Sulfated Glycoprotein Research Articles

DEVELOPMENT OF THE CONDITIONALLY IMMORTALIZED TESTICULAR SERTOLI CELL LINE TTE3 EXPRESSING SERTOLI CELL SPECIFIC GENES FROM MICE TRANSGENIC FOR TEMPERATURE SENSITIVE SIMIAN VIRUS 40 LARGE T ANTIGEN GENE

We developed and characterized a conditionally immortalized testicular Sertoli cell line from transgenic mice bearing the temperature sensitive simian virus 40 large T antigen gene pSVtsA58. Established cells from 8-week-old male transgenic mice were cultured at a permissive (33C) or nonpermissive (39C) temperature on a collagen type I pre-coated culture vessel. The expression of Sertoli cell specific proteins was analyzed by reverse transcriptase-polymerase chain reaction, immunocytochemical testing and Western blot analysis. The Sertoli cell line TTE3 grew at 33C but not at 39C. Large T antigen was expressed only in the nuclei at 33C, indicating that the temperature sensitive growth phenotype of the cells arose as a result of the function of temperature sensitive simian virus 40 large T antigen. The cells did not show any colony forming activity in soft agar or form tumors in subcutaneous tissue in nude mice, showing that TTE3 cells were not transformed. The cells expressed messenger RNAs encoding steel factor, inhibin-alpha, transferrin, follicle-stimulating hormone receptor and sulfated glycoprotein-2. Moreover, expression of vimentin and zonula occludens-1 was observed in the cytoplasm and on the boundaries of the cells, respectively. Interestingly expression levels of transferrin and zonula occludens-1 were significantly elevated at 39C. TTE3 cells with these unique characteristics should serve as a useful model to study the regulation of Sertoli cell function.

Changes in the exocrine pancreas secondary to altered small intestinal function in the CF mouse.

The exocrine pancreas of the cystic fibrosis (CF) mouse (cftr(m1UNC)) is only mildly affected compared with the human disease, providing a useful model to study alterations in exocrine function. The CF mouse pancreas has approximately 50% of normal amylase levels and approximately 200% normal Muclin levels, the major sulfated glycoprotein of the pancreas. Protein biosynthetic rates and mRNA levels for amylase were not altered in CF compared with normal mice, and increases in Muclin biosynthesis and mRNA paralleled the increased protein content. Stimulated pancreatic amylase secretion in vitro and in vivo tended to be increased in CF mice but was not statistically significant compared with normal mice. We show for the first time that the CF mouse duodenum is abnormally acidic (normal intestinal pH = 6.47 +/- 0.05; CF intestinal pH = 6.15 +/- 0.07) and hypothesize that this may result in increased signaling to the exocrine pancreas. There were significant increases in CF intestinal mRNA levels for secretin (310% of normal, P < 0.001) and vasoactive intestinal peptide (148% of normal, P < 0.05). Furthermore, CF pancreatic cAMP levels were 147% of normal (P < 0.01). These data suggest that the CF pancreas may be chronically stimulated by cAMP-mediated signals, which in turn may exacerbate protein plugging in the acinar/ductal lumen, believed to be the primary cause of destruction of the pancreas in CF.

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.

A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens.

A protein isolated from the supernatant of cryopreserved rooster sperm was found to increase the capability of cryopreserved rooster sperm to bind in vitro to the perivitelline membrane of a chicken egg and substantially raise fertility after artificial insemination (AI). That activity was partially purified and termed universal primary sperm-egg binding protein (UPSEBP). Insufficient protein remained from 6 x 10(11) sperm, despite retention of bioactivity, to allow sequencing. We deduced that the protein may be related to prosaposin (also termed SGP-1, for sulfated glycoprotein-1), and we used published amino acid sequences of prosaposin as a guide for synthesis of peptides. Certain peptides were found to increase in vitro sperm-egg binding and increase fertility of frozen-thawed or fresh rooster sperm, in a manner similar to semipurified UPSEBP. Active epitopes were in a 60 amino acid sequence, reflecting the intervening sequence between saposins A and B, plus short extensions into saposins A and B. Highest activity was found when this synthetic peptide was oxidized to form a disulfide bond between terminal cysteines. Antibody against a synthetic peptide consisting of 58 of these 60 amino acids bound to a 7-9 kilodalton protein in UPSEBP. Collectively, the data support the conclusion that UPSEBP is a fragment of prosaposin. Because prosaposin is in semen in humans and animals, these observations have broad implications for possible cause and therapy of one type of subfertility.

