Biomedical subjects

C Y Cheng

Publications and source records attributed to C Y Cheng.

At least 217 records · Page 12Linked to original sources

PubMedDate not supplied

The heterogeneity of rat androgen binding protein (rABP) in the vascular compartment differs from that in the testicular tubular lumen. Further evidence for bidirectional secretion of rABP.

Fractionation of testicular extracts and serum on a Concanavalin A-Sepharose column resolved two peaks of immunoreactive rat androgen binding protein. The rat androgen binding protein in the first peak, designated Form I, was present in the void volume; the other, designated Form II rat androgen binding protein, was bound by the column and specifically eluted by alpha-methylmannoside. In the course of studying the heterogeneity of rat androgen binding protein on Concanavalin A-Sepharose, it was observed that the distribution of the two forms of this protein was similar in the fluid obtained by micropuncture from the seminiferous tubule and the rete testis, that is, the ratios of Form I to Form II were 1:1 and 1:1.8, respectively. By contrast, Form I rat androgen binding protein in blood, interstitial fluid, and thoracic duct lymph of adult rats was reduced relative to Form II; the ratios of Form I:Form II in these fluids were 1:4.4, 1:3.1, and 1:4.6, respectively. since previous studies indicated that the reduced amount of Form I relative to Form II observed in the blood of adult rats was not the result of more rapid clearance of Form I, these results suggest that Form I rat androgen binding protein is preferentially secreted into the lumen of the seminiferous tubule rather than into the interstitial fluid and blood. We conclude that Sertoli cells in adult rats may partition rat androgen binding protein between the interstitial and luminal compartments of the testis based on the carbohydrate composition of this protein.

Androgen-Binding Protein↗

PubMedDate not supplied

The differential effects of the indazole-carboxylic acid derivative, tolnidamine, on Sertoli cell protein secretion.

The indazole-carboxylic acid derivative tolnidamine (TOL) has marked antispermatogenic activity in rats. Previous morphological and biochemical studies indicate that Sertoli cells are one of the targets of this compound. The aim of this study was to assess the effect of TOL on the in vitro secretory functions of primary Sertoli cell-enriched cultures prepared from rats of different ages by monitoring the changes of three known Sertoli cell proteins, androgen binding protein (rABP), transferrin (rTF), and testibumin (rTB). The addition of TOL at the beginning of the culture period reduced the plating efficiency of Sertoli cells; however, TOL did not induce a significant change in cell number if it was added 24 h after plating of the cells. Sertoli cell-enriched cultures prepared from tests of 10-day-old rats were highly sensitive to TOL as evidenced by a marked inhibition in secretions of rABP, rTB, and rTF in all experiments. In cultures prepared from 15- and 20-day-old rats, TOL had no apparent effect on rABP secretion, but reduced rTF and increased rTB secretion. Thus, TOL has a differential effect on the secretion of individual proteins in Sertoli cells cultured from rats between 10 and 20 days of age. This phenomenon is presumably a consequence of the progressive maturation of Sertoli cells in the seminiferous epithelium.

Age Factors↗

PubMedDate not supplied

Changes in gene expression and DNA methylation in adrenocortical cells senescing in culture.

Recent experiments in cultured bovine adrenocortical cells show that the previously observed phenotypic switching of CYP17 (steroid 17 alpha-hydroxylase) expression is preceded at a much earlier time by changes in methylation in the CYP17 5' flanking region. Two CpG sites that are methylated in the adrenal cortex in vivo were observed to undergo rapid demethylation when adrenocortical cells were placed in culture. Two adjacent CpG sites that are also methylated in vivo did not demethylate; these two sites are completely nonmethylated in fibroblasts. All CpG sites downstream, in the promoter or coding region, are always methylated in all tissues and in bovine adrenocortical cells even after many population doublings in culture. In contrast to the specific and rapid demethylation of sites in CYP17, satellite I shows a slower and apparently random loss of methylation that extends over the entire replicative life span. These changes in methylation provide examples of genetic instability in cells that undergo senescence in culture. Future experiments will focus on the relationship of these events to the phenotypic switching process.

