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Biomedical subjects

A Steinberger

Publications and source records attributed to A Steinberger.

At least 37 records · Page 2Linked to original sources
PubMed1989

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures.

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.

Animals↗
PubMed1989

Sertoli cells in culture: views from the perspectives of an in vivoist and an in vitroist.

Culturing Sertoli cells has proven to be a valuable technique to study the responses of these cells under a variety of conditions. This minireview focuses on the culture system and how it has contributed to our knowledge of the capabilities of isolated Sertoli cells. In addition, it focuses on the limitations of the system and the gaps in our knowledge about Sertoli cells in culture and what the results of in vitro studies mean in the intact animal. Caution is urged in interpreting in vitro results to the intact animal without their validation in vivo.

Animals↗
PubMed1989

Age-dependent Sertoli cell responsiveness to germ cells in vitro.

This study investigated the effect of germ cells (greater than 80% mid- and late-pachytene spermatocytes) on the secretion of androgen binding protein (ABP) and transferrin by monolayer cultures of Sertoli cells isolated from rats aged 10, 18 or 26 days. There was an age-dependent increase in secretion of ABP and transferrin. Treatment of the Sertoli cell monolayers with hypotonic buffer to remove residual germ cells reduced this increase significantly. On the other hand, addition of germ cells to hypotonic-treated Sertoli cell monolayers increased both basal and FSH + testosterone-stimulated ABP and transferrin secretion at all three ages, although Sertoli cells from 10-day-old animals showed the greatest response. Moreover, addition of germ cells reduced responsiveness to FSH + testosterone in Sertoli cell monolayers obtained from rats aged 18 or 26 days. In monolayers obtained from 10-day-old rats, the opposite effect was noted in the case of ABP secretion. The stimulatory effect of germ cells on ABP and transferrin secretion was proportional to their number, and was reversed 48 h after the germ cells added previously were removed by hypotonic treatment. Whereas the reversal was complete with cultures of Sertoli cells isolated from 18- and 26-day-old rats, approximately 40% of the stimulatory effect remained after removal of germ cells from cultures from the 10-day-old age group. Adhesion of germ cells to Sertoli cell monolayers was also found to be age-dependent, with the largest proportion of added germ cells adhering to Sertoli cells isolated at 18 and 26 days of age. It is concluded that germ cells can significantly and differentially modulate the basal and hormone-stimulated secretory activity of Sertoli cells in vitro and that Sertoli cell responsiveness to germ cells (pachytene spermatocytes) is age-dependent and seems to appear early during the maturation process, before these germ cells appear in the testis.

Aging↗
PubMed1988

Experimental pitfalls in evaluating vectorial protein secretion in vitro; Sertoli cell secretion of androgen-binding protein and transferrin in two-compartment culture chambers.

We examined the influence of various Millipore filter pretreatments on the amounts of androgen-binding protein (ABP) and transferrin (Trf) found in the outer (OC) and inner (IC) compartment of two-compartment Sertoli cell (Sc) cultures. When Sc were cultured on untreated Millipore filters, less than 10% of ABP was found in OC during 3 initial culture days compared to similar cultures on pretreated filters. Most of the glycoprotein was shown to be bound by the filter. Pretreatment of Millipore filters with 5% bovine serum albumin (BSA) or 2% fetal bovine serum (FBS) maximally saturated the nonspecific protein-binding sites resulting in OC:IC ratio of ABP similar to that found in cultures on polycarbonate membranes, which exhibit very low protein-binding capacity. In contrast to ABP, about 40% of Trf was bound by the Millipore filter on Day 1, with only trace amounts bound thereafter. These differences were due to much higher secretion rate of Trf than ABP, resulting in a relatively smaller fraction of Trf bound to the filter. Again, the nonspecific binding of Trf was greatly reduced by filter pretreatment with 5% BSA or 2% FBS. It is concluded that complete saturation of protein-binding sites of cellulose ester supports is necessary for reliable evaluation of vectorial protein secretion by Sc and other polarized epithelial cells maintained in this type of culture. The implications of partial saturation of protein-binding sites of culture support in interpreting experimental results are discussed.

Androgen-Binding Protein↗
PubMed1987

Study of the dynamics of Sertoli cell secretions in a new superfusion, two-compartment culture system.