Cancer anorexia and cachexia

Patients with cancer cachexia experience a profound wasting of adipose tissue and lean body mass. Anorexia, although often present, is insufficient to account for tissue wasting because 1) cachexia involves massive depletion of skeletal muscle that does not occur during anorexia, 2) nutritional supplementation cannot replenish the loss of lean body mass, 3) cachexia can occur without anorexia, and 4) food intake might be normal for the lower weight of the cancer patient. Anorexia can arise from 1) decreased taste and smell of food, 2) early satiety, 3) dysfunctional hypothalamic membrane adenylate cyclase, 4) increased brain tryptophan, and 5) cytokine production. Appetite stimulants such as cyproheptadine, medroxyprogesterone acetate, and megestrol acetate do not significantly improve lean body mass. Tumor products might be more important in the development of cachexia. Cachectic patients excrete in their urine a lipid-mobilizing factor that directly stimulates lipolysis in a cyclic AMP–dependent manner and increases energy expenditure. Loss of skeletal muscle in cachexia is caused by upregulation of the ubiquitin–proteasome catabolic pathway. Cachexia-inducing tumors elaborate a sulfated glycoprotein, which directly initiates protein catabolism in skeletal muscle. The action of this proteolysis-inducing factor is attenuated by the polyunsaturated fatty acid eicosapentaenoic acid, which is also effective in preventing loss of skeletal muscle in cancer patients. Antagonists of tumor catabolic factors will provide important new agents in the treatment of cancer cachexia.

Morphological analysis of endocytosis in efferent ductules of estrogen receptor‐α knockout male mouse

Lack of estrogen receptor (ER) results in fluid accumulation and dilation of the efferent ductules, suggesting that the role of estrogen and ER in the male reproductive tract is related to fluid reabsorption in the ductules. In the present study, endocytosis of the nonciliated cells of the efferent ductules was compared morphologically between wild type (WT) and estrogen receptor-alpha knockout (alpha ERKO) male mice. The epithelial cells lining the WT efferent ductules were tall columnar in shape, whereas those of the alpha ERKO were low columnar. Immunocytochemically, the nonciliated cells of both genotypes showed positive reactions of sulfated glycoprotein-2, but the reaction products were reduced in amount in the alpha ERKO. Electron microscopy revealed that the nonciliated cells of the WT had numerous organelles for endocytosis such as coated pits and vesicles, tubules, endosomes, multivesicular bodies and lysosomes in the apical cytoplasm. These organelles were less developed in the nonciliated cells of the alpha ERKO. Morphometric analysis indicated that there was a significant reduction in area of endocytotic apparatus in the nonciliated cells of the alpha ERKO compared with that of the WT. A tracer study using gold particles demonstrated that the nonciliated cells of both WT and alpha ERKO efferent ductules were capable of taking up luminal contents. These results suggest that reabsorption of the luminal contents via endocytosis takes place in the efferent ductules but is greatly reduced in amount in the absence of ER alpha.

Acinar phenotypes in salivary pleomorphic adenoma: unusual differentiation or disordered functional activity?

To explain the occurrence in salivary pleomorphic adenoma of structures with an arrangement and appearance of tumour cells resembling acini, two tumours showing such structures and, for comparison, a tumour showing goblet cells were examined with the use of histochemistry and immunocytochemistry for constituents of the salivary secretory process. One tumour consisted mainly of slightly granular cells with an acinar arrangement, which contained neutral and carboxylated glycoproteins, -SH groups and cytoplasmic epithelial membrane antigen. The second tumour showed a minor component of structures resembling mucous acini, which contained neutral and carboxylated glycoproteins, -SS- groups and fucoglycoconjugates. The goblet cells of the third tumour contained sulphated glycoproteins and were associated with cystic lumina. Acinar phenotypes in salivary pleomorphic adenoma could reflect either an unusual line of differentiation or luminal cells with increased synthesis and/or retention of variably mature glycoproteins different from those of goblet cells. Disordered secretion and externalization of glycoproteins are possible factors influencing phenotypes in this tumour.

The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor

The mannose receptor (MR) binds foreign and host ligands through interactions with their carbohydrates. Two portions of MR have distinct carbohydrate recognition properties. One is conferred by the amino-terminal cysteine-rich domain (Cys-MR), which plays a critical role in binding sulfated glycoproteins including pituitary hormones. The other is achieved by tandemly arranged C-type lectin domains that facilitate carbohydrate-dependent uptake of infectious microorganisms. This dual carbohydrate binding specificity enables MR to bind ligands by interacting with both sulfated and non-sulfated polysaccharide chains. We previously determined crystal structures of Cys-MR complexed with 4-SO4-N-acetylglucosamine and with an unidentified ligand resembling Hepes (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]). In continued efforts to elucidate the mechanism of sulfated carbohydrate recognition by Cys-MR, we characterized the binding affinities between Cys-MR and potential carbohydrate ligands using a fluorescence-based assay. We find that Cys-MR binds sulfated carbohydrates with relatively high affinities (KD=0.1 mM to 1.0 mM) compared to the affinities of other lectins. Cys-MR also binds Hepes with a KD value of 3.9 mM, consistent with the suggestion that the ligand in the original Cys-MR crystal structure is Hepes. We also determined crystal structures of Cys-MR complexed with 3-SO4-Lewisx, 3-SO4-Lewisa, and 6-SO4-N-acetylglucosamine at 1.9 Å, 2.2 Å, and 2.5 Å resolution, respectively, and the 2.0 Å structure of Cys-MR that had been treated to remove Hepes. The conformation of the Cys-MR binding site is virtually identical in all Cys-MR crystal structures, suggesting that Cys-MR does not undergo conformational changes upon ligand binding. The structures are used to rationalize the binding affinities derived from the biochemical studies and to elucidate the molecular mechanism of sulfated carbohydrate recognition by Cys-MR.

Loss of oocytes in Dazl knockout mice results in maintained ovarian steroidogenic function but altered gonadotropin secretion in adult animals.

Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (-/-) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from -/- females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor alpha (ER alpha) and ER beta together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ER beta, inhibin alpha, and betaB-subunits, and -/- mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P < 0.01) lower LH and FSH in intact +/+ and +/- females. However, significantly (P < 0.05) increased plasma inhibin B together with significantly (P < 0.05) lower FSH were observed in the +/- females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the +/- mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle.

Loss of Oocytes in Dazl Knockout Mice Results in Maintained Ovarian Steroidogenic Function but Altered Gonadotropin Secretion in Adult Animals

Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (−/−) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from −/− females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor α (ERα) and ERβ together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ERβ, inhibin α, andβ B-subunits, and −/− mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P < 0.01) lower LH and FSH in intact +/+ and +/− females. However, significantly (P < 0.05) increased plasma inhibin B together with significantly (P < 0.05) lower FSH were observed in the +/− females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the +/− mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle.

Alteration of follicle-stimulating hormone and testosterone regulation of messenger ribonucleic acid for Sertoli cell proteins in the rat during the acute phase of spinal cord injury.

The detrimental effects of spinal cord injury (SCI) on spermatogenesis in the rat can be attenuated by exogenous testosterone (T) but enhanced by exogenous follicle-stimulating hormone (FSH). These results suggest that T-dependent cellular events may be involved in testicular injury after SCI and that such events may be associated with modification of FSH effects on Sertoli cell function. The current study compared the responses of Sertoli cells to exogenous T and FSH after SCI or sham surgery using steady-state levels of Sertoli cell protein mRNA transcripts as markers of responsiveness. Rats underwent sham surgery or SCI and then were treated for 7 or 14 days with T-filled silastic capsules (2 x 5 cm) and/or daily injections of 0.1 units of porcine FSH. Vehicle-treated control rats received 5-cm empty capsules and daily injections of saline vehicle. Two weeks after sham surgery, levels of mRNA for the androgen receptor (AR), FSH receptor (FSHR), androgen-binding protein (ABP), or sulfated glycoprotein (SGP)-2 in the testis were unaffected by T or FSH alone. Testosterone alone, however, significantly decreased transferrin (Trf) mRNA levels in the testis (P: < 0.01). The combination of T and FSH treatments resulted in significant decreases in levels of the above transcripts (P: < 0.05; P: < 0.01). Seven days after SCI, the testes of vehicle-treated SCI rats had higher levels of AR and SGP-2 mRNA than did those of sham control rats (P: < 0.01); such effects were transient and disappeared by Day 14 post-SCI. Testosterone treatment of SCI rats for 7 days resulted in decreases in mRNA levels for AR and Trf in the testes (P: < 0.01) but increased testicular levels of mRNAs for FSHR and SGP-2 in SCI rats. Follicle-stimulating hormone treatment for 7 days prevented the increase in AR mRNA that was seen in the testis of untreated SCI rats and increased levels of ABP and SGP-2 mRNAs in SCI rats (P: < 0.01). Follicle-stimulating hormone treatment of SCI rats did not affect FSHR mRNA levels by itself, but it blocked the stimulatory effect of T on FSHR and SGP-2 mRNAs. Fourteen days after SCI, testicular AR mRNA levels were not affected by T alone, but they increased in those rats that received FSH with or without concurrent T treatments (P: < 0.05). In contrast to their effects in sham control rats, T or FSH alone or in combination resulted in significant increases in testicular levels of ABP, SGP-2, and FSHR mRNAs (P: < 0.05). At this time, Trf mRNA in the testis of SCI rats was also suppressed by T (P: < 0.05), as it did in sham control rats, but Trf mRNA was increased by the FSH (P: < 0.01) that had inhibited this transcript in the testes of sham control rats. The effects of FSH on the Sertoli cell transcripts in SCI rats were either attenuated or blocked when T was given concurrently. In addition, testicular and serum T levels in those SCI rats that received FSH (alone or in combination with T) for 14 days were significantly increased, an effect that was not seen after sham surgery. These findings demonstrate that hormonal regulation of both Sertoli and Leydig cells was altered during the acute phase of SCI. Such changes may modify the functions of both cell types, thereby affecting the endocrine and/or paracrine microenvironment within the seminiferous epithelium. These effects could impair the functional capacity of Sertoli cells and contribute to impairment of spermatogenesis after SCI.