Adrenal Cortex↗

PubMedDate not supplied

Effects of lonidamine on testicular and epididymal proteins in the rat.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

Acute-Phase Proteins↗

PubMedDate not supplied

Partial amino acid sequencing of 80-kDa human sperm antigen (80-kDa HSA).

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.

Amino Acid Sequence↗

PubMedDate not supplied

The expression of rac1 pseudogene in human tissues and in human brain tumors.

BACKGROUND: Recent studies have demonstrated that Rac is a regulator of cell morphology and growth. Rac1 gene appears to have involvement in tumorigenesis and metastatic potential. In our previous study of rac1 gene in 45 human brain tumors, rac1 pseudogene was found. The rac1 pseudogene is an intronless pseudogene and has a similarity of 86% with rac1 nucleotide sequence. The rac1 pseudogene contains 579 nucleotides and only 46 amino acids can be translated. Little is known about the expression of rac1 pseudogene in human tissues or tumors. MATERIALS AND METHODS: The expression of rac1 gene and rac1 pseudogene in different human tissues and brain tumors was investigated by the use of reverse transcriptase-polymerase chain reaction and Northern blotting. RESULTS: The rac1 gene is apparently expressed in these 8 human tissues. The rac1 pseudogene is also apparently expressed in human tissues except for brain tissue. The overexpression of rac1 gene in brain tumors was 8% (2/25) and the overexpression of rac1 pseudogene was 76.9% (20/26). Only two astrocytomas had overexpression of rac1 gene, compared with normal brain tissues. The overexpression of rac1 pseudogene was 6 of 9 in meningiomas, 7 of 9 in astrocytomas, and 7 of 8 in pituitary adenomas. CONCLUSIONS: High frequency of overexpression of rac1 pseudogene was detected in the human brain tumors when compared with that expressed in the normal brain tissues. Our study suggested that the rac1 pseudogene may play an important role of the tumorigenesis of brain.

Astrocytoma↗

PubMedDate not supplied

Human testis vitamin D binding protein involved in infertility.

Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman containing antisperm antibodies that induced head-to-head agglutination of human sperm. The antibodies in the IgG fraction interacted with a 60-kD protein found in human testes determined by Western blot. The 60-kD protein was purified from human testis by isoelectric focusing (IEF), affinity chromatography on blue sepharose column, and preparative electrophoresis with electroelution. The purified 60-kD protein migrated as a single homogeneous band when analyzed by SDS-PAGE. The amino acid sequence of the N-terminus was determined. The initial 10 amino acid residues were identical to the human serum vitamin D binding protein (VDBP). Polyclonal antibodies were raised against the 60-kD protein. The polyclonal anti-60-kD antibodies and the anti-VDBP antibodies obtained from a commercial source immobilized human sperm in vitro. The interacting antigens were located on the postacrosomal region and midpiece of human sperm, as determined by an immunofluorescence method. The IgG fraction prepared from the serum of an infertile woman interacted with the human testis 60-kD protein but failed to stain serum VDBP. The results suggest that the 60-kD and VDBP are related proteins but not identical entities and that the 60-kD protein contains a unique structural group lacking in serum VDBP. Production of antibodies against the unique structure of the 60-kD protein may be the cause of the infertility.

Antibodies↗

PubMedDate not supplied

Obstruction of the vas deferens alters protein secretion by the rat caput epididymidal epithelium in vivo.

