Publications (15)37.13 Total impact
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Article: A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.
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ABSTRACT: When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH(2)-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209-230 in the second extracellular loop of rat occludin, at 0.2-4 microM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH(2)-TKVNERYELCDTYPALLAVPAN-COOH (residues 156-177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5-10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8-16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [(125)I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [(125)I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.Biology of Reproduction 12/2001; 65(5):1340-51. · 4.01 Impact Factor -
Article: Two new male contraceptives exert their effects by depleting germ cells prematurely from the testis.
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ABSTRACT: The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.Biology of Reproduction 09/2001; 65(2):449-61. · 4.01 Impact Factor -
Article: Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis.
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ABSTRACT: The full-length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three approximately 2.9-kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln(421) and Phe(422) within the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X-linked myotubular myopathy. A 22 amino acid peptide NH(2)-TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66-kDa protein was indeed detected in both Sertoli and germ-cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter-Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady-state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell-cell interactions in the testis.Journal of Cellular Physiology 01/2001; 185(3):366-85. · 3.87 Impact Factor -
Article: Identification and purification of proteins from germ cell-conditioned medium (GCCM).
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ABSTRACT: Germ cells are known to regulate Sertoli cell and testicular function possibly through released factor(s) or via cell-cell contact. However, the identities of many of these putative biological factors are not known. The aim of this study is to present a strategy to identify and purify germ cell-derived proteins found in germ cell-conditioned medium (GCCM) at a quantity sufficient to permit protein microsequencing. The purification scheme of a novel germ cell-derived protein from GCCM designated GC-26 is presented along with several germ cell proteins using a combination of high pressure liquid chromatography (HPLC) columns. The purity of GC-26 and other germ cell proteins were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver staining. The identities of GC-26, a 26-kDa polypeptide, and other proteins were determined by direct protein microsequencing. These partial NH2-terminal amino acid sequences were compared with the existing databases at Protein Identification Resource (PIR), GenBank, and BLAST. These analyses revealed that these proteins are unique. This strategy should be useful for the micropurification of proteins from other biological samples and/or fluids.Biochemistry and molecular biology international 04/1999; 47(3):479-91. -
Article: Quantification of prostaglandin D synthetase in cerebrospinal fluid: a potential marker for brain tumor.
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ABSTRACT: Prostaglandin D synthetase (PGD-S; prostaglandin-H2 D-isomerase, EC 5,3,99,2), a 30 kDa glycoprotein also known as beta-trace protein that catalyzes the formation of prostaglandin D2 (PGD2) from PGH2, was purified to apparent homogeneity from human cerebrospinal fluid (CSF) using a two-step procedure involving HPLC on a Vydac C8 reversed-phase column and high performance electrophoresis chromatography (HPEC) using a 10% T SDS-polyacrylamide gel. The purity of PGD-S isolated from CSF was confirmed by silver stained SDS-polyacrylamide gel and direct protein microsequencing (NH2-APEAQVSVQPNFQ). A highly specific polyclonal antibody was prepared against this protein for immunoassay development. Using an ELISA, it was found that the concentration of PGD-S in CSF did not alter significantly in different pathological conditions of the central nervous system (CNS). These include dementia (n = 9), hydrocephalus (n = 4), neuropathy (n = 11), optic neuritis (n = 4), multiple sclerosis (n = 11), and demyelinating syndrome (n = 11), when compared to normal individuals (n = 12); however, the level of PGD-S in the CSF obtained from patients with brain tumor (n = 11), was reduced by as much as 2-fold when compared to control samples (n = 12) illustrating PGD-S is a potentially useful marker for brain tumor.Biochemistry and molecular biology international 12/1998; 46(4):643-56. -
Article: Rat testicular extracellular superoxide dismutase: its purification, cellular distribution, and regulation.
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ABSTRACT: Using multiple HPLC steps, we have identified and purified a 68-kDa polypeptide (as estimated by gel permeation HPLC) to apparent homogeneity, from primary Sertoli cell-enriched culture medium, that consisted of two monomers of 35 (alpha chain) and 33 kDa (ss chain) on SDS-polyacrylamide gel running under reducing conditions. Partial N-terminal amino acid sequence analysis of these two monomers revealed sequences of NH2-DXGESGVDLADRL (SODEX-alpha) and NH2-XXDTGESGVDLADXL (SODEX-ss), which are identical to rat extracellular superoxide dismutase (SODEX) with the exceptions that SODEX-alpha and SODEX-ss are missing, respectively, four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids from their N-termini, compared to rat SODEX, suggesting that the cleavage sites of the SODEX gene in the testis are different from that of other organs. Studies by sequential use of reverse transcription and polymerase chain reaction (PCR) using two SODEX primers have demonstrated the expression of SODEX in the heart, brain, lung, kidney, epididymis, testis, Sertoli, and germ cells, with low expression in the liver and ovary and no expression in the uterus, spleen, or thymus. Nucleotide sequence analysis of this 447-base pair PCR product from Sertoli cells revealed that its sequence is equivalent to the sequence of previously published rat SODEX. During testicular maturation, the SODEX steady-state mRNA level increased significantly from 20 to 60 days of age and then declined at 90 days of age. Such an increase in the testicular SODEX expression during maturation is not likely a result of an up-regulation by germ cells, since germ cells isolated from either 20- or 60-day-old rats when cocultured with Sertoli cells failed to elicit an increase in SODEX expression in the cocultures. Using primary Sertoli cell cultures in vitro, it was found that Sertoli cell SODEX expression was stimulated by interleukin-1alpha but not by either interferon-gamma or basic fibroblast growth factor. These results illustrate that Sertoli cells as well as germ cells synthesize and/or secrete a testicular variant of SODEX that may provide essential clues to understanding superoxide radical-mediated damage in the gonad.Biology of Reproduction 09/1998; 59(2):298-308. · 4.01 Impact Factor -
Article: Regulation of alpha2-macroglobulin expression in rat Sertoli cells and hepatocytes by germ cells in vitro.
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ABSTRACT: Germ cells isolated from rat testes by trypsinization have been shown to yield unwanted artifacts in biological assays, since conditioned media derived from these germ cells (germ cell-conditioned media [GCCM]) can modulate Sertoli cell secretory function because of the presence of residual trypsin. To determine whether germ cells themselves can modulate Sertoli cell function, we isolated germ cells from tubules by a mechanical procedure and assessed the effect of these cells on Sertoli cell alpha2-macroglobulin (alpha2-MG) steady-state mRNA level. It was found that germ cells indeed could stimulate Sertoli cell alpha2-MG expression. This effect is probably mediated by a soluble factor(s) released from germ cells, since GCCM fractionated by HPLC contained multiple fractions that can stimulate Sertoli cell alpha2-MG expression dose-dependently. These results illustrate that germ cells play a role in regulating testicular alpha2-MG expression. Since Sertoli cells synthesize and secrete many of the serum proteins behind the blood-testis barrier that are also produced by hepatocytes, we sought to ascertain whether germ cells can affect hepatic alpha2-MG expression. When germ cells were cocultured with hepatocytes isolated from adult rats, the hepatocyte alpha2-MG steady-state mRNA level was shown to be stimulated by germ cells dose-dependently. Using different pools of fractions derived from GCCM after their fractionation by a preparative anion-exchange HPLC column, GCCM was found to contain a factor(s) that stimulated hepatocyte alpha2-MG expression dose-dependently. More importantly, the fractions that stimulated hepatocyte alpha2-MG expression had a retention time different from that of the factor(s) that affected Sertoli cell alpha2-MG expression. These data illustrate that germ cells secrete multiple biological factors capable of regulating alpha2-MG expression in the testis and the liver. In summary, this study reveals a possible physiological link between the testis and the liver in that germ cells may release a factor(s) capable of modulating alpha2-MG expression in both organs.Biology of Reproduction 08/1998; 59(1):111-23. · 4.01 Impact Factor -
Article: An increase in the carbohydrate moiety of alpha 2-macroglobulin is associated with systemic lupus erythematosus (SLE).
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ABSTRACT: Using lectin blots in conjunction with peptide mapping, alpha 2-macroglobulin micropurified from systemic lupus erythematosus (SLE) patients was shown to become abnormally glycosylated suggesting the occurrence of complex glycosylation in this pathological condition. To confirm there is indeed a quantitative increase in specific monosaccharides in this protein; alpha 2-macroglobulin was micropurified from a battery of 37 serum samples which included 6 normal donors (3 male and 3 female), 23 SLE patients, 6 rheumatoid arthritis patients, 1 mixed connective tissue disease patient, and 1 Sjogren's syndrome patient; for carbohydrate analysis. It was noted that the concentration of total monosaccharides in alpha 2-macroglobulin micropurified from serum samples of SLE patients is significantly higher than normal donors with a mean +/- SD of 188 +/- 410 micrograms/mg protein (SLE, n = 23) versus 14.5 +/- 4 micrograms/mg protein (normal, n = 6) even though there was a high variation in the level of monosaccharides among the SLE patients. An increase in oligosaccharides in alpha 2-macroglobulin from SLE patients compared to normal subjects was confirmed by concanavalin A (Con A) blots using peptide fragments derived from the micropurified protein. Since the interaction of peptide fragments derived from alpha 2-macroglobulin with Con A requires the presence of mannose and/or glucose residues, we have also examined if there are any correlations between the levels of mannose and glucose in alpha 2-macroglobulin and SLE. The concentration of mannose (38 +/- 60 micrograms/mg protein) in alpha 2-macroglobulin derived from SLE patients was significantly higher than normal donors (mannose, 4.8 +/- 1 micrograms/mg protein) however, the concentration of glucose in alpha 2-macroglobulin derived from SLE patients when compared to normal donors was not statistically significant, 18 +/- 20 micrograms/mg protein in SLE versus 2 +/- 0.5 micrograms/mg protein in normal donors due to high variation between samples. Also, the concentration of galactose in alpha 2-macroglobulin from SLE patients was significantly higher than normal donors (45.7 +/- 173 micrograms/mg protein versus 0.13 +/- 0.03 microgram/mg protein). These results illustrate quantification of carbohydrate in selected glycoproteins such as alpha 2-macroglobulin may be a novel and alternative clinical marker for SLE.Biochemistry and molecular biology international 01/1998; 43(6):1305-22. -
Article: Identification, isolation, and characterization of a 41-kilodalton protein from rat germ cell-conditioned medium exhibiting concentration-dependent dual biological activities.
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ABSTRACT: In this report, we describe the purification of a novel protease with dual biological actions from germ cell-conditioned medium (GCCM) where germ cells were isolated from adult rat testes using a mechanical procedure. Using multiple HPLC columns and two sequential high performance electrophoresis chromatography steps in association with an [125I]-collagen film assay to detect protease activity, a 41-kDa polypeptide (41-kDa-P) was purified to apparent homogeneity from GCCM. Partial N-terminal amino acid sequence analysis of the purified protein revealed a sequence of NH2-KYEFYEIXLL that, when compared with the existing database at Protein Identification Resource (PIR), GenBank, and BLAST revealed that this is a unique protein. The purified protein, when incubated with [125I]-testin, a Sertoli cell secretory product that is localized at the intertesticular cell junction and is resistant to tryptic digest, was found capable of hydrolyzing testin dose dependently. The proteolysis of [125I]-testin by this 41-kDa protein was inhibited by alpha2-macroglobulin (a Sertoli cell secretory product) also in a dose-dependent manner. A study on the interactions between different classes of protease inhibitors and the purified 41-kDa protein revealed that it is a serine protease. At doses ranging between 0.5 and 50 ng/ml, 41-kDa-P induced a dose-dependent inhibition of Sertoli cell secretory function using testin and clusterin as markers without any apparent proteolytic activity. However, at doses greater than 0.5 microg/ml, 41-kDa-P was found to cleave [125I]-collagen and [125I]-testin at physiological pH, indicating that this 41-kDa protein has dual biological activities whose primary action is concentration dependent. In view of the biological activities of this protease, it is postulated that this protein may be involved in facilitating germ cell migration in the epithelium.Endocrinology 09/1997; 138(8):3259-68. · 4.46 Impact Factor -
Article: Differential expression of multiple cathepsin mRNAs in the rat testis during maturation and following lonidamine induced tissue restructuring.
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ABSTRACT: In the seminiferous epithelium, germ cell development behind the blood-testis barrier involves continual degradation and renewal of inter-testicular cell junctions. This allows: (i) the translocation of developing germ cells from the basal lamina to the adluminal compartment during spermatogenesis, and (ii) the eventual release of mature spermatids into the tubular lumen during spermiation. Throughout spermatogenesis, cellular debris must also be removed from the epithelium Thus, it is conceivable that proteases, protease inhibitors, and cell junctional components are involved in these events. The present study sought to examine whether testicular cells can express multiple cathepsin mRNAs given that these proteases are involved in the degradation and processing of proteins as well as in tissue regeneration. By using total RNA isolated from primary cultures of Sertoli, Leydig, and germ cells for reverse-transcription and polymerase chain reaction (RT-PCR), the mRNAs of cathepsin B, C, D, H, L, and S were shown to be expressed by Sertoli and Leydig cells, whereas germ cells isolated from adult rats expressed all of the above cathepsin mRNAs except cathepsin D. Throughout postnatal development and maturation, the testicular steady-state mRNA levels of cathepsin B, C, D, L, and S remain relatively unchanged with the exception of cathepsin H whose mRNA level increased during maturation and peaked at 45-60 days of age. Using lonidamine, an anti-spermatogenic drug which is known to induce premature release of germ cells without affecting Leydig cell function by disrupting the inter-Sertoli-germ cell junctions, we have examined the differential expression of these cathepsin mRNAs in the testis at the time of extensive tissue restructuring. It was noted that the expression of cathepsin L and S in the testis increased significantly concomitant with the disappearance of elongate spermatids whereas the expression of cathepsin B, C, D, and H increased significantly when most of the round spermatids and spermatocytes were depleted. These results illustrate the intricate inter-relationship between these proteases in the testis during maturation and tissue restructuring.Biochemistry and molecular biology international 07/1997; 42(2):217-33. -
Article: The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study.
