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Biomedical subjects

C W Bardin

Publications and source records attributed to C W Bardin.

At least 19 recordsLinked to original sources
PubMed1992

A germ cell factor(s) modulates preproenkephalin gene expression in rat Sertoli cells.

Within the seminiferous tubule, both Sertoli and specific germ cells express opioid genes. Little is known about the paracrine regulation or role of opioid gene expression in the tubule. The present study shows that interactions among cells within the tubule may play a role in regulating preproenkephalin (PPenk) gene expression. Rat pachytene spermatocytes (PS) and round spermatids (RSd) were purified by centrifugal elutriation and established as primary cultures or co-cultured with Sertoli cells. The effects of germ cells or germ cell-conditioned media were studied to determine the expression of one of the opioid precursor genes in rat Sertoli cells, the PPenk gene. Following a 24 h co-culture with either PS or RSd, the expression of PPenk gene in Sertoli cells was increased 6.4- and 1.9-fold, respectively. Conditioned media obtained from either PS or RSd cultured for 20 h stimulated PPenk mRNA levels in Sertoli cells from as early as 2 h after exposure; maximum increases of 3.5- and 7.6-fold were observed at 12 h, respectively. The molecular weight of the germ cell factor(s) is greater than 30 kDa. 2 h after the addition of either PS- or RSd-conditioned media to Sertoli cells, small (2- to 2.6-fold, respectively) but significant (p less than 0.02) increases in extracellular cAMP levels were observed. Although both FSH and forskolin activated c-fos and PPenk gene expression in Sertoli cells, the germ cell factor(s) that stimulated PPenk mRNA levels did not affect the expression of this oncogene. These results indicate that germ cells interact with Sertoli cells, possibly by a protein(s) that acts as a short-loop paracrine factor, which regulates the expression of PPenk gene in Sertoli cells. These data suggest that stage-specific regulation of PPenk levels in Sertoli cells may occur in vivo.

Animals↗
PubMed1992

Binding of sex-hormone-binding globulin (SHBG) to testicular membranes and solubilized receptors.

UNLABELLED: Sex-hormone-binding globulin (SHBG) binds to a specific protein on the surface of prostate, epididymis, and a human breast cancer cell line (MCF-7), and is internalized by these cells. The present study demonstrated specific binding of SHBG to receptor on membranes prepared from rat testes. The binding was saturable, specific, and time and temperature dependent. Scatchard analysis of these binding studies suggested that SHBG binds to a single class of sites on testicular membranes with a Kd at 37 degrees C of 5 x 10(-8) M and a binding capacity of 30 +/- 0.6 pmol/mg protein. These binding characteristics are similar to the SHBG receptor on human prostate and MCF-7 cells. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (158 +/- 0.3 pmol/mg protein) and a 10-fold decrease in binding affinity (Kd at 37 degrees C = 6.5 x 10(-7) M). The apparent molecular weight of the testicular SHBG receptor, as estimated by gel filtration, was M(r) = 174,000. CONCLUSION: a specific binding site for SHBG was identified on testicular membranes. This binding site has been tentatively identified as a SHBG receptor based on its physical properties in testicular membrane preparations and following solubilization.

Animals↗
PubMed1992

Rat Sertoli cell clusterin, alpha 2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells.

Clusterin, alpha 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, alpha 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 (PH) and 66,000 (PL) were detected in the Sertoli cell cytosol in addition to the alpha (M(r) 43,000) and beta (M(r) 35,000) subunits of the mature protein. However, the precursors were not secreted into the medium since only the alpha and beta subunits of clusterin were detected. For alpha 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, alpha 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 alpha 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 alpha 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 micrograms 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 alpha 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.

Animals↗
PubMed1992

Testins are localized to the junctional complexes of rat Sertoli and epididymal cells.