Obstruction of epididymal lumen fluid flow alters the intraluminal environment and potentially changes epididymal epithelial cell function when those functions are dependent on intraluminal regulatory molecules. This investigation tested the hypothesis that obstruction of the rat vas deferens alters caput epididymidal protein synthesis and secretion In vivo. Adult male rats were subjected to vasal obstruction or sham operation. Fourteen days later, caput epididymides were subjected to in vivo microperfusion with medium containing a [35S]-amino acid mixture. At the end of a 3-hour perifusion, micropuncture was used to obtain caput lumen fluid (LF). Tubule extract (TE) was obtained as supernatant after homogenization and centrifugation of caput tubules. Tubule extract contained all [35S]-proteins synthesized within the 3-hour experiment, and LF contained the secreted [35S]-proteins. Radioactivity of trichloroacetic acid (TCA)-precipitable proteins in LF and TE was determined, and two-dimensional electrophoresis and autoradiography of each sample were carried out. The resultant autoradiograms were evaluated densitometrically. A protein synthesis index calculated from the TCA-precipitable radioactivity data demonstrated that a significant decline in overall protein synthesis was induced by vasal obstruction. Densitometry of autoradiograms demonstrated that the total number of radiolabeled proteins detected in both the LF and TE of obstructed animals was significantly smaller than the same number in control animals (P < 0.05). Autoradiography revealed seven major, consistently appearing gene products in LF, and these were subjected to amino acid sequence analysis. Cysteine-rich secretory protein (CRISP)-1 proteins were significantly reduced in the LF of obstructed animals, which implies that these proteins are dependent on luminal regulatory molecules for their normal production.

Amino Acid Sequence↗

PubMedDate not supplied

Changes in the expression of junctional and nonjunctional complex component genes when inter-sertoli tight junctions are formed in vitro.

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.

Animals↗

PubMedDate not supplied

Regulation of Sertoli cell myotubularin (rMTM) expression by germ cells in vitro.

Recent studies have shown that rat myotubularin (rMTM), the homolog of human myotubularin, which is a putative protein tyrosine phosphatase (PTP), is expressed by Sertoli cells in the rat testis. In addition, a significant increase in its steady-state mRNA level was detected in Sertoli cells at the time of inter-Sertoli tight junction (TJ) assembly in vitro. Since the interplay of protein kinases and phosphatases that determines the intracellular phosphoprotein content can, in turn, regulate the assembly and maintenance of TJ and anchoring junctions (AJ) in vitro, as demonstrated in different cell types, such as Madin-Darby canine kidney (MDCK) cells, endothelial cells, and Sertoli cells, rMTM may be an important molecule in regulating the assembly and maintenance of inter-Sertoli TJs during spermatogenesis. We thus sought to characterize its regulation. During testicular maturation, it was shown that the rMTM steady-state mRNA level increased drastically with aging. The expression of rMTM increased by as much as 2-4-fold in the rat testis at 45-60 days of age versus 20 days of age, coinciding with the onset of spermiation. This result seemingly suggests that rMTM may participate in the release of spermatids by disassembling the Sertoli-spermatid AJs, since PTP inhibitor was shown to perturb the inter-Sertoli TJ permeability barrier in vitro. Unexpectedly, when Sertoli cells were isolated from 20-, 45-, and 90-day-old rats and the steady-state rMTM level was quantified, it was shown that there is a drastic reduction in rMTM expression in adult Sertoli cells. Studies that used Sertoli-germ cell cocultures and Sertoli cells incubated with increasing germ cell-derived proteins have shown that the high level of testicular rMTM expression in the testis might be maintained by germ cells. Although work remains to be done to delineate the role of rMTM in the testis, these results illustrate that germ cells play a very active role in regulation testicular function by altering the phosphoprotein content.

Animals↗

PubMedDate not supplied

The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study.

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.

Animals↗

PubMedDate not supplied

Local interstitial emphysema caused by meconium aspiration: report of one case.

A neonate of meconium aspiration syndrome presented with respiratory distress and bilateral pneumothorax. The image studies, including chest X-ray, chest computed tomography and operation finding all showed cystic changes in the right lung. Therefore congenital cystic lesions of lung was the first impression. However, the pathologic report disclosed meconium aspiration with interstitial emphysema.

Biopsy, Needle↗

PubMedDate not supplied

Basal and FSH-stimulated steady state levels of SGP-2, alpha 2-macroglobulin, and testibumin in culture media of rat seminiferous tubules at defined stages of the epithelial cycle.

Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium. The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin. Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05). Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05). Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle. Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I. Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05). The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.

Animals↗

PubMedDate not supplied

Matrix metalloproteinase-8 in sera and from polymorphonuclear leucocytes in rheumatoid arthritis: in vitro characterization and correlation with disease activity.

OBJECTIVES: To determine matrix metalloproteinase-8 (MMP-8) secretion from rheumatoid arthritis (RA) peripheral blood polymorphonuclear leucocytes (PMNs), in response to immune complexes (IC), cytokines and their combinations, and to study correlation of serum MMP-8 with disease activity. METHODS: PMNs from RA patients and controls were stimulated in vitro with interleukin-15 (IL-15), IL-18, adherent immune complexes, rabbit anti-human immunoglobulin G (anti-HIgG), human immunoglobulin G (HIgG), and their F (ab') 2 prongs, phorbol myristate acetate (PMA) or combinations of above. Supernatants from these experiments and sera from both groups were assayed for MMP-8 using ELISA and correlated with disease activity measures in patients. RESULTS: MMP-8 secretion from stimulated PMNs was compared to unstimulated PMNs. Immune complexes elicited significant MMP-8 secretion (p = 0.006 and 0.001, control and RA respectively). Unlike HIgG and its F (ab')2 fragment, very high secretion was elicited by anti-HIgG (242.37 +/- 10.85 ng/ml) and its F (ab')2 prong (195.85 +/- 28.67 ng/ml). IL-15 did not elicit any secretion. IL-18 with PMA increased secretion significantly only from RA PMNs (p = 0.003). Serum MMP-8 correlated positively with serum CRP (p = 0.017) and not with disease activity score (p = 0.199). CONCLUSIONS: We for the first time demonstrate that immune complexes elicit MMP-8 secretion from PMNs. Except for higher secretion from RA PMNs in response to combination of IL-18 and PMA, both control and RA PMNs respond similarly to various stimuli. Secretion by anti-HIgG occurs by a mechanism independent of Fc receptor. Correlation with CRP suggest that serum MMP-8 may be an indicator of acute inflammatory activity.

Adult↗

PubMedDate not supplied

Chronic cough.

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Chronic Disease↗

PubMedDate not supplied

The immunohistochemical localization of alpha 2-macroglobulin in rat testes is consistent with its role in germ cell movement and spermiation.

alpha 2-Macroglobulin (alpha 2-MG) is a nonspecific protease inhibitor and binding protein for peptide hormones that was recently isolated from Sertoli cell-enriched culture medium and shown to be the same protein as alpha 2-MG in serum. The present study was conducted to determine the localization of alpha 2-MG in the seminiferous epithelium in order to gain insight into its possible site(s) of action. Immunostainable alpha 2-MG was present in the lumen of the tubules consistent with its proposed role as a protease inhibitor needed to inactivate the protease released from defective spermatozoa in the male reproductive tract. Immunoreactive alpha 2-MG was also localized adjacent to the heads of elongated spermatids, the most mobile cells in the seminiferous epithelium; immunostainable alpha 2-MG was not observed adjacent to round spermatids and spermatocytes, which are relatively less mobile. The intensity of the staining around the elongated spermatids was dependent on the stage of the spermatogenic cycle. Stainable alpha 2-MG was present adjacent to the spermatids in stage XI soon after the elongation process began. Immunoreactive product was in stages XI-XIV but only faintly visible. The most intense staining reaction for alpha 2-MG was in stages I-VI; it was reduced in stage VII; and virtually no alpha 2-MG was detectable in stages VIII-X at and just after spermiation. The postnatal changes of alpha 2-MG in the testis was also examined. During the first 2 weeks after birth, alpha 2-MG was not detected in the seminiferous epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferrin by Sertoli cells.