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

Ultrastructural and Histochemical Characterization of Secretory Granules in Human Lung Adenocarcinoma Cells

Abstract Histochemical methods offer an excellent research tool for the characterization of glycoproteins in the secretory cells, thus contributing to the elucidation of the pathophysiology of different diseases. The different staining characteristics of mucins can be useful in diagnostic histopathology. It has been proposed that the reduction in sulphated glycoproteins and an increase in sialomucins in intestinal mucosa was an indicator of premalignant changes in carcinoma of the bowel. It has subsequently argued that this change may be consequence rather than a precursor of neoplasia. It may still be of some value as a marker of a premalignant change although it is somewhat variable. Previous studies have demonstrated the characterization of glycoproteins in different type of epitheloid cells in normal and pathologic conditions by using histochemical and electron microscopic techniques. However; little information has been available concerning the exact features of secretory granules in both normal and tumoral cells in lung.

Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent.

A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross-linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion. At this stage of development, the sulfhydryl reagent 5, 5'-dithio-bis(2-nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis. Treated post-inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells. Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family. Cross-linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state. Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors.

Crystal structure of the cysteine-rich domain of mannose receptor complexed with a sulfated carbohydrate ligand.

The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH(2)-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 A resolution, respectively. Cys-MR folds into an approximately three-fold symmetric beta-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other beta-trefoil proteins.

Lectin histochemical detection of sulfoglycans in the zona pellucida of mammalian antral oocytes

Sulphated esters are important to increase effectiveness of specific biological activities of carbohydrates. Biochemical studies revealed the presence of distinct sulphated glycoproteins in mammal zona pellucida (ZP) that bind proacrosin and thus participate in the sperm-egg fusion processes. In the present study, 6 lectin-horseradish peroxidase conjugates (SBA, PNA, RCA-I, GSA-IB4, GSA-II and DBA) were used in combination with desulphation and sialidase digestion to identify sulphocarbohydrates in the terminal and/or subterminal position of oligosaccharide side chains of glycoproteins in the ZP of bovine, ovine, caprine and porcine antral oocytes. In particular, we identified the following terminal sulphoglycans located in the outer layer of the ZP only: SO4-GalNAc in bovine ZP; SO4-Galbeta1,3GalNAc in bovine and ovine ZP; SO4-Galbeta1,4GlcNAc in bovine, ovine and caprine ZP; SO4-alpha-Gal in bovine, caprine and porcine ZP. Subterminal sulphoglycans linked to sialic acid residues were evenly distributed throughout the entire thickness of the ZP: Neu5Ac-SO4-Galbeta1,3GalNAc in bovine and porcine ZP; Neu5Ac-SO4-Galbeta1,4GlcNAc in caprine ZP; Neu5Ac-SO4-alpha-Gal in porcine ZP; Neu5AcSO4-GlcNAc in bovine ZP. The results demonstrate that the chemical composition of the ZP differs among species determining the species-specificity of gamete interactions.

Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta.

Mice lacking estrogen receptors alpha and beta were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of alphabeta estrogen receptor knockout (alphabetaERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult alphabetaERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Müllerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.