UNLABELLED: Testin I and Testin II were originally identified as Sertoli cell products with similar NH2-terminal amino acid sequences. Secretion of testins is stimulated by testosterone in Sertoli cell-enriched cultures. By contrast the secretion of testins from intact seminiferous tubules appears to be inversely related to germ cell number. In the present study testin antiserum that recognized both Testin I and Testin II ("testin") was used to localize these proteins in tissue secretions by immunofluorescence. Testin was localized at the base of the seminiferous epithelium at Sertoli-Sertoli junctions. Fluorescence also appeared to be located at the sites of interaction between spermatoids and Sertoli cells. A punctate pattern of fluorescence was also present in the cytoplasm of Leydig cells; without electron microscopic studies it was not possible to determine which structures the antibodies bound to in these cells. In the epididymis the reaction product was localized at the apices of the epithelial cells adjacent to the lumen at the sites of known junctional complexes. A variety of positive and negative controls indicated that staining was specific for testins. CONCLUSIONS: This is the first study to associate testins with junctional complexes. Relative to other junctional proteins, testins are unusual because of their small size and because they are secreted proteins.

Animals↗
PubMed1992

Effects of follicle-stimulating hormone on inhibin release by different testicular compartments in the adult ram.

The aim of this study was to determine the bidirectional release of immunoreactive inhibin-alpha (irINH-alpha) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-alpha was measured by RIA in fluid samples collected concurrently from the three testicular compartments--the seminiferous tubules, the interstitium, and the vascular system--through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively. Overall, the concentration of irINH-alpha in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-alpha concentration in rete testis fluid was inversely related to that in testicular lymph, but i.v. administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibin levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-alpha concentration so that, overall, the total output of inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-alpha in the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an FSH-induced release of a series of proteins in the M(r) range of 30,000-32,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Binding of an extracellular steroid-binding globulin to membranes and soluble receptors from human breast cancer cells (MCF-7 cells).

Studies of MCF-7 breast cancer cells demonstrated that sex hormone-binding globulin (SHBG) is internalized by receptor-mediated endocytosis. The present study demonstrated specific binding of SHBG to receptor on membranes isolated from MCF-7 cells. Scatchard analysis of these binding studies suggested that SHBG binds to a single class of sites on membranes. The analysis yielded a dissociation constant (Kd) at 37 C of 3 x 10(-8) M and a binding capacity of 48 +2- 0.12 pmol/mg protein. A procedure for solubilizing the SHBG receptor from MCF-7 membranes used buffers containing protease inhibitors, 10% glycerol, and 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (246 +/- 14 pmol/mg protein) and a 10-fold decrease in binding affinity (Kd at 37 C = 2 x 10(-7) M).

Binding, Competitive↗
PubMed1992

The biological activity of 7 alpha-methyl-19-nortestosterone is not amplified in male reproductive tract as is that of testosterone.

Based on the premise that testosterone, but not 7 alpha-methyl-androgens, is reduced at the 5 alpha-position in the prostate and seminal vesicles, the differential bioactivities of these androgens were investigated in castrated rats. The ability of 7 alpha-methyl-19-nortestosterone acetate (MENT) to increase the weights of ventral prostate and seminal vesicles of castrated rats was four times higher than that of testosterone, while its effect on the weights of bulbocavernosus plus levator ani muscles (muscle), was 10 times that of testosterone. MENT was also approximately 12 times more potent than testosterone in the suppression of serum gonadotropin levels. A dose of testosterone that maintains serum gonadotropin levels and muscle mass also maintains prostate and seminal vesicle weights in castrated rats. By contrast, a dose of MENT that maintains muscle and gonadotropins does not maintain prostate and seminal vesicles. The action of other 7 alpha-methylated androgens were similar to that of MENT. The importance of 5 alpha reductase in the differential action of testosterone and MENT on prostate was confirmed by using a 5 alpha-reductase inhibitor. The activity of testosterone was significantly suppressed in the ventral prostate and seminal vesicles but not on muscle by the 5 alpha-reductase inhibitor (N,N-diethyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide). The enzyme inhibitor, however, had no influence on the activity of MENT on either tissue. In contrast, cyproterone acetate, an antiandrogen that competitively binds to the androgen receptors, inhibited the action of MENT and of testosterone on the prostate as well as on the muscle. In conclusion, these observations show that 7 alpha-methylated androgens can maintain muscle mass and normal gonadotropin levels in androgen deficient rats without hyperstimulating the prostate. These findings suggest that 7 alpha-methylated androgens may offer some health benefits to men who require androgen treatment.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Clusterin production in the obstructed rabbit kidney: correlations with loss of renal function.

