Stereospecific toxicokinetics of bromochloro- and chlorofluoroacetate: effect of GST-zeta depletion.

The chloro- and bromohaloacetates are drinking water disinfection by-products and rodent carcinogens. Chloro-bromo dihaloacetates are also mechanism-based inhibitors of glutathione S-transferase-zeta (GSTZ1-1). We studied the stereospecific toxicokinetics and in vitro metabolism of two chiral dihaloacetates in male F344 rats: (-),(+)-bromochloroacetate (BCA) and racemic chlorofluoroacetate (CFA), a non-GST-zeta-inhibiting dihaloacetate. These experiments were repeated in animals that had previously been treated with dichloroacetate (DCA) to deplete GST-zeta activity. Results indicated that the elimination half-life of (-)-BCA was 0.07 compared to 0.40 h for (+)-BCA in naive rats. A comparable difference in elimination half-life was also observed for the CFA stereoisomers (0.79 vs 0.11 h). In GST-zeta-depleted rats, stereospecific elimination of (-),(+)-BCA was absent, with both stereoisomers having an elimination half-life of approximately 0.4 h. This finding was in contrast to results for CFA, which still maintained the same relative difference in elimination rate between its stereoisomers, although overall elimination was diminished in GST-zeta-depleted rats. Results of in vitro metabolism experiments indicated (-)-BCA was affected by modulating GST-zeta activity, with the intrinsic metabolic clearance decreasing from 2.81 to 0.15 ml h(-1) mg.protein(-1) (naive, GST-zeta depleted) compared with values for (+)-BCA (0.30 and 0.31 ml h(-1) mg.protein(-1)). Incubations with 350 microM diethyldithiocarbamate preferentially decreased (+)-BCA metabolism in naive and GST-zeta-depleted cytosol. These results indicate (+)-BCA is a poor substrate for GST-zeta and its metabolism is controlled by an additional GST isoenzyme.

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

The testicular iron shuttle: a "nurse" function of the Sertoli cells.

The techniques of cell culture and molecular biology first revealed that Sertoli cells synthesize transferrin. Accumulated biological information led to a plausible model for the role of testicular transferrin in an iron shuttle system designed to transport ferric ions around the cellular tight junctions to the germ cells inside the blood-testis barrier. Experiments done in culture and in vivo have supported many aspects of this model. A mutant mouse model that lacks the ability to synthesize transferrin is defective in spermatogenesis and may help to delineate the nature of the iron requirement by germ cells. The levels of seminal transferrin, possibly of Sertoli cell origin, are proportional to sperm production in humans and cattle and may be an effective indicator of Sertoli cell function. The testicular iron shuttle thus represents an important "nurse cell" function of the Sertoli cells.

Localization and molecular heterogeneity of sulfated glycoprotein-2 (clusterin) among ventral prostate, seminal vesicle, testis, and epididymis of rats.

In the rat reproductive tract, sulfated glycoprotein 2 (SGP-2) is present in the ventral prostate, seminal vesicle, testis, and epididymis. In the ventral prostate, SGP-2 is associated with the process of programmed cell death, while in the testis and epididymis a role for SGP-2 in sperm maturation has been proposed. Available information suggests that there are both inter- and intra-organ variations in SGP-2 localization, molecular forms, and response to androgen ablation. In the present study, localization of SGP-2 within the ventral prostate, seminal vesicle, and epididymis was compared by immunohistochemistry. In the ventral prostate of intact rats, immunoreactive SGP-2 was confined to a discrete population of epithelial cells lining the proximal ducts. Epithelial cells in other regions of the ventral prostate did not stain for SGP-2. A similar staining pattern was observed for the seminal vesicle; a small population of SGP-2-expressing epithelial cells was found in epithelium that did not stain for SGP-2. The epididymis also demonstrated a non-uniform staining pattern. The caput displayed strong immunoperoxidase reaction over the apical membrane and stereocilia of all principal cells. Principal cells also showed variable degrees of cytoplasmic staining ranging from weak to strongly positive. The corpus and cauda showed a similar staining pattern. After castration, all epithelial cells in the ventral prostate and seminal vesicle became intensely positive for SGP-2 staining. In the caput and cauda epididymis there was an increase in the number of principal cells demonstrating strong intracellular staining for SGP-2. These results suggest that as observed previously in the regressing ventral prostate, increased intracellular SGP-2 staining may also be associated with the regressing epididymis and seminal vesicle. Differences in molecular forms of SGP-2 were investigated by two-dimensional Western and lectin blots. Molecular forms of SGP-2 differed between testis and epididymis but were similar between ventral prostate and seminal vesicle. Prostate and seminal vesicle forms of SGP-2 differed from those of both testis and epididymis. Analysis of terminal carbohydrate present on the various SGP-2 molecular forms also confirmed the existence of heterogeneity. These results demonstrate the presence of multiple molecular forms of SGP-2 in various organs of the male reproductive tract in rats and suggest a possible variation in functional activity and/or half-life of SGP-2 in these organs.