A new superfusion, two-compartment culture system recently developed in our laboratory was used to investigate the dynamic changes in bidirectional secretion of transferrin (Trf) and androgen binding protein (ABP) by rat Sertoli cells (Sc) cultured for up to 12 d under various experimental conditions. The system is unique in that the cells are grown on porous substrate and can be superfused independently at the apical (A) and basal (B) surfaces. The Sc formed confluent monolayers with tight junctions and were highly polarized, morphologically resembling their normal appearance in vivo. The bidirectional secretion patterns (total amount and A:B ratio) of both Trf and ABP were affected by the addition of hormones (testosterone, 10(-6) M; follicle stimulating hormone, 0.1 microgram/ml; and fetal bovine serum 2%), but not by changes in the medium flow rate (0.8 to 3.2 ml/h). The superfusion, two-compartment culture system provides a very useful model for culture of polarized cell monolayers and for the study of bidirectional secretions under more "physiologic" conditions than those provided by static cultures.

Androgen-Binding Protein↗
PubMed1987

Identification of a new Sertoli cell metabolite of testosterone: 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol, by HPLC and GC-MS.

Testosterone metabolism by isolated rat Sertoli cells cultured in vitro was investigated using HPLC and GC-MS techniques. Monolayer cultures of Sertoli cells (greater than 90% pure and free of Leydig cells) were incubated for 3-day periods with a stable labeled [2,2,4,6,6-d5]testosterone prepared and used in a 1:1 proportion with unlabeled testosterone as the substrate (5 X 10(-7)M). After incubation, the metabolites were extracted from the media and reacted with oestradiol-antibodies. The antibody-bound components were separated on reverse phase HPLC and the fraction corresponding to oestradiol was analyzed by GC-MS in the form of TMS-ether. One of the metabolites whose mass spectrum contained d0 + d5 species was detected and interpreted to be a triol with a mol. wt of 308. Mass spectra data indicated that this testosterone metabolite is one of the sixteen possible isomers of 3,16,17-trihydroxy androstane. This substance was identified based on the Vm value (27.81) closely resembling that of 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol TMS-ether (Vm reported = 27.78) [1] and when compared directly with synthesized compounds [2-3]. Recently we have demonstrated that similar Sertoli cell preparations contain two 16 alpha-hydroxylases by their ability to convert oestradiol to oestriol [4] and 5 alpha-androstane-3 alpha,17 beta-diol to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol [3], where the former conversion is not affected by FSH, the latter is significantly stimulated by the presence of FSH. Presence of this new product represents the first example of testosterone conversion to 5 alpha-androstane-3 alpha,16 alpha,17 beta-triol and confirms our previous observation that 16 alpha-hydroxylation of 5 alpha-reduced androgens can occur in the rat testis.

Androstane-3,17-diol↗
PubMed1987

Vectorial secretion of transferrin and androgen binding protein in Sertoli cell cultures: effect of extracellular matrix, peritubular myoid cells and medium composition.

We examined the effect of various culture conditions on the polarized secretion of androgen binding protein (ABP) and transferrin (Trf) by Sertoli cells (Sc) in vitro. Sc from 18-day-old rats were cultured as confluent monolayers on permeable membranes in two-compartment chambers for up to 11 days. Coating of the membranes with extracellular matrix (ECM) components: collagen IV + laminin (CL) or reconstituted basement membrane (RBM) enhanced ABP and Trf secretion (200% and 150%, respectively), with RBM being more effective than CL in stimulating ABP but not Trf secretion. Neither CL nor RBM significantly influenced the relative amounts of ABP and Trf secreted into the outer (OC) and inner (IC) compartments of the culture chamber (OC/IC ratio). All of these effects were not significantly influenced by the presence of testosterone and serum. Co-culture of Sc with peritubular myoid cells (Pc) significantly increased the secretion of both ABP and Trf, although the magnitude of stimulation and the time-response patterns were different for each protein. Co-culture with Pc also dramatically increased the OC/IC ratios for ABP and Trf. Testosterone (10(-6) M) enhanced the Pc effects. In cultures of Sc alone, presence of 2% fetal bovine serum increased the OC/IC ratios, whereas testosterone had no effect. Based on these results, we suggest a possible role of Pc in the regulation of Sc polarized secretions.

Androgen-Binding Protein↗
PubMed1987

Bipolar secretion of androgen-binding protein and transferrin by Sertoli cells cultured in a two-compartment culture chamber.