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ABSTRACT: The timely opening and closing of inter-Sertoli cell tight junctions in the rat testis are essential cellular events in the completion of spermatogenesis. They permit the passage of preleptotene and leptotene spermatocytes to cross the blood-testis barrier from the basal compartment to the adluminal compartment of the seminiferous epithelium so that these cells can continue their further development into spermatids. However, the mechanism by which these events is regulated remains a mystery in male reproductive physiology. As part of our long-term goal of understanding the biology of this event and its regulation, transepithelial electrical resistance (TER) across the Sertoli cell epithelia when inter-Sertoli tight junctions were being assembled in vitro was quantified to assess the effects of different inhibitors of phosphatases and kinases on the inter-Sertoli tight junction permeability barrier. It was shown that inhibitors of protein tyrosine phosphatases (PTPi) and inhibitors of protein Ser/Thr phosphatases (PPi) could perturb the assembly and maintenance of the inter-Sertoli tight junction permeability barrier. Moreover, the inhibitory effects of PTPi were abolished by pretreating Sertoli cells with protein tyrosine kinase inhibitor (PTKi), which illustrates the specificity of the PTPi treatment. A cyclic adenosine monophosphate-dependent protein kinase A (PKA) activator and inhibitors of calcium-diacylglycerol-dependent protein kinase C (PKC) can also perturb the inter-Sertoli tight junction permeability barrier, which suggests that opening and closing of the inter-Sertoli tight junctions during spermatogenesis is likely regulated, at least in part, by the PKA/PKC pathways. Needless to say, these results illustrate that the interplay of protein kinases and phosphatases, which regulate the intracellular phosphoprotein content of Sertoli cells possibly via PKA and PKC signal transduction pathways, plays a crucial role in modulating the assembly and maintenance of inter-Sertoli tight junctions in the testis.Journal of Andrology 22(5):847-56. · 2.97 Impact Factor -
Article: Changes in the expression of junctional and nonjunctional complex component genes when inter-sertoli tight junctions are formed in vitro.
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ABSTRACT: Throughout spermatogenesis, germ cells move progressively from the basal to the adluminal compartment, which is accompanied by continual disassembly and reassembly of intercellular junctions suggesting germ cell movement is composed of intermittent phases of junction disassembly and reassembly. A study was performed to correlate the expression of junctional-complex components (such as zonula occludens-1 [ZO-1], a tight-junction component protein) and nonjunctional complex components (such as urokinase-type plasminogen activator [uPA], a serine protease; cathepsin L, a cysteine protease; alpha2-macroglobulin, a nonspecific protease inhibitor; and cystatin C, a cysteine protease inhibitor) at the time when inter-Sertoli tight junctions were established in vitro. This is an attempt to investigate whether the expression of nonjunctional component genes also correlates with the formation of inter-Sertoli tight junctions in vitro. This is part of an effort to understand the physiologic elements of germ cell movement in the epithelium. Sertoli cells cultured in vitro are known to undergo programmed cell death. To ensure that the changes in target gene expression were not the result of apoptosis, Sertoli cells were cultured in vitro at densities of 0.25, 0.75, and 3 x 10(6) cells/cm2 for up to 7 days on bicameral culture units coated with Matrigel (Collaborative Research) and were assessed by morphologic analysis and agarose gel electrophoresis. It was noted that many of the Sertoli cells cultured at 3 x 10(6) cells/cm2 underwent apoptosis by day 7, in contrast to cultures at 0.25 and 0.75 x 10(6) cells/cm2 illustrating the Sertoli cell number per unit of area may be an important parameter to be considered when studying Sertoli cell function in vitro. Also, it was shown that the expression of ZO-1 increased significantly between days 2 and 3 prior to the establishment of inter-Sertoli tight junctions assessed by transepithelial resistance measurement (TER), which illustrates that ZO-1 can be used as a marker to monitor this cellular event. More interestingly, there was also a transient increase in the expression of uPA and cathepsin L between days 2 and 3 at the time preceding the formation of tight junctions. In Sertoli cells cultured at low density (2 x 10(4) cells/cm2), when a confluent monolayer of cells could not form, there were no changes in the expression of either ZO-1, uPA, or cathepsin L throughout the 7-day culture period. These results show that the establishment of specialized junctions, such as tight junctions between Sertoli cells in vitro, may require the participation of both junctional and nonjunctional complex components.Journal of Andrology 21(2):227-37. · 2.97 Impact Factor -
Article: Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro.
