Sertoli Cell Proteins Research Articles

Serum factors induce messenger ribonucleic acid levels for cellular retinol-binding protein in rat Sertoli cells.

This report shows that serum factors dramatically increase the levels of mRNA for cellular retinol-binding protein (CRBP) in cultured rat Sertoli cells. Incubation of rat Sertoli cells (0-24 h) with 10% fetal calf serum (FCS) was associated with a time-dependent increase in CRBP mRNA levels. A significant increase (6-fold) was observed after 3 h of incubation. Maximal levels (15- to 50-fold) were reached after 9-12 h and were maintained for as long as serum was present. The effect was concentration dependent, with maximal induction at 10% FCS. Removal of FCS resulted in a decline in the CRBP mRNA levels, with a t1/2 of approximately 7 h. The CRBP mRNA stimulating activity (CMSA) was completely removed from FCS by precipitation with 5% trichloroacetic acid, but was only partly (50%) inhibited by heating at 100 C or trypsin treatment. Removal of retinol from FCS by repeated ether extractions did not alter the CMSA of FCS. Both the induction and degradation of CRBP mRNA were inhibited by the protein synthesis inhibitor cycloheximide. A nuclear protein binding to the 5'-flanking region of the CRBP gene was detected in nuclear extracts from untreated Sertoli cells, but not in nuclear extracts from Sertoli cells treated with 10% FCS for 3 h. Thus, serum factors, different from retinoids, dramatically stimulate the levels of CRBP mRNA in rat Sertoli cells. This is associated with the loss of protein binding to the 5'-flanking region of the CRBP gene.

Serum factors induce messenger ribonucleic acid levels for cellular retinol-binding protein in rat Sertoli cells

Serum factors induce messenger ribonucleic acid levels for cellular retinol-binding protein in rat Sertoli cells

Relative roles of testosterone and the germ cell complement in determining stage-dependent changes in protein secretion by isolated rat seminiferous tubules.

This study has compared the effect of withdrawal of testosterone (+/- replacement) with that of selective depletion of pachytene spermatocytes (PS) or round/elongating spermatids (RS), or both PS and RS, on the level of overall protein secretion by seminiferous tubules (ST) isolated at particular stage-groups of the spermatogenic cycle. Testosterone withdrawal was induced by destroying the Leydig cells with a single injection of ethane-dimethane sulphonate (EDS), with or without concomitant replacement of testosterone by injection; ST at stages II-V, VI-VIII or IX-XII were then isolated from control and treated rats at 4 days after treatment. Methoxyacetic acid (MAA) was administered, in either one or two doses, to selectively destroy 80-100% of pachytene and later spermatocytes; ST at stages I-V, VI-VIII or IX-XIV were then isolated at specific times after treatment such that ST were depleted selectively of either PS, RS or PS+RS. Isolated ST (5 cm) were then cultured for 22 h at 34 degrees C with 35S-methionine and its incorporation into secreted proteins then quantified. Based on the incorporation of 35S-methionine, ST at stages VI-VIII showed a significantly higher level of protein secretion than did ST at earlier or later stages. This difference was abolished following testosterone withdrawal but was maintained by testosterone replacement. The normal increase in protein secretion by ST at stages VI-VIII was also prevented if either PS or RS were depleted, whereas depletion of either PS or RS alone had no significant effect on protein secretion by ST at stages I-V, and only the depletion of RS significantly reduced protein secretion by ST at stages IX-XIV. Depletion of both PS+RS reduced protein secretion significantly by ST at all stages. In contrast to the data for total protein secretion, the levels of sulphated glycoprotein (SGP)-1 and SGP-2 secreted by ST at stages VI-VIII showed that these two Sertoli cell proteins were unaffected by germ cell depletion except after co-depletion of PS+RS when secretion of SGP-1 was halved.(ABSTRACT TRUNCATED AT 400 WORDS)

Tri-iodothyronine effects on Sertoli cell protein turnover in the prepubertal pig

The direct effect of Tri-iodothyronine (T(3): 0.1-100 nM) on protein turnover was studied using primary cultures of Sertoli cells isolated from immature piglet testis. The results demonstrate that T(3) significantly increases protein synthesis without altering the protein degradation rate. These data and previous ones, showing the presence of specific T(3) receptors in Sertoli cell nuclei, indicate that T(3) plays a fundamental role in the early regulation of porcine Sertoli cell growth and maturation.

