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J A Grootegoed

Publications and source records attributed to J A Grootegoed.

At least 73 records · Page 4Linked to original sources
PubMed1991

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.

Androgens↗
PubMed1991

Characterization of the testicular cell types present in the rat by in vivo 31P magnetic resonance spectroscopy.

Testes of vitamin A-deficient Wistar rats before and after vitamin A replacement, of rats irradiated in utero, and of control rats were investigated by in vivo 31P magnetic resonance (MR) spectroscopy. The testicular phosphomonoester/ATP (PM/ATP) ratio ranged from 0.79 +/- 0.05 for testes that contained only interstitial tissue and Sertoli cells to 1.64 +/- 0.04 for testes in which spermatocytes were the most advanced cell types present. When new generations of spermatids entered the seminiferous epithelium, this ratio decreased. The testicular phosphodiester/ATP (PD/ATP) ratio amounted to 0.16 +/- 0.06 for testes in which Sertoli cells, spermatogonia, or spermatocytes were the most advanced cell type present. When new generations of spermatids entered the seminiferous epithelium, the PD/ATP ratio rapidly increased and finally reached a value of 0.71 +/- 0.06 for fully developed testes. Taken together, specific patterns of the PM/ATP ratio, the PD/ATP ratio, and pH were obtained that were correlated to the presence of spermatogonia, spermatocytes, round spermatids, and elongated spermatids or to the absence of spermatogenic cells. Hence, a good impression of the status of the seminiferous epithelium in the rat can be obtained by in vivo 31P MR spectroscopy.

Adenosine Triphosphate↗
PubMed1990

The rat androgen receptor gene promoter.

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcription.

Animals↗
PubMed1990

Regulation of inhibin alpha- and beta B-subunit mRNA levels in rat Sertoli cells.

The hormonal regulation of the expression of the inhibin alpha-subunit and beta B-subunit genes was studied in cultured rat Sertoli cells. The alpha-subunit mRNA level increased during incubation of the cells in the presence of follicle-stimulating hormone (FSH), reaching maximal levels within 1.5 h. This stimulation was mimicked by addition of dibutyryl-cyclic AMP, indicating that FSH action on the alpha-subunit gene is exerted via cyclic AMP. Inhibition of translation by cycloheximide (CX) caused upregulation of the alpha-subunit mRNA, and did not block the effect of FSH on the level of this mRNA. In FSH-stimulated cells, the half-life of the alpha-subunit mRNA was 6 h, and this half-life was prolonged by inhibition of transcription using actinomycin D (AD). It is concluded that the effect of FSH on alpha-subunit mRNA expression represents a direct effect on the alpha-subunit gene, and that alpha-subunit mRNA levels are influenced by a short-lived mRNA destabilizing protein. The levels of two beta B-subunit mRNAs (4.2 kb and 3.5 kb) were not affected by FSH or dbcAMP. However, these mRNAs were also upregulated by CX treatment. Experiments using AD showed that the 4.2 kb mRNA is less stable than the 3.5 kb mRNA. The differential regulation of the inhibin alpha- and beta B-subunit mRNAs is discussed.

Animals↗
PubMed1990

Effects of glucose and adenosine on the ATP content of hamster spermatids.

Effects of glucose and adenosine on ATP metabolism were studied using isolated round spermatids from hamsters. The ATP content of the spermatids was strongly decreased after 1 h of incubation of the cells in the presence of 0.1 mM D-glucose. Glucose (1 mM) had no effect during 18 h of incubation in the presence of 12 mM sodium DL-lactate. However, 10 mM glucose caused an almost complete loss of cellular ATP in the presence of lactate. The effect of adenosine was estimated in the absence of glucose with lactate as the energy-yielding substrate. The cellular ATP content was approximately 4 and 8 nmol/10(6) cells, after 18 h of incubation in the absence and presence of 0.1 mM adenosine, respectively. This two-fold increase was prevented by inhibitors of adenosine uptake and phosphorylation and was slowly reversed after removal of the exogenous adenosine. Treatment of the cells with adenosine had no effect on the energy charge, which was higher than 0.90, and did not alter the cellular cyclic AMP content. The suggestion that the physiological ATP content of the round spermatids is probably stabilized in the region of 4 nmol/10(6) cells is discussed.

Adenine↗
PubMed1990

Glutathione-dependent defence mechanisms in isolated round spermatids from the rat.

The different mechanisms for glutathione-dependent inactivation of a number of oxidizing compounds and other xenobiotics were studied using isolated round spermatids from rats. For the estimation of cellular GSH a flow cytometric assay was used. The cells were exposed to the oxidizing compounds cumene hydroperoxide and diamide, to study the activity of the GSH redox cycle. Incubation of the isolated cells with these compounds showed that the cells had a limited capacity to withstand the oxidative stress associated with their inactivation. The GSH level of the spermatids was maintained during 18 h of incubation in the presence of low concentrations of cumene hydroperoxide and diamide, whereas spermatids exposed to higher concentrations showed a loss of both GSH and ATP. No partial loss of GSH from individual cells was observed. Diethyl maleate and 1,2-epoxy-p-(nitrophenoxy)propane (ENPP) were used to study the effect of glutathione S-transferase-catalysed GSH conjugation on the GSH content of spermatids. Exposure of the cells to low concentrations of diethyl maleate and ENPP resulted in a decrease in GSH content. The flow cytometric analysis showed that this was a partial loss of GSH from all cells, rather than GSH depletion in a part of the cell population. This diminