, human gamma-globulin, bovine serum albumin or human seminal plasma. Seminal plasma was at least 10 times more effective than the other three proteins, when expressed on a per mg protein basis. Attempts were made to purify the active fraction from human seminal plasma. The purification steps included gel filtration, ammonium sulphate precipitation, centrifugal microconcentration and fast-performance liquid chromatography. A single active protein of Mr = 16,000 was purified to a final yield of 0.18%. The 16 kd protein was not observed in male blood plasma. The protein was found to be heat-stable and leucine-rich (16% of the molecule), and has been designated 'gossact'. The inhibitory effect of gossypol on the LDH reaction was completely blocked by the addition of gossact (5 micrograms/ml); human blood plasma (25 micrograms/ml) and human serum albumin (200 micrograms/ml) were far less potent in this assay. In addition, gossact bound 1.4 mol of gossypol/mol of protein with the dissociation constant (Kd) = 3.06 x 10(-5) M. The role of gossact in the protection of LDH from gossypol is discussed.

A new membrane-associated Ca(2+)-binding protein of rat spermatogenic cells: its purification and characterization.

A Ca(2+)-binding protein of Mr = 52000, estimated by SDS-PAGE, was purified to a final yield of 0.04% from rat spermatogenic cells. Purification steps included gel filtration, ammonium sulfate precipitation and HPLC. Amino acid analysis showed the content of 34% acidic residues and 15% basic residues. The isoelectric point of this protein was 4.7. Dot-blot analysis indicated that the Ca(2+)-binding protein bound 2 mol of calcium per mol of protein. This protein had two binding sites with dissociation constants of 4.8 microM and 0.2 microM. No appreciable amount of hexose was observed (less than 1 microgram of hexose/70 micrograms of protein). This protein may play an important role such as the Ca(2+)-transport in the plasma membrane of spermatogenic cells.

Fractionation of jelly substance of the sea urchin egg and biological activities to induce acrosome reaction and agglutination of spermatozoa.

Jelly coat substance surrounding the egg of the sea urchin, Hemicentrotus pulcherrimus, was fractionated by gel filtration and three fractions designated A, B, and C were obtained which mainly consisted of fucose sulfate, sialic acid, and protein, respectively. The biological activities of the fractions were examined for induction of acrosome reaction (AR) and agglutination of spermatozoa. Only fraction A, a fucose-rich glycoprotein fraction, had activities for both AR and agglutination. Fraction A was found to lose activity for AR but to retain activity for agglutination after pronase digestion. Pronase-digested fraction A was further fractionated by the same gel filtration and three fractions designated P1, P2, and P3 were obtained, which contained mainly fucose sulfate, sialic acid, and proteinous material, respectively. These fractions had no activity for AR but activity for agglutination resided in fraction P1, a fucose sulfate fraction. Furthermore, beta-elimination of the jelly substance was carried out to separate protein and fucose sulfate polysaccharide and three fractions designated E1, E2, and E3 were obtained by gel filtration, of which the fucose-rich fraction (E1) exhibited activities for both AR and agglutination, and the sialoprotein fraction (E2) retained activity only for AR. However, the activity for AR of both fractions was destroyed by pronase digestion. These results suggest that activity to induce AR resides in the protein moiety of fucose-rich glycoprotein and activity for agglutination resides in the fucose sulfate polysaccharide moiety of the same glycoprotein of the jelly substance.

Amino acid sequence of exogastrula-inducing peptide C from the sea urchin, Anthocidaris crassispina.

The complete amino acid sequence of exogastrula-inducing peptide C from embryos of the sea urchin, Anthocidaris crassispina has been determined by analysis of the amino acid sequences in the S-pyridylethylated peptide C and the peptides generated after digestion of the peptide C with arginyl endopeptidase. Exogastrula-inducing peptide C was composed of 58 amino acid residues and its molecular weight was calculated to be 6464. The sequence was DTKGGCERATNNCNGHGDCVQGRWGQYYCKCTLPYRVGGSESSCYMPKDKEEDVEIET.

The exogastrula-inducing peptides in embryos of the sea urchin, Anthocidaris crassispina--isolation and determination of the primary structure.

Four exogastrula-inducing peptides, A, B, C, and D have been isolated from the homogenates of embryos of the sea urchin, Anthocidaris crassispina, with successive chromatographic fractionations. The complete amino acid sequences of the peptides A and D were determined by analysis of the peptides generated by their digestion with lysyl endopeptidase. They were composed of 52 and 53 amino acid residues, and their molecular weights were calculated to be 5754 and 5737, respectively. The sequences of peptides A and D were DSVYQCNRDTNSCDGFGKCEKSTFGRTTGQYICNCDDGYRNNAYGGCSPRTE, and DTVARCERDTKNCDGHGTCQLSTFGRRTGQYICFCDAGYRKPNSYGGCSPSSA, respectively. The biological significance of the exogastrula-inducing peptides was discussed.

Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni.

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.

Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization.

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.

Specific binding sites for natural glucocorticoids in plasma membranes of rat liver.

The presence of sites specifically binding natural glucocortocoids in plasma membrane (PM) preparations (PM0, density=1.13-1.16; PM1, density=1.16-1.18) from rat liver was elucidated by equilibrium dialysis as well as by centrifugal methods. Equilibrium dialysis showed the presence of binding sites having a higher affinity for [3-H]cortisol (Kd=1.4 times 10- minus 9M at 4C) in PM0, and that of the binding sites having a lower affinity for [3H] cortisol (Kd=1.3 times 10- minus 8M at 4C) in PM1, while centrifugal analysis showed the presence of higher affinity binding sites (Kd=1.5-1.9 times 10- minus 97 at 0 C) in both PM0 and PM1, and also of intermediate affinity binding sites (Kd=4.1 times 10- minus 9M at 0 C) in PM1. The discrepancy in the cortisol binding parameters obtained by the two different methods seems to be due mainly to the lability of some binding sites, especially the PM1. The glucocorticoid-binding sites in the plasma membranes of rat liver appear to have the highest affinity of corticosterone, followed by cortisol and cortisone. A synthetic glucocorticoid [3H]-dexamethasone, did not show any specific binding to the liver plasma membranes. Neither dexamethasone nor nonglucocorticoids such as estradiol given simultaneously affected [3H] cortisol binding to the plasma membranes.