Basic fibroblast growth factor (bFGF) gene expression and protein production during pubertal development of the seminiferous tubule: follicle-stimulating hormone-induced Sertoli cell bFGF expression.

The potential role of basic fibroblast growth factor (bFGF) as a mediator of cell-cell interactions in the growth and development of the testis was examined. Nuclease protection analysis was used to evaluate bFGF gene expression in the testis and other male reproductive tract tissues. bFGF expression was evident in seminal vesicle, prostate, epididymis, and, at low levels, testis of 20-day-old rats. The developmental expression of bFGF in whole testis and isolated somatic cells types was determined. Mesenchymal-derived peritubular cells and epithelial-like Sertoli cells were isolated from prepubertal, midpubertal, and late pubertal rat testes. In whole testis, bFGF expression is predominant early in prepubertal testicular development and decreases with sexual maturity. Both freshly isolated peritubular and Sertoli cells express bFGF at relatively constant levels during pubertal development, with a slight suppression at the late pubertal stages. Freshly isolated mature Leydig cells also expressed low levels of bFGF. Cultured Sertoli and peritubular cells produced bFGF-like proteins, including 18- and 24-kilodalton forms. Interestingly, FSH increased Sertoli cell bFGF gene expression and protein production. Previously, FSH and bFGF have been shown to stimulate immature Sertoli cell growth. The results of the current study suggest that the ability of FSH to regulate testis and Sertoli cell proliferation may in part be indirectly mediated through the local production and action of bFGF. bFGF has also previously been shown to localize in developing germinal cells. Therefore, FSH-induced Sertoli cell bFGF expression may mediate Sertoli-germinal cell interactions involved in the control of the spermatogenic process. Observations demonstrate the presence of bFGF at a time coinciding with active growth of the somatic cell populations of the seminiferous tubule. Potential roles for bFGF in the seminiferous tubule to consider include angiogenesis of the tubule, prepubertal Sertoli cell proliferation, and mediating Sertoli-germinal cell interactions.

Rat Sertoli cell clusterin, [formula omitted], and testins: Biosynthesis and differential regulation by germ cells

Clusterin, α 2- macroglobulin and testins are three novel Sertoli cell proteins whose physiological functions may be related to cell-cell interactions in the seminiferous epithelium of the testis. We have demonstrated the biosynthesis of clusterin, α 2- macroglobulin , and testins by Sertoli cells in vitro using pulse-chase labeling analysis. For clusterin, two precursors with an apparent molecular weight ( M r ) of 72,000 (P H) and 66,000 (P L) were detected in the Sertoli cell cytosol in addition to the α ( M r 43,000) and β ( M r 35,000) subunits of the mature protein. However, the precursors were not secreted into the medium since only the α and β subunits of clusterin were detected. For α 2- macroglobulin and testins, no precursor molecules were detected either in the Sertoli cell cytosol or culture medium. The polarized secretory pattern of these proteins and their regulation by follicle stimulating hormone (FSH) and testosterone (T) were examined using a bicameral culture chamber that mimics the in vivo physiological conditions. Clusterin was secreted almost exclusively into the apical chamber of the bicameral culture unit with an apical:basal ratio of 30:1. In contrast, α 2- macroglobulin and testins had an apical:basal ratio of 1:1 and 1.5:1, respectively. Thus, the polarized secretory pattern for clusterin is different from α 2- macroglobulin and testins. It was noted that FSH and T, the known Sertoli cell regulators, did not affect the secretion of either clusterin or α 2- macroglobulin . Due to the morphological intimacy between Sertoli cells and germ cells in the adluminal compartment of the testis, the effects of germ cell-conditioned medium were investigated. Addition of germ cell-conditioned medium (1–30 μg protein) to the apical chamber of the bicameral culture unit caused a dose-dependent inhibition of clusterin and testins apical secretion and a slight but statistically significant stimulation of their basal secretion. In contrast, the secretion of α 2- macroglobulin by Sertoli cells was stimulated both apically and basally. These observations suggest that germ cell-conditioned medium contains a biological factor(s) that differentially regulates the bidirectional secretion of Sertoli cell proteins. These studies therefore reveal the complicated regulatory processes involved in cell-cell interactions in the seminiferous epithelium.

Follicle-stimulating hormone-regulated Sertoli cell proteins SCc1 and SCc2 are phosphorylated and mitochondrially associated

Sertoli cell intracellular protein 1 (SCc1) and 2 (SCc2) are polypeptides found in rat Sertoli cell cultures incubated with either FSH or (Bu)2cAMP. They were first identified in [35S]methionine-labeled Sertoli cell lysates using two-dimensional gel electrophoresis. Here we extend these observations by showing that SCc1 and SCc2 are present in rat seminiferous tubules, ovaries, and granulosa cells incubated with either FSH or (Bu)2cAMP and in testicular peritubular cells incubated with (Bu)2cAMP. Peritubular cells do not, however, respond to FSH with the production of SCc1 and SCc2. Peptide mapping with N-chlorosuccinimide revealed that SCc1 and SCc2 have similar cleavage patterns, suggesting a common primary amino acid sequence that is modified posttranslationally. Metabolic labeling with [32P]orthophosphate provided direct evidence that SCc1 and SCc2 are phosphoproteins. A shift in mobility of SCc1 and SCc2 toward the basic region of the gel to positions designated SCc1' and SCc2' occurred when cell lysates were treated with alkaline phosphatase before electrophoresis, providing additional evidence that SCc1 and SCc2 are phosphoproteins. SCc1 and SCc2 are also shown to be mitochondrially-associated in the Sertoli cell. Peptide maps of SCc1, SCc2, SCc1', and SCc2' obtained by treatment with alpha-chymotrypsin, are identical to proteolytic maps of proteins pp30', p30, and pp30 from adrenocortical cells. SCc1, SCc2, SCc1', and SCc2' are homologous with regard to their regulated expression, electrophoretic mobility, and mitochondrial localization to the adrenal proteins pp30' and pp30 as well as a series of 30 kilodalton proteins from MA-10 Leydig tumor cells. Both the adrenal cell proteins and the Leydig tumor cell proteins are thought to participate in cholesterol transport to the inner mitochondrial membrane, providing substrate for the cholesterol side-chain cleavage enzyme complex, an activity which the Sertoli cell does not perform, suggesting that alternative functions must be sought for SCc1 and SCc2 in Sertoli cells.

Human sertoli cells in vitro: Morphological features and androgen-binding protein secretion

Sertoli cells play a pivotal role in the regulation of spermatogenesis as they provide the anatomical basis of the blood-testis barrier. In the present paper we report some results of our studies on the ultrastructural features, the responsiveness to FSH, and the ability to secrete androgen-binding protein (ABP) of human Sertoli cells in vitro. The nucleus showed the characteristic foldings of the nuclear membrane, scattered chromatin, and a fibrillar nucleolus. In the cytoplasm Charcot-Boettcher crystals were present and active phagocytic activity was documented by the presence of vacuoles containing lipids and cellular debris. Human Sertoli cells in culture responded to FSH with a maximal rise in cAMP that was approx. 3-fold. This response to FSH is comparable to that reported for the adult rat but lower than that of the immature rat, and suggests that human as well as rat Sertoli cells could have a reduced response to FSH since sexual maturation was achieved. As no evidence has been reported on ABP secretion by human Sertoli cells in culture we evaluated the concentration of this protein in the Sertoli cell spent media. Human Sertoli cells in culture produced ABP and the response to FSH was dose-related. The K d value of human ABP (hABP) was approx. 7.5 nM, being slightly higher than that of the rat ABP and an order of magnitude different from that of sex hormone-binding globulin (SHBG) present in human plasma. We also measured the association and dissociation rates of dihydrotestosterone-hABP complexes and the K d K a ratio was very close to the value of K d of the Scatchard analysis. The differences between hABP and SHBG may open the way to the selective measurement of ABP in many conditions of male infertility.

Beta-nerve growth factor influences the expression of androgen-binding protein messenger ribonucleic acid in the rat testis.

The effect of infusion of nerve growth factor (NGF) into the rat testis on the expression of androgen-binding protein (ABP) mRNA was studied. A major 1.7-kb and a minor 3.7-kb ABP mRNA were present at all stages of the seminiferous epithelium with maximal levels at stages VIII-XI and the lowest levels at stages IV-VI. Infusion of 15 ng/h of NGF with a mini-osmotic pump for 14 days resulted in a 2-fold increase of ABP mRNA as revealed by Northern blots, whereas the mRNA level of another Sertoli cell protein, urokinase-type plasminogen activator, remained unchanged. Image analysis of autoradiograms obtained by in situ hybridization of sections from treated testes showed a similar increase in APB mRNA compared to noninfused or PBS-infused testes. However, at the cellular level the labeling intensity for ABP mRNA over Sertoli cells of different stages of the seminiferous epithelium was the same in NGF-infused and control testes. This suggests that the increase of ABP mRNA in NGF-infused testes was caused by prolongation of stages VII-VIII with maximal ABP mRNA expression; the suggestion is supported by an increase of 30 percent in frequency of these stages in histological sections from NGF-infused testes.

The low molecular weight heat shock protein (hsp27) in rat Sertoli cells: evidence for identity of hsp27 with a germ cell-responsive phosphoprotein

The low mol wt heat shock protein (hsp27) exists as three forms. hsp27a is the most basic and is not phosphorylated. The other two forms are phosphorylated, with hsp27b of intermediate isoelectric point and hsp27c as the most acidic form. We hypothesized that a Sertoli cell protein, previously discovered as being responsive to germ cells and referred to as GC1, is hsp27c. To investigate this hypothesis, cultured Sertoli cells were treated with CdCl2 or were heat shocked and then labeled with H3(32)PO4, [35S]methionine, or 3H-labeled amino acids. Sertoli cell proteins were then prepared for two-dimensional polyacrylamide gel electrophoresis and autoradiography or fluorography. The 27K protein did not incorporate [35S] methionine. After CdCl2 or heat shock treatments, hsp27b and hsp27c (GC1) showed increasing 32P incorporation with time. To test if this result was due to increased synthesis of hsp27 Sertoli cell proteins were labeled with 3H-labeled amino acids. Heat shock and CdCl2 resulted in time-dependent increases in 3H label incorporation into all three forms of hsp27. Shortly after heat shock, a greater proportion of hsp27b and hsp27c was observed in the pellet fraction of Sertoli cell homogenates, with more of these hsp27 forms being found in the supernatant fraction after 4 h recovery from heat shock. Treatment of CdCl2-exposed Sertoli cells with germ cells increased 32P labeling of hsp27c (GC1). The location on two-dimensional gels of the most acidic form of hsp27, hsp27c, was exactly coincident with the location of GC1. Taken together, the results support the conclusion that GC1 in Sertoli cells is hsp27c.

The low molecular weight heat shock protein (hsp27) in rat Sertoli cells: evidence for identity of hsp27 with a germ cell-responsive phosphoprotein.

The low mol wt heat shock protein (hsp27) exists as three forms. hsp27a is the most basic and is not phosphorylated. The other two forms are phosphorylated, with hsp27b of intermediate isoelectric point and hsp27c as the most acidic form. We hypothesized that a Sertoli cell protein, previously discovered as being responsive to germ cells and referred to as GC1, is hsp27c. To investigate this hypothesis, cultured Sertoli cells were treated with CdCl2 or were heat shocked and then labeled with H3(32)PO4, [35S]methionine, or 3H-labeled amino acids. Sertoli cell proteins were then prepared for two-dimensional polyacrylamide gel electrophoresis and autoradiography or fluorography. The 27K protein did not incorporate [35S] methionine. After CdCl2 or heat shock treatments, hsp27b and hsp27c (GC1) showed increasing 32P incorporation with time. To test if this result was due to increased synthesis of hsp27 Sertoli cell proteins were labeled with 3H-labeled amino acids. Heat shock and CdCl2 resulted in time-dependent increases in 3H label incorporation into all three forms of hsp27. Shortly after heat shock, a greater proportion of hsp27b and hsp27c was observed in the pellet fraction of Sertoli cell homogenates, with more of these hsp27 forms being found in the supernatant fraction after 4 h recovery from heat shock. Treatment of CdCl2-exposed Sertoli cells with germ cells increased 32P labeling of hsp27c (GC1). The location on two-dimensional gels of the most acidic form of hsp27, hsp27c, was exactly coincident with the location of GC1. Taken together, the results support the conclusion that GC1 in Sertoli cells is hsp27c.