Basic fibroblast growth factor is a testicular germ cell product which may regulate Sertoli cell function.

Previously, a proteinacious factor secreted by a mixture of rat testicular spermatocytes and round spermatids was shown to stimulate transferrin mRNA and protein levels in Sertoli cells. To identify the germ cell-secreted proteins which affect Sertoli cell functions, concentrated germ cell-conditioned medium was fractionated by reverse-phase HPLC. The fraction which eluted at 35% acetonitrile increased transferrin secretion in Sertoli cell cultures 2.4-fold above the basal level. Both the active fraction and a protein extract from cultured germ cells were positive for basic fibroblast growth factor (bFGF) as determined by Western blot analysis and immunoprecipitation. The apparent molecular sizes of the immunoreactive proteins were 30, 27, and 24 kilodaltons (kDa). By immunohistochemistry, bFGF was shown to be present in pachytene spermatocytes and Leydig cells. The bFGF receptor was also examined by immunohistochemistry and found to be present in Leydig cells, round and elongated spermatids, and Sertoli cells. The presence of receptors was more pronounced in stages I-VIII. Western blot analysis confirmed that the receptors were expressed in isolated round spermatids, elongated spermatids, and Sertoli cells. Two major receptor species with apparent molecular sizes of 120 and 145 kDa were detected in the rat testis. Germ cells contained both of these receptors, but Sertoli cells possessed only the 120-kDa receptor. From these experiments, it is evident that bFGF is a germ cell product which may regulate Sertoli cell function. The expression of bFGF and its receptor may be an important component of germ cell-Sertoli cell and/or germ cell-germ cell communication during spermatogenesis.

Differential expression of c-erbA mRNAs in the developing cerebellum and cerebral cortex of the rat.

Expression of c-erbA genes, which encode thyroid hormone receptors, has been shown to be tissue and age specific. The differential regulation of thyroid hormone receptor mRNAs in rat brain was examined in the present study using Northern hybridization techniques. Ontogenic profiles of two known mRNAs for rat thyroid hormone receptors (rTR), rTR alpha and rTR beta, were determined in the rat cerebellum and cerebral cortex. Three bands of approximately 6, 5 and 2.6 kb in size were detected at 1, 6, 12 and 21 days of age (preweanling period) and in the adult, in both brain regions using a rTR alpha-common DNA probe. The 2.6-kb band was expressed at much higher levels than either the 5- or 6-kb band at all ages tested. The 6- and 5-kb bands were expressed at similar levels throughout the preweanling period in the cerebellum, but only at 1 and 6 days of age in the cerebral cortex. The 6-kb transcript was expressed at a much lower level than the 5-kb mRNA by postnatal day 12 in the cerebral cortex. A similar change in the relative level of 6- to 5-kb mRNA expression was noted in cerebellar mRNA isolated from adult animals. The overall expression of rTR alpha mRNA was less in the adult cerebellum and cerebral cortex compared to that in the preweanling period. The rTR beta transcripts were present in very low levels in the cerebral cortex and were undetectable in the cerebellum. These results are consistent with the fact that the developing brain is more sensitive to thyroid hormone than the mature rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat.

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.

Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.