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ABSTRACT: The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.Journal of Andrology 18(6):612-22. · 2.97 Impact Factor -
Article: Haptoglobin is a Sertoli cell product in the rat seminiferous epithelium: its purification and regulation.
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ABSTRACT: Using multiple HPLC steps, a protein of 67 kDa (estimated by gel permeation HPLC) was purified from Sertoli cell-enriched culture medium that consisted of two dissimilar subunits of 9 (alpha chain) and 24 (beta chain) kDa on SDS-polyacrylamide under reducing conditions. Direct protein sequence analysis of the 9-kDa subunit revealed a sequence of NH2-VELGNDATDIEXD, which is identical to the alpha subunit of the rat haptoglobin (Hp). Hp is a 67-kDa tetrameric serum acute-phase protein consisting of two alpha and two beta subunits (alpha2beta2) of 8.5 kDa and 24.5 kDa, respectively. Using a 351-bp cDNA coding for Hp for northerns and two Hp primers for RT-PCR, we have demonstrated the expression of Hp in Sertoli and Leydig cells, germ cells, and the testis, but not in the epididymis. In contrast to the hepatic haptoglobin, an acute-phase protein whose steady-state mRNA level increased by as much as fivefold during induced inflammation, the testicular homolog reduced by fourfold within 24 hours following induced inflammation, suggesting that this gene is regulated differently in the testis and in the liver. Moreover, the testicular steady-state Hp mRNA level increased steadily after birth during maturation, suggesting its involvement in spermatogenesis. Using primary Sertoli cell cultures in vitro, it was found that the Sertoli cell Hp expression was not regulated by either FSH, testosterone, estradiol, dexamethasone, interleukin-1beta (IL-1beta), IL-6, interferon-gamma (INF-gamma), transforming growth factor-beta (TGF-beta), lymphocyte inhibitory factor (LIF), or germ-cell-conditioned medium (GCCM). Since transferrin secreted by Sertoli cells is an important molecule in maintaining the crucial iron level necessary for spermatogenesis, the identification of haptoglobin as a Sertoli and germ cell product adds a new member to the growing family of metal transporters in the testis that are likely to play an important role in iron metabolism in the testis.Journal of Andrology 18(6):637-45. · 2.97 Impact Factor -
Article: Partial amino acid sequencing of 80-kDa human sperm antigen (80-kDa HSA).
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ABSTRACT: An 80-kDa human sperm antigen (80-kDa HSA) has been identified as a sperm protein responsible for inducing immunoinfertility. Immunization with the purified protein induced infertility in male and female rats. Immunohistochemical and immunofluorescent studies have demonstrated that the antigen is specific to spermatozoa. The present study describes the partial amino acid sequencing of 80-kDa HSA. The homogeneous protein was electrophoretically transferred onto a PVDF membrane and the excised band of 80-kDa HSA was used to determine the partial N-terminal amino acid sequence. The protein was then subjected to enzymatic digestion with endoproteinase Lys-C and endoproteinase Glu-C. The partial amino acid sequence of the major peptides thus obtained was determined. The digestion with endoproteinase Lys-C generated 4 major peptides, two of which showed partial sequence homology with lactoferrin. Endoproteinase Glu-C digestion produced 3 major peptides. The sequences of the 2 peptides were determined for which no matches were found in the databank. These results confirmed earlier observations that 80-kDa HSA is a sperm-specific protein that is chemically distinct from any other protein involved in normal physiological process. Earlier studies have demonstrated that it is antigenic, efficacious, conserved, and could be a promising candidate for the development of an antifertility vaccine.Archives of Andrology 47(3):227-33. · 0.89 Impact Factor
Top co-authors
Top Journals
Institutions
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1998–2001
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Population Council
- Center for Biomedical Research
New York City, NY, USA -
The Rockefeller University
New York City, NY, USA
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1997–2001
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Biomedical Research Alliance of New York
Lake Success, NY, USA
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