The influence of various medium supplements on Sertoli cell (Sc) monolayer permeability to [3H]inulin and polarized secretion of transferrin (Trf) and androgen-binding protein (ABP) was investigated in a two-compartment culture chamber. Sc from 14- and 18-day-old rats were maintained for 13 days in one of the following media: Modified Eagle's Medium-Ham's F-12 (DFM), DFM plus insulin, epidermal growth factor, progesterone, hydrocortisone, and vitamins A and E (6F), 6F plus testosterone (7F), 7F plus FSH (8F), 6F plus 2% fetal bovine serum (6F2S), 6F plus 5% fetal bovine serum (6F5S). The monolayer permeability to [3H]inulin decreased rapidly during the initial 3-5 days of culture for the Sc isolated from 18- and 14-day old animals, then remained stable in all media except DFM. Morphological examination revealed the presence of tight junctions between neighbouring Sc in both age groups, indicating their de novo formation. Secretion of Trf was lowest in DFM and steadily declined. In all other media, Trf secretion peaked on day 5 and remained relatively constant after day 7. Medium 7F only slightly and inconsistently increased the secretion, whereas 8F was always highly stimulatory compared to 6F. Supplementation of 6F with serum resulted in the greatest Trf secretion. In the case of ABP, three different secretion patterns were noted depending on the medium composition; secretion was greatest in the presence of 5% fetal bovine serum. The medium supplements also differentially affected the polarity of Trf and ABP secretion. The ratio of Trf secreted to the outer and inner compartments (OC/IC) was approximately 2.0 in DFM and was not influenced by supplements in 6F, 7F, and 8F. However, in serum-containing media, the OC/IC ratio gradually increased with time to about 5.0 on day 13. The average OC/IC ratio of ABP was 1.7 in DFM and, in contrast to Trf, declined to 0.7 in other serum-free media. Serum supplementation reversed and increased the ABP ratio to about 6.0 on day 13. These data indicate that Sc grown on permeable supports form confluent monolayers that limit the diffusion of macromolecules, most likely due to the formation of tight junctions. The monolayer permeability as well as the total and polarized secretion of Trf and ABP are differentially regulated by hormones and serum factors.

Age Factors↗
PubMed1986

Effect of hypotonic treatment on Sertoli cell purity and function in culture.

Commonly used enzymic methods for the isolation of rat Sertoli cells yield populations containing approximately 15% germ cells. Although the germ cells become eliminated after several media changes, they could interfere with the use of Sertoli cells for critical studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4) was found to eliminate most of the residual contaminating germ cells. The duration of this treatment varied from 1.0 to 10 min, depending on cell density in the culture, the degree of germ cell contamination, and the age of animals used for Sertoli cell isolation. In a study of 95% pure, 7-d Sertoli cell cultures, the hypotonic treatment did not alter the DNA or RNA content per dish or the incorporation of [3H]uridine into total and poly A+ RNA. Also, the hypotonic treatment did not alter specific Sertoli cell functions, i. e., secretion of Sertoli cell factor (inhibin) and stimulation of cAMP levels by follicle stimulating hormone in 2-d cultures. Androgen receptor concentration per dish was also not changed. Changes in several general metabolic parameters observed after hypotonic treatment of 2-d cultures were attributed primarily to loss of contaminating germ cells. Consequently, hypotonic treatment can be used to eliminate contaminating germ cells from the Sertoli cell cultures without apparent detrimental effects on a number of Sertoli cell biochemical parameters. This may be of considerable importance when the purity of Sertoli cells is critical for the interpretation of experimental data.

Animals↗
PubMed1986

Specific irritability of acupuncture points as an early symptom of multiple sclerosis.

28 patients suffering from multiple sclerosis were treated by acupuncture. The age of the patients ranged from 24 to 60 yr. Results of the treatment were satisfactory, but remarkable spontaneous remissions, characteristic for this illness, make any treatment difficult to evaluate. In this article the peculiar behaviour of all treated patients is described: increased sensibility of the skin over the acupuncture loci, while insertion of the acupuncture needles provoke spasms, clonus and even tonic-clonic contractions of the muscles of the extremities. This phenomenon is very specific and can be used as an early sign of this ill-fated illness.

Acupuncture Therapy↗
PubMed1986

Maturational and hormonal influences on Sertoli cell function.