The rat prostate is a complex ductal system with branches and subbranches extending from one end to another. Owing to the relative distance of various regions from the urethra, the entire length of the ductal system can be divided into three segments, i.e. the proximal, intermediate, and distal segments. The present study was carried out to examine the pattern of localization of sulfated glycoprotein-2 (SGP-2), a marker protein associated with programmed cell death, in various regions of the prostatic ductal system under normal conditions and during castration-induced regression. SGP-2 has been considered an androgen-repressed gene product in the rat prostate and has previously been known as castration-induced protein or TRPM-2. In the normal rat prostate, immunoreactive SGP-2 was localized in epithelial cells lining the proximal segment in which signs of programmed cell death were apparent. Cells lining the distal and intermediate segments were, however, devoid of SGP-2. This observed regional variation in SGP-2 localization did not support an earlier hypothesis which stated that SGP-2 was constitutively expressed by all prostatic epithelial cells in the normal rat prostate. After castration in adult rats, there was a shift in the location of cells containing SGP-2 from the proximal segment toward the distal segment. Therefore, there is a regional variation in the distribution of SGP-2 in the rat prostate both before and after castration in the host. These findings are likely to be associated with a regional variation in cellular responsiveness to androgen stimulation and androgen depletion in the prostatic ductal system. Results also support the view that SGP-2 localization is associated with an early manifestation of programmed cell death in the rat prostate.

Increased expression of c-myc and c-Ha-ras in dichloroacetate and trichloroacetate-induced liver tumors in B6C3F1 mice.

The expression of c-myc and c-H-ras in hyperplastic nodules and hepatocellular carcinomas induced in male B6C3F1 mice after chronic administration of dichloroacetate (DCA) and trichloroacetate (TCA) was studied using in situ hybridization. Expression of c-myc and c-H-ras mRNA was increased in both nodules and carcinomas relative to surrounding tissue and tissues obtained from control animals. Myc expression was similar in hyperplastic nodules and carcinomas induced by DCA, but was significantly higher in TCA-induced carcinomas than in hyperplastic nodules and carcinomas produced by DCA. In carcinomas from animals whose TCA treatment was suspended at 37 weeks, c-myc expression remained high relative to control and surrounding liver tissue at 52 weeks. In contrast, the expression of c-H-ras was consistently elevated in carcinomas from both treatments relative to hyperplastic nodules and non-tumor tissue. Within carcinomas from both treatments, focal areas could be located which expressed even higher levels of c-myc. This heterogeneity was not observed in carcinomas hybridized to c-H-ras-probes. These data suggest that elevated expression of c-H-ras and c-myc might play an important role in the development of hepatic tumors in B6C3F1 mice. Elevated expression of c-H-ras was closely associated with malignancy. Increased c-myc expression does not seem necessary for progression to the malignant state. On the other hand, the increased expression of c-myc appears related to the earlier progression of TCA-induced tumors to the malignant state.

Synthesis of transferrin and transferrin mRNA in bovine Sertoli cells in culture and in vivo: sequence of partial cDNA clone for bovine transferrin.

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.

Seminal transferrin and spermatogenic capability in the bull.

The objective of this study was to determine the relationship between seminal transferrin and sperm output in ejaculates from mature dairy bulls. Caudal sperm reserves in mature Holstein bulls (n = 15) were depleted by 8 successive ejaculations during a 50-70-min period (depletion phase). Bulls were then ejaculated 6 times per week for a period of 4 weeks (6X phase). Weekly sperm output (WSO) and weekly transferrin output (WTfO) were the sums of sperm and transferrin levels in 6 ejaculates taken in 1 week of the study. Mean WSO ranged from 20.7 billion to 39.6 billion and mean WTfO ranged from 334 micrograms to 1872 micrograms among the bulls. Regression analysis of sperm and transferrin levels in ejaculates collected during the depletion phase indicated that approximately 40% of seminal transferrin was not related to sperm output and probably was from accessory fluids. A relationship between total seminal transferrin and total sperm in ejaculate was observed (p less than 0.01, r = 0.54). This relationship was stronger when the transferrin was corrected for accessory fluid contribution (p less than 0.01, r = 0.65). The relationship between WSO and WTfO corrected for accessory fluid transferrin contribution (cWTfO) was significant (p less than 0.01, r = 0.64). The relationship between WSO and cWTfO can be interpreted to reflect the relationship between actual testicular sperm production and transferrin from testicular or epididymal origin.