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.

HPLC analysis of free amino acids and amino acids of total proteins in cultured cells: An application to the study of rat sertoli cell protein metabolism

A simple, rapid and, sensitive HPLC method, coupled with fluorometric detection, has been worked out and employed to determine the intracellular free amino acid concentrations and the amino acid composition of total proteins in rat Sertoli cell primary culture. Sertoli cells were isolated enzymatically from testes of 20- and 28-day-old rats and cultured at 32°C in Eagle's minimum essential medium. On the second day of culture, cell monolayers were quickly rinsed with ice-cold saline, immediately frozen in liquid nitrogen, accurately harvested, and homogenized in 10% trichloroacetic acid. Tissue free amino acids were determined in the acidic soluble fraction following neutralization, while the precipitate was hydrolyzed for the evaluation of the fractional content of amino acids into total proteins. Amino acid samples were derivatized with o-phthaldialdehyde/3-mercaptopropionic acid and resolved by a linear one-step acetonitrile gradient in 12.5 m m sodium phosphate buffer, pH 7.2, employing a 5-μm particle size reversed-phase column. Fluorescence was monitored with excitation at 330 nm and emission at 450 nm. Under these conditions all major physiological amino acids could be satisfactory separated, identified, and subsequently quantified with the aid of standards. The run time was about 50 min; the linearity was excellent over a large range of concentrations (1–800 pmol) and the lower limit of sensitivity appeared to be 0.5 pmol. This method permits us to demonstrate age-dependent modifications in the intracellular amino acid pool and to adequately evaluate the process of protein synthesis in cultured Sertoli cells. The precise evaluation of the specific activity in the amino acid precursor pool allows us the quantitative estimation of protein synthesis by incorporation of a radioactive amino acid; the fractional content of the amino acid precursor into total cell protein was used for the calculation of the rate of protein synthesis.

Identification and partial characterization of laminin binding proteins in immature rat Sertoli cells

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [ 125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the M r 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the M r 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32 67 laminin binding proteins.

Immunocytochemical demonstratin of c‐fos protein in sertoli cells and germ cells in rat testis

Immunocytochemical demonstratin of c‐fos protein in sertoli cells and germ cells in rat testis

Modulation of Sertoli cell secretory function by rat round spermatid protein(s)

The influence of rat round spermatid protein(s) (RSP) on protein synthesis and secretory function of Sertoli cells was used in the bicameral chamber system. Round spermatids (RS) were purified from 90-day-old rats by centrifugal elutriation. RS were incubated in a supplement-enriched culture medium that lacked exogenous proteins. The RS-conditioned media were dialysed and lyophilized to obtain RSP. Most de novo protein synthesized under basal conditions by Sertoli cells (18-day-old) was secreted into the apical chamber (apical/basal ratio: 3.42). Follicle-stimulating hormone (FSH, 100 ng/ml) stimulated total protein secretion from Sertoli cells by a factor of 1.54. The RSP (100 μg/ml) stimulated total protein secretion from Sertoli cells by a factor of 2.33. The enhancement of total Sertoli cell protein secretion by FSH and RSP additively increased by a factor of 2.82. The combined effect of FSH and RSP on total protein secretion from Sertoli cells was dose dependent and saturated at approximately 200 μg/ml of RSP. Polarity of total protein secretion from Sertoli cells (apical/basal ratio: 3.42) was stimulated by RSP predominantly in the apical direction (apical/basal ratio: 8.48). The modulation of radiolabeled Sertoli cell secretory proteins (ceruloplasmin, CP; sulfated glycoprotein-2, SGP-2; testins and transferrin, Tf) by cold (non-labeled) RSP was investigated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secretion of CP, SGP-2 and Tf was stimulated in a dose-dependent manner by the addition of RSP up to a saturating concentration of between 200 and 300 μg/ml, whereas the secretion of Sertoli cell testins did not reach saturation at 300 μg/ml RSP. These results indicate that FSH and RSP independently modulate Sertoli cell protein secretion, and that Sertoli cell secretory proteins may differentially respond to RSP stimulation.

Laminin promotes formation of cord-like structures by sertoli cells in vitro

Basement membranes are thin extracellular matrices which contact epithelial cells and promote their adhesion, migration, differentiation, and morphogenesis. These matrices are composed of collagen IV, heparan sulfate proteoglycan, laminin, and entactin as well as other minor components. Sertoli cells, like most epithelial cells, are in contact at their basal surface with a basement membrane. When cultured within three-dimensional basement membrane gels (Matrigel), Sertoli cells reorganize into cords that resemble testicular seminiferous cords found in the in vivo differentiating testis. Anti-laminin and anti-entactin antisera inhibit this cord morphogenesis by Sertoli cells whereas antisera against type IV and type I collagen, heparan sulfate proteoglycan, fibronectin, and preimmune sera had no effect. The RGD (RGDS-NH 2) sequence, found in the cell binding domain of the integrin family of cell adhesion molecules as well as in the A chain of laminin and in entactin, effectively inhibited Sertoli cell cord formation at a concentration of 1.0 mg/ml but was unable to prevent Sertoli cell attachment at concentrations as high as 2.0 mg/ml. A synthetic pentapeptide from a cell-binding domain of the B1 chain of laminin, YIGSR-NH 2, inhibited cord formation at a concentration of 0.25 mg/ml, but Sertoli cells were still adherent to the basement membrane matrix. At concentrations greater than 0.50 mg/ml, Sertoli cells detached. Antiserum against the YIGSR-NH 2-containing sequence was also effective in inhibiting cord formation by Sertoli cells. Ligand (YIGSR-NH 2 peptide) blot analysis of Sertoli cell lysates revealed an interaction with a major band at 60 kDa and with minor bands at 39 and 127 kDa. Furthermore, in Western blot analysis the anti-67-kDa laminin-binding protein antibody recognized a 59- to 60-kDa protein in Sertoli cells. The data indicate that laminin is involved in both Sertoli cell attachment and migration during formation of histotypic cord structures by these cells in culture. Two separate laminin cell-binding domains appear to be involved in Sertoli cell cord morphogenesis in vitro and are likely to participate in the formation of seminiferous cords in vivo.

Testins Are Structurally Related Sertoli Cell Proteins Whose Secretion Is Tightly Coupled to the Presence of Germ Cells

Previous studies from this laboratory have shown that Sertoli cell-enriched culture medium contained two immunologically and structurally related proteins designated CMB-22 and CMB-23 with Mr of 37,000 and 40,000, respectively. We have now demonstrated that both CMB-22 and CMB-23 are monomeric proteins with the following NH2-terminal amino acid sequences: CMB-22, NH2-TPDPSLDVEWNEWRTKHGKTYNMNEERLKR; CMB-23, NH2-XAPXPDPSLDVEXNEXRTK. These sequences are virtually identical except that CMB-23 has three extra NH2 terminus amino acids of X-A-P. Comparison of these sequences with those in the Protein Identification Resource revealed that they are unique proteins. CMB-22 and CMB-23 are highly concentrated in testes and their levels in this tissue increase with age. Studies using [35S]methionine incorporation and immunoprecipitation demonstrated that Sertoli cells synthesize and secrete these proteins in vitro. Because they seem not to have been isolated previously, are concentrated in and synthesized by the testes, and are structurally related, we propose that CMB-22 and CMB-23 be designated testin I and testin II, respectively. The distribution of these proteins in biological fluids were compared with those of testibumin and rat androgen binding protein (rABP), two other Sertoli cell proteins. The results suggest that testins, unlike testibumin and rABP, are not transported to the epididymis. Although the amount of testins secreted by Sertoli cells in vitro is similar to that of testibumin and rABP, the concentrations in testis and rete testis fluid are several orders of magnitude less than that of testibumin and rABP. These observations suggest that the secretion of these proteins in vivo might be suppressed by germ cells. The fact that 10 times more testins are secreted by tubules from immature rats than by those from adult rats and that there is an increase in the testicular content of testins following a single dose of busulfan, which depleted the germ cells from the seminiferous epithelium, supports this hypothesis. Thus, the secretion of testins by Sertoli cells appears to be tightly coupled to the presence of germ cells; there is an inverse relationship between the amount of testins in the testis and the number of germ cells. These results suggest that testins are unique testicular proteins that can be used to study Sertoli cell-germ cell interactions in the seminiferous epithelium.

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.