Species-specificity of the acrosome reaction in starfish

Animal eggs are generally encased in extracellular investments. These structures are not simply a protective barrier against infectious microbes, parasites and various small predators: in starfish, three components of the egg jelly, the outermost egg investment, are responsible for triggering the acrosome reaction. These components are a highly sulphated glycoprotein of an extremely large molecular size named acrosome reaction-inducing substance (ARIS), a steroid saponin named Co-ARIS, and asteroidal sperm-activating peptides (asterosaps) (Matsui et al., 1986a, b; Nishigaki et al., 1996). ARIS can induce the acrosome reaction in homologous spermatozoa with asterosaps or Co-ARIS in normal seawater. Specificity at the genus or order level was found for sperm activation by asterosaps, whereas the acrosome reaction by jelly components was species-specific. The main sugar saccharide chain of ARIS, composed of the pentasaccharide repeating units [Xyl-Gal-Fuc(SO3−)-Fuc(3−)-Fuc-], has been observed to induce the acrosome reaction in starfish sperm at high calcium concentrations (Koyota et al., 1997). Recently, we cloned cDNAs encoding asterosaps and elucidated their nucleotide sequences (Matsumoto et al., 1999). The mRNA encoding asterosaps was transcribed only in the oocytes but not in the follicle cells, and the length was 3.7 kb. The cDNA clones contained multiple isoforms of asterosaps. We assume that asterosap cursors are large prepolypeptide chains with an unusual ‘rosary-type’ structure composed of 10 successive similar stretches of 51–55 residues. Each stretch ends with a ‘spacer’ of 17–21 residues immediately followed by the sequence of one asterosap isoform. The amino terminal of this precursor has 19–21 successive glutamine-rich repeating units.

Collagen fibril aggregation-inhibitor from sea cucumber dermis.

Collagen fibrils from the dermis of the sea cucumber Cucumaria frondosa are aggregated in vitro by the dermal glycoprotein stiparin (Trotter et al., 1996). Under physiological ionic conditions stiparin appears to be both necessary and sufficient to cause fibrils to aggregate (Trotter et al., 1997). We report here the initial biochemical and biophysical characterization of a sulfated glycoprotein from C. frondosa dermis that binds stiparin and inhibits its fibril-aggregating activity. This inhibitory glycoprotein, which has been named 'stiparin-inhibitor,' has the highest negative charge density of all the macromolecules extracted from the dermis. SDS-PAGE reveals three approximately 31-kDa bands that stain with alcian blue but not with Coomassie blue. Analytical ultracentrifugation indicates a native molecular weight of 62 kDa. Transmission electron microscopy of rotary-shadowed molecules shows curved rods about 22 nm long. The glycoprotein does not bind collagen fibrils, but does bind stiparin with a 1:1 stoichiometry. The binding of stiparin-inhibitor to stiparin prevents the binding of stiparin to collagen fibrils. The carbohydrate moiety produced by papain-digestion of the glycoprotein retains all of its inhibitory activity. The carbohydrate moiety of the inhibitor is dominated by galactose and sulfate.

Up-regulation of clusterin (sulfated glycoprotein-2) in pancreatic islet cells upon streptozotocin injection to rats.

Clusterin is a heterodimeric glycoprotein which has been shown to play important roles in programmed cell death and/or in tissue reorganization not only during embryonic development but also in damaged tissues. Recently, we reported the transient induction of clusterin in pancreatic endocrine cells during early developmental stages of islet formation. In the present study, we have investigated the expression of clusterin in pancreatic tissue of streptozotocin-treated rats which were undergoing extensive islet tissue reorganization due to degeneration of insulin beta cells. Clusterin was found in endocrine cells identified as glucagon-secreting alpha cells at the periphery of the islet. Using immunoelectron microscopy, clusterin-positive cells showed the typical ultrastructural features of pancreatic alpha cells. In addition, colocalization of clusterin and glucagon in the same secretory granules was shown by double immunogold labeling. These results imply that clusterin is a secretory molecule having endocrine and/or paracrine actions in parallel with glucagon. Further, we noted that clusterin expression was increased in pancreatic alpha cells during the process of beta cell death upon streptozotocin injection. The increase was significant as early as 1-3 h after streptozotocin treatment prior to any morphological alteration of islet beta cell and any manifestation of hyperglycemia. The expression of clusterin was steady-stately up-regulated during the process of islet reorganization caused by streptozotocin-induced cytotoxic injury. Therefore, we suggest that clusterin might be considered as a molecule induced by both embryonic development and drug-induced reorganization of the endocrine pancreas. Since clusterin expression is up-regulated in alpha cells, but not in beta cells undergoing degeneration, it may play a protective role against the cytotoxic insult.