Clusterin, a protein associated with cell death, has been suggested as a marker of renal injury. Correlation of clusterin gene expression with changes in renal function and quantitative measurement of clusterin protein levels after ureteral obstruction have not been previously reported. With unilateral ureteral obstruction in rabbits as the experimental model, the time course of alterations in renal function, clusterin mRNA accumulation, and concentrations of clusterin protein in serum, urine, and renal tissue were investigated. RBF, GFR, and renal concentrating ability (percent sodium reabsorption and urine osmolarity) all decreased (P < 0.05) in the obstructed kidney from control values within 1 day of ureteral obstruction. Clusterin mRNA levels started to rise in the ipsilateral kidney within 12 h of ureteral obstruction and increased up to 10-fold above control levels after 3 days of obstruction. Hybridization histochemistry showed that clusterin mRNA was initially detectable in collecting ducts and distal tubules within 12 h of ureteral obstruction. After 7 days of obstruction, increased accumulation of clusterin mRNA was also detectable in proximal tubular epithelial cells. Clusterin gene expression remained elevated in collecting ducts after 60 days of obstruction. Clusterin expression in the contralateral kidney was increased twofold over control values after 12 h of obstruction. No increase in clusterin mRNA accumulation was detectable after 24 h in the contralateral kidney. Total clusterin protein in the obstructed kidney increased from 0.59 +/- 0.66 (mean +/- 1 SD) to 2.5 +/- 1.3 micrograms after 7 days of ureteral obstruction (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Measurement of urinary clusterin as an index of nephrotoxicity.

Measurements of tissue immunoassayable clusterin, a protein associated with programmed cell death and tissue reorganization, were performed in rats treated with nephrotoxic doses of gentamicin sulfate. Adult Lewis rats were treated with 100 mg/kg/day of gentamicin sulfate for 12 days. Urine, serum, and tissue levels of clusterin protein were measured, as were urinary N-acetyl beta-glucosaminidase (NAG) and serum creatinine levels. Induction of renal injury by gentamicin was detectable within 4 days by increased levels of urinary N-acetyl beta-glucosaminidase (from 280 +/- 66 (mean +/- SD) to 910 +/- 210 nmol/mg creatinine), and within 9 days of initiating gentamicin treatment by increased serum creatinine (from 0.5 +/- 0.1 to 1.2 +/- 0.4 mg/dl). Paralleling these changes, renal, urinary, and serum levels of clusterin increased 10-, 116-, and 3-fold (P less than 0.05). Treatment with gentamicin sulfate did not increase clusterin levels in the seminal vesicle, ventral prostate, testis, or epididymis. The measurement of urinary or serum clusterin may play a role in the early detection of renal injury.

Acetylglucosaminidase↗
PubMed1992

Experience with 19-nortestosterone in the therapy of systemic lupus erythematosus: worsened disease after treatment with 19-nortestosterone in men and lack of improvement in women.

Three men and 4 women with systemic lupus erythematosus (SLE) received 100 mg of 19-nortestosterone decanoate in weekly intramuscular injections over a period of 3 to 24 months. During therapy in the men plasma luteotrophic hormone and free testosterone levels decreased while estrogen levels increased. Anti-DNA antibodies also increased for unknown reasons. Serological changes in two men coincided with an overall increase in the clinical activity of SLE and the appearance of new onset Raynaud's phenomenon and pleuropericardial disease. In contrast, women treated with 19-nortestosterone showed clinical stability despite the persistence of high titer antibodies to DNA, and were able to continue therapy for as long as 16 months in one case. These data indicate that men and women with SLE respond differently to synthetic androgen therapy. Additionally, dissociation of clinical signs and symptoms from serological variables seems possible.