In view of the evidence showing that germ cells regulate Sertoli cell (SC) function, the aim of this study was to examine if germ cell (GC)-conditioned media contained multiple biological factors that affect SC secretory functions. Total GC were isolated from adult rat testes. Pachytene spermatocytes (SPC) and early spermatids (SPT) were enriched to about 90% pure by centrifugal elutriation. GC, SPC, and SPT were cultured in serum-free medium for 20 hours with a viability greater than 95%. Conditioned media derived from these cells were fractionated by anion-exchange high-performance liquid chromatography (HPLC). An aliquot from each of these fractions was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were visualized by silver staining. The patterns of protein staining using media from GC, SPC, and SPT were similar. Bioassays of these column fractions on SC showed that the transferrin stimulatory, the testins inhibitory, and the clusterin inhibitory activities were eluted from the anion-exchange HPLC column in overlapping fractions. To determine whether these activities were confined to one or several molecules, further fractionations were performed. Eleven liters of GC-conditioned medium were fractionated by sequential HPLC using anion-exchange, gel permeation, and reversed-phase columns. Throughout the entire fractionation scheme, the HPLC fractions were bioassayed using primary SC-enriched cultures prepared from 20-day-old rats by incubating SC with aliquots of these fractions for 24 hours to monitor their effects on SC secretory function. The concentrations of transferrin, clusterin, and testins were quantified by specific radioimmunoassays. These studies showed that the transferrin stimulatory activity can be fractionated into four peaks (I, IIa, IIb, and IIc); clusterin inhibitory activity into three peaks (A, B, and C); and testins inhibitory activity into two peaks (1 and 2). Some of these bioactivities were eluted in overlapping fractions such as I and B, IIb and 1, and IIc and 2, whereas A, C, and IIa were not associated with any other assayed activities. In summary, additional GC modulators of SC function were identified for the first time, these include clusterin and testins inhibitors. The previously identified transferrin stimulatory activity was also resolved into multiple molecular forms.

Animals↗

PubMedDate not supplied

Purification, cDNA cloning, and developmental changes in the steady-state mRNA level of rat testicular tissue inhibitor of metalloproteases-2 (TIMP-2).

Using multiple high-performance liquid chromatography (HPLC) steps and high-performance electrophoresis chromatography (HPEC) in conjunction with an [125I]collagen film assay to identify inhibitors of metalloproteases, we have purified a 22-kDa polypeptide to apparent homogeneity from primary Sertoli cell-enriched culture medium. Partial N-terminal amino acid sequence analysis revealed that this protein is similar to the human tissue inhibitor of metalloproteases-2 (TIMP-2). To determine the similarity of rat testicular TIMP-2 to the human homolog, a full-length cDNA coding for rat testicular TIMP-2 was isolated from a rat Sertoli cell cDNA expression library and sequenced. Analysis of the nucleotide sequence and the deduced amino acid sequence of the rat testicular TIMP-2 cDNA revealed an 84 and 98% homology with the human TIMP-2 nucleotide and amino acid sequences, respectively. A survey of its mRNA transcripts in different tissues by northern blots revealed the presence of two mRNA species of 3.7 and 1.3 kb in the testis and brain but not in the kidney, spleen, epididymis, and liver in adult male rats. Studies using polymerase chain reaction (PCR) and Southern blot to detect the TIMP-2 mRNA using total RNA isolated from germ cells, Sertoli cells, and Leydig cells have shown that only Sertoli and Leydig cells expressed TIMP-2 mRNA. These results indicate that Sertoli cells are the major source of TIMP-2 in the testis behind the blood-testis barrier (seminiferous tubule barrier). During testicular development from 3 to 60 days of age, the testicular steady-state TIMP-2 mRNA level increased steadily with an advancement of age. Such an increase in the steady-state testicular TIMP-2 mRNA level apparently is not the result of an up-regulation by germ cells because germ cells cocultured with Sertoli cells failed to elicit an increase in the Sertoli cell steady-state TIMP-2 mRNA level. The results of this study suggest that TIMP-2 secreted by Sertoli cells may play a role in tissue restructuring and germ cell migration during spermatogenesis.

Animals↗