High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes.

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.

Sulfated glycoprotein-1 (saposin precursor) in the reproductive tract of the male rat.

Sulfated glycoprotein-1 is one of the major protein secretion products of rat Sertoli cells in culture. This 70,000 Mr protein shares substantial sequence similarity with human prosaposin, the precursor of lysosomal saposins. Saposins are known to enhance the activity of lipid modifying enzymes presumably by solubilizing the lipids. We report here the immunolocalization of sulfated glycoprotein-1 in the cells and fluid of the male reproductive tract. The protein is present in secondary lysosomes of Sertoli cells and also in the luminal fluid of seminiferous tubules and epididymis. The highest concentrations of the protein are in seminiferous tubule fluid and rete testis fluid, while relatively low amounts are found in cauda epididymal fluid and serum. Sulfated glycoprotein-1 is believed to be involved in degradation of lipids in residual bodies and may also assist in modification of membrane lipids during sperm maturation.

Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator.

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.

Molecular biology of the Sertoli cell.

The "nurse cell" concept developed as a result of the morphological relationships between germ cells and Sertoli cells, and because of the junctional barrier defined by Sertoli cells which allowed the creation of a compartment in which the constituents could be regulated by Sertoli cells. The molecular approach to studies of the role of Sertoli cells in spermatogenesis has already led to a better understanding of the "nurse cell" concept. Molecular studies on the function of Sertoli cells have for the most part been dependent on the Sertoli cell culture techniques which were first published in 1975 (Dorrington and Fritz 1972; Steinberger et al. 1975a; Welsh and Wiebe 1975). As a result of this technical innovation Sertoli cells which remained responsive to hormones and which continued to carry on secretory activities could be obtained relatively free of other cell types. In the spent medium from cultured Sertoli cells specific secretion products could be detected such as ABP, transferrin, and SGP-2; components with inhibin-like activity; and metabolic products such as lactate. Thus, for the first time some molecular correlates to the "nurse cell" role of Sertoli cells have been described. In addition to the close morphological relationships between germ cells and Sertoli cells it has now been demonstrated that protein products of the Sertoli cells directly interact with germ cells. The Sertoli cell-mediated iron transport to germ cells via transferrin is the clearest demonstration of this interaction. Both SGP-1 and SGP-2 interact with spermatozoa and can be found tightly associated with the plasma membrane of these cells. As more of the secretion products of Sertoli cells are characterized the important role of these cells in spermatogenesis and spermiogenesis will be underscored. The identification and characterization of the Sertoli cell secretion products is important information to obtain, but an understanding of the function of these products also requires temporal knowledge about their synthesis. The interdependence of the morphological observations of the testis and the new technology of molecular biology is most clearly illustrated by the studies involving quantitative in situ hybridization. This technique has been utilized to quantify the amount of transferrin and SGP-2 mRNA present in Sertoli cells associated with different stages of the cycle of the seminiferous epithelium and to establish positively the Sertoli cell location of a specific mRNA (Morales et al. 1987). The technique is potentially applicable to any Sertoli cell specific protein product for which a cDNA probe becomes available.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport of iron and transferrin synthesis by the seminiferous epithelium of the rat in vivo.

The transport of radioactive iron across the seminiferous tubules was analyzed in vivo by light-microscope quantitative radioautography. At 5 min after a single intratesticular injection of 55Fe-transferrin, a strong labeling of the basal aspect of the seminiferous epithelium was observed. Between 30 min and 2 h, the labeling on the basal aspect of the seminiferous epithelium decreased. This decrease was accompanied by a substantial increase of the radioautographic reaction over the cellular elements in the adluminal compartment. These results were consistent with the demonstration of 59Fe associated with meiotic spermatocytes and differentiating spermatids isolated by velocity sedimentation from testes injected with 59Fe-transferrin. Furthermore, after a single intratesticular injection of 59Fe-labeled human transferrin, radiolabeled rat transferrin was immunoprecipitated from homogenates of isolated tubules with a specific antibody and appeared as a single radioactive band on fluorographs of urea/polyacrylamide gels. Similarly, 59Fe-labeled rat transferrin but not 125I-transferrin was immunoprecipitated from rete testis fluids of testes infused with either 59Fe- or 125I-labeled human transferrin. Finally, the synthesis of testicular transferrin in vivo was demonstrated in fluorographs of immunoprecipitated transferrin after an intratesticular injection of 35S-methionine in rats whose livers were excluded from the general circulation by ligation of both the hepatic artery and the portal vein. Thus, our results demonstrated a unidirectional system of iron transport from the basal compartment of the seminiferous epithelium to the germ cells in the adluminal compartment involving two distinct transferrins, i.e., a serum transferrin and a testicular transferrin synthesized by the seminiferous epithelium.

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity.

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.

A sulfated glycoprotein synthesized by Sertoli cells and by epididymal cells is a component of the sperm membrane.

We report here the purification, partial characterization and immunofluorescent localization of a dimeric acidic glycoprotein (DAG-protein) secreted by cultures of Sertoli cells of rat testes. Partially purified protein was obtained after chromatography over Sepharose 4B under conditions which favored a soluble, nonaggregated form of the protein. Rechromatography over the same column under reducing conditions yielded very pure monomers of 41,000 daltons and 29,000 daltons. Antibodies were prepared against the mixed monomers and used to immunoprecipitate proteins in spent medium from cultures incubated with [35S] methionine, 35SO4 = or tunicamycin. DAG-protein and another protein (Band 4, 70,000 daltons) were coprecipitated by the antiserum and all contained 35SO4 = in their structures. It was shown by Western blotting that the antiserum cross-reacted very weakly with Band 4 protein. The DAG-protein polypeptides secreted in the presence of tunicamycin were assumed to lack N-glycosylation and exhibited apparent molecular weights of 27,000 and 21,000 daltons. Immunoprecipitations of media from organ cultures of testis and epididymis yielded DAG-protein of slightly lower molecular weight than the protein secreted in Sertoli cell cultures. Indirect immunofluorescence of DAG-protein in paraffin sections of testis and epididymis revealed that the protein was concentrated in the cytoplasm of Sertoli cells, on the stereocilia of epididymal principal cells, in the cytoplasm of epididymal halo cells, and was associated with late spermatids and mature sperm. Sperm were specifically labeled on the acrosome, at the neck, and on the endpiece of the tail. No enzymatic or structural function has been ascribed to DAG-protein as yet, but the protein must play a pivotal role in spermatogenesis because it is secreted by both the testis and epididymis and becomes an integral component of sperm.

Localization of transferrin and transferrin receptors in rat testes.

One of the major proteins secreted by rat Sertoli cells in culture is a transferrin-like protein (Skinner and Griswold, 1980). The purpose of this study was to quantitate the amount of testicular transferrin in fluids isolated from the testis by the use of a radioimmunoassay and to determine the location of transferrin and transferrin receptors in the testis by indirect immunofluorescence. Seminiferous tubule fluid, rete testis fluid, and testicular lymph were collected from rat testes and were found to contain 141 micrograms, 47 micrograms and 3.7 mg transferrin per ml of fluid, respectively. Serum was found to contain 3.7 mg/ml transferrin. Paraffin sections of rat testis were incubated with rabbit anti-rat transferrin, biotinylated goat anti-rabbit and fluorescein-conjugated avidin. Immunoreactive transferrin was thus localized on the proacrosome and nuclear cap of developing spermatids. Late spermatids showed transferrin over the entire region of the head but mature testicular spermatozoa exhibited little fluorescence. The interstitial tissue between seminiferous tubules fluoresced brightly, indicating a large amount of transferrin in this area. By pretreating sections with rat transferrin, the receptor for the protein was localized on and in spermatocytes and early round spermatids. Dividing germ cells were brightly fluorescent.