Adult↗
PubMed1991

Clinical trial with 3-keto-desogestrel subdermal implants.

The study was done to assess the clinical performance and in vivo steroid release rate of 3-keto-desogestrel subdermal implants designed to deliver 5 different doses of the progestin. Volunteers were healthy women of proven fertility who provided blood samples at scheduled intervals during treatment. No pregnancy occurred in 514 woman-months in users of implants delivering 30 and 40 micrograms per day of 3-keto-desogestrel. Three pregnancies, one ectopic, were observed in 109 woman-months recorded with implants delivering 20 micrograms per day or less. Ovulation was inhibited, as judged by depressed progesterone levels, in 57 of 59 (97%) blood samplings in women whose 3-keto-desogestrel plasma levels were greater than 0.28 nmol/L and in 39 of 75 (52%) of cases with lower levels. Users of 4 cm implants manufactured by The Population Council, New York, showed mean levels above 0.28 nmol/L until 18 months of use. Levels achieved with 4.4 cm implants manufactured by Organon, Oss, Holland, were less consistent. No changes were observed in the plasma lipoprotein pattern or clinical chemistry during treatment. The main complaint was the occurrence of bleeding irregularities, particularly with the lower doses. Ovarian cysts found during pelvic examination in 11 (22%) subjects disappeared spontaneously within 7-90 days. 3-keto-desogestrel implants releasing around 40 ug/day and providing plasma levels around 0.28 nmol/L afford efficient contraceptive protection.

Adolescent↗
PubMed1991

Alpha 2-macroglobulin is not an acute-phase protein in the rat testis.

Earlier studies from this laboratory have shown that Sertoli cells actively synthesize and secrete a nonspecific protease inhibitor in vitro; N-terminal sequence analysis, subunit structural analysis, and other biological studies revealed that this protein is the homolog of serum alpha 2-macroglobulin. We have now quantified the relative distribution of alpha 2-macroglobulin in the reproductive compartments and their comparison with nonreproductive organs. In serum and all nonreproductive tissues examined, the concentration of alpha 2-macroglobulin progressively decreased with advancing age. However, in both the testis and epididymis, the levels of this protein increased with the age of the animals. Serum alpha 2-macroglobulin levels were consistently higher than those in any other tissues until 60 days when the concentrations of this protein were the highest in the epididymis. The distribution of alpha 2-macroglobulin in various nonreproductive tissues from female rats was similar to that observed for male rats in that its levels tended to decrease with age. However, uterine levels of alpha 2-macroglobulin increased progressively with advancing age, whereas ovarian levels of alpha 2-macroglobulin remained relatively stable with an increase in animal age. As serum alpha 2-macroglobulin is an acute-phase protein in the rat, the response of this protein in the testis to induced inflammation was examined. The concentration of alpha 2-macroglobulin in serum rose about 150-fold after injection of fermented yeast. By contrast, the levels of this protein in rete testis fluid, which is derived exclusively from seminiferous fluid, did not change in response to inflammation. These results suggest that there might be distinctive mechanisms that regulate this protein in the systemic circulation vs. the microenvironment behind the blood-testis barrier in the seminiferous epithelium.

Acute-Phase Proteins↗
PubMed1991

Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells.

Previous studies suggested that an extracellular steroid-binding protein, testosterone-estradiol-binding globulin (TeBG), can enter a variety of cells. Experiments were conducted to determine whether uptake of TeBG occurs by nonspecific fluid phase endocytosis or via a specific receptor-mediated process. In human breast carcinoma cells (MCF-7) maintained on serum-free medium, exposure to radiolabeled TeBG resulted in cellular uptake, which reached a plateau by 6 h and could be inhibited 80% by competition with unlabeled TeBG. Uptake was temperature dependent with cell-associated radioactivity at 37 C being 1.6-fold higher than at 4 C. Subsequent exposure of cells to pronase resulted in release of the cell-associated TeBG by 88% and 44% at 4 C and 37 C, respectively. After transfer to media devoid of TeBG, approximately 35% of cell-associated radioactivity was release into the medium at 37 C; it was not possible to distinguish whether this was released from the cell surface or from inside the cell. Investigation of the localization of TeBG-gold complexes by electron microscopy revealed that TeBG first binds to the plasmalemma. Within 15 min label appears in receptosomes, which fuse to form multivesicular endosomes. By 1 h all label is observed in multivesicular endosomes and lysosomes, most of which are in the Golgi zone. Localization of the internalized radioactivity using classical cell fractionation techniques showed it appears in a symmetrical band exhibiting the same buoyant density as the lysosomal marker acid phosphatase. The observations reported here show that: 1) TeBG binds to MCF-7 cells; 2) some of the bound TeBG is taken up via a mechanism with all the characteristics of receptor-mediated endocytosis; and 3) within these cells TeBG is localized in endosomes and lysosomes.

Breast Neoplasms↗
PubMed1990

Sertoli cell synthesizes and secretes a protease inhibitor, alpha 2-macroglobulin.

The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial component of the seminiferous epithelium, release a protease inhibitor, testicular alpha 2-macroglobulin, in vitro. Immunoprecipitation using [35S]methionine and a monospecific polyclonal antibody prepared against purified testicular alpha 2-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that alpha 2-macroglobulin rather than alpha 1-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of alpha 2-macroglobulin to inactivate proteases and growth factors such as TGF-beta by a common mechanism suggests that this protein may have a dual function in the testis.

Amino Acid Sequence↗
PubMed1990

A 90-day subcutaneous toxicity and fertility study of a LHRH antagonist in rats.

[Ac-D2Nal1,4Cl-DPhe2,D3Pal3,Arg5,DGlu6+ ++ (anisole adduct),DAla10]-GnRH (Nal-Glu) is an antagonist of LHRH and has the potential to be utilized as an antigonadal agent. A study was undertaken to evaluate the toxicological effects of Nal-Glu in rats. Nal-Glu, dissolved in 5% mannitol in water containing 9 ml/liter benzyl alcohol, was administered subcutaneously. In subchronic studies, groups of 12 male and 12 female rats received 0, 50, 250, or 1250 micrograms/kg body weight (BW) Nal-Glu for 90 days and were killed on Day 91. Additional groups of male and female rats were given the high dose of Nal-Glu (1250 micrograms/kg BW) or vehicle for either 30 or 90 days. Their fertility was assessed by mating them with normal animals. Unlike some other LHRH antagonists, Nal-Glu exhibited a low potency for causing in vitro histamine release from rat peritoneal mast cells. Furthermore, in acute in vivo studies, Nal-Glu was less active in the induction of peripheral edema. In the subchronic study, all doses of Nal-Glu were well tolerated and there were no apparent systemic toxic effects. The pharmacological effects of Nal-Glu were quite evident, however. Nal-Glu treatment led to a significantly decreased body weight gain in the males and a significantly increased body weight gain in the females. There was a dose-dependent decrease in weights of gonads and reproductive organs in both the sexes. Some of the hematological and serological parameters were significantly different in Nal-Glu-treated animals. However, most of the values were within the normal range and are considered to be of no toxicological significance. Histopathological evaluations were made in the control and high-dose groups only. In the male, a seminiferous tubular degeneration and atrophy of the interstitial cells was seen. The prostate and seminal vesicles were also atrophied and the epididymides were devoid of spermatozoa. In the females, the ovaries and uteri were atrophic. The injection site of Nal-Glu-treated rats had inflammatory changes indicative of a local irritating action of the drug. All other tissues had normal histomorphology. Both male and female rats became infertile when 1250 micrograms/kg Nal-Glu was administered for 30 days. Normal fertility was restored 8 weeks after cessation of 90-day treatment. It is concluded that repeated administration of Nal-Glu leads to reversible infertility in both male and female rats. Although it was irritating at the site of injection. Nal-Glu had no systemic toxicological effects.

Animals↗