The maturation of Sertoli cell function was studied by observing properties of cells obtained from 15-, 25-, and 35-day-old rats after 3 days in culture gamma-Glutamyl transpeptidase activity, expressed per mg DNA or per mg protein, and total and soluble protein to DNA ratios increased with age. In contrast, lactate dehydrogenase activity was unchanged between 15 and 35 days of age. Secreted protein to DNA ratios and the secretion of lactate, androgen-binding protein (ABP), and transferrin per mg DNA also increased with age. Two-dimensional electrophoresis maps of [35S]methionine-labeled secretory proteins indicated the appearance of two specific bands [mol wt, 66,000; pI 6.0-6.8 (band 1); mol wt, 56,000; pI 5.3-6.0 (band 2)] between 15 and 35 days of age. The hormone dependence of these parameters was studied in Sertoli cells isolated from rats hypophysectomized at 20 days of age and subsequently treated with oil, testosterone propionate, or testosterone propionate plus FSH for 15-21 days. Hypophysectomy decreased total, soluble, and secreted protein to DNA ratios; hormone treatment in vivo increased these ratios compared to oil treatment. gamma-Glutamyl transpeptidase activity was significantly decreased by hypophysectomy and increased, compared to oil treatment, by hormone treatment. In contrast, lactate dehydrogenase activity per mg DNA was also decreased by hypophysectomy, but was unaffected by hormone treatment. [35S]Methionine incorporation into secreted protein and secretion of ABP and lactate per mg DNA were all decreased by hypophysectomy, whereas transferrin secretion per mg DNA was unaffected. While the hormone treatments increased ABP secretion, they had no effect on lactate or transferrin secretion, expressed per mg DNA. The [35S]methionine-labeled secretory proteins (bands 1 and 2) were not visible in two-dimensional electrophoresis maps after hypophysectomy of the donor animals. Treatment with testosterone propionate or testosterone propionate plus FSH in vivo resulted in the appearance of the acidic components of band 2. These data demonstrate that changes reflecting in vivo maturational patterns and hormonal influences on Sertoli cell function persist, at least in a relative sense, after 3 days in culture. Although the majority of [35S]methionine-labeled Sertoli cell cellular and secretory proteins were present under all conditions, maturation- and hormone-dependent changes in a number of specific functions were demonstrated.

Animals↗
PubMed1985

Localization of ornithine decarboxylase in rat testicular cells and epididymal spermatozoa.

We have employed a monospecific, polyclonal antibody to ornithine decarboxylase (ODC) for the immunocytochemical localization of ODC in freshly isolated testicular cells, epididymal spermatozoa, and cultured Sertoli cells. Antigenically detectable material was present in the cytoplasm of all cell types tested and was highly concentrated in the acrosomal vesicle of round spermatids and in the acrosome region of epididymal spermatozoa. The specific enzymatic activity of ODC, as measured biochemically, was much higher in the interstitial cells than in the other testicular cell types, and no ODC activity was detected in the epididymal spermatozoa or in the Sertoli cells after 5 days in culture. These studies showed that, while all testicular cell types studied contained ODC-like immunoreactive molecules, only testicular germ cells and interstitial cells exhibited detectable ODC activity.

Animals↗
PubMed1984

Leishmania tropica: protective response in C3H mice vaccinated with excreted factor crosslinked with the synthetic adjuvant, muramyl dipeptide.

Excreted factor, an immunosuppressive, acidic polysaccharide released by promastigotes of Leishmania tropica major in culture, was chemically crosslinked to the synthetic adjuvant muramyl dipeptide via the bifunctional imidoester dimethyladipimidate and poly-L-lysine. This conjugate, an uncrosslinked mixture of the components, or each of the components alone were injected one to three times into different groups of 8- to 12-week-old C3H mice. The mice were challenged 2 weeks after the last injection with 2 X 10(6) promastigotes of L. t. major in the base of the tail. For the next 5 weeks, the animals were monitored for number of parasites and size of the lesion which developed at the site of the challenge. Mice receiving one intraperitoneal injection of the conjugate were partially protected against challenge. Treated animals had higher initial parasite numbers but showed a more rapid clearing of the parasites. Furthermore, the treated animals developed smaller lesions that healed quicker than did those of the control groups. Multiple injections, or injection into a footpad, rather than intraperitoneally, reduced the ability to elicit a protective response. On the other hand, muramyl dipeptide injected into a footpad was partially protective. Antibody production to excreted factor, which was measured by indirect hemagglutination of sensitized erythrocytes, was detected after challenge in mice which had received conjugate or conjugate components. A delayed hypersensitivity reaction (measured by skin testing) was not detected in any of the groups prior to challenge.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Control of androgen cytosol receptor concentrations in Sertoli cells: effect of androgens.

Androgen receptor concentrations were measured in Sertoli cells under a variety of conditions. After brief (30- to 60-min) incubation of cultured cells with [3H]R1881 (methyltrienolone), cytosol receptor concentrations were greatly diminished, and nuclear bound steroid was elevated. Removal of the exogenous steroid was accompanied by a return of cytosol receptor to preincubation concentrations by 1 h and a slower decline in nuclear bound steroid. In the continued presence of R1881 or testosterone, cytoplasmic receptor concentrations declined and then returned to or above preincubation concentrations by 6-17 h. Actinomycin-D and cycloheximide did not alter this pattern. Over this same interval, nuclear bound steroid concentrations remained elevated. Exposure of the cells to R1881 or testosterone for the entire 72-h culture period did not alter cytoplasmic receptor concentrations. Cytoplasmic androgen receptor concentrations were decreased in Sertoli cells from hypophysectomized rats 15-22 days after surgery compared to those in cells from intact controls. Treatment with testosterone propionate (0.5 mg/day) or FSH (75 micrograms/day) prevented the decline in receptor concentrations. Cryptorchidy (33 days) also decreased cytosol receptor concentrations. These data indicate that exposure to androgens can influence Sertoli cell cytosol androgen receptor concentrations in vitro and in vivo. The Sertoli cell adapts to the continual presence of androgens with a return of cytosol receptor while maintaining elevated concentrations of nuclear bound steroid. In vivo, androgen treatment can maintain cytoplasmic receptor concentrations in the absence of pituitary hormones in the immature rat.

Animals↗
PubMed1984

Pituitary binding of 3H-labeled Sertoli cell factor in vitro: a potential radioreceptor assay for inhibin.

We have previously demonstrated that binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The present study further characterized the binding of [3H]SCF to rat anterior pituitary in vitro and explored its potential use as a radioreceptor assay for SCF and other inhibin preparations. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 mu Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of approximately 20,000 dpm/micrograms protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing one pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37 C; 30 min) or heat (100 C; 10 min). Storage of the pituitary glands at -20 C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high mol wt fraction of ram testis fluid (mol wt, greater than 10,000), low mol wt fraction of ram testis fluid (mol wt, less than 5,000), ovine testicular lymph, and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low mol wt fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). In contrast, various noninhibin substances tested [rat androgen-binding protein, bovine LH, BSA, native GnRH, or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-EA and D-Ala6-des-Gly10-GnRH-N-EA] did not significantly compete for the [3H]SCF binding. The binding ability correlated well with inhibin biological activity in vitro. These results provide additional evidence for the presence of SCF-binding sites in the rat anterior pituitary which interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. Our results also indicate that the pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations.

Activin Receptors↗
PubMed1984

Transient neurological deficits secondary to saccular vertebrobasilar aneurysms. Report of two cases.

Aneurysms have rarely been implicated as a possible cause of transient neurological deficits, and most reports of this phenomenon describe aneurysms in the anterior circulation. There is only one previous report of a saccular posterior circulation aneurysm associated with transient ischemic attacks. The authors document two cases of giant saccular vertebrobasilar artery aneurysms associated with transient neurological deficits.

Adolescent↗
PubMed1984

Testicular peptides and their endocrine and paracrine functions.

A number of testicular peptides appear to have endocrine and/or paracrine functions. Most, if not all, of these peptides are secreted by the Sertoli cells into the tubular fluid. Although their physiological role has not been fully clarified, these secretory products are believed to play an important role in the local regulation of testicular functions, particularly the spermatogenic process. Presence of high affinity, specific binding sites for transferrin and androgen binding protein, both secreted by the Sertoli cells, has been demonstrated on the round spermatids and/or pachytene spermatocytes. Since within the seminiferous tubules, only the Sertoli cells respond directly to follicle stimulating hormone and testosterone, they could mediate the hormonal effects on spermatogenesis by providing certain products which may be essential during specific stages of germ cell differentiation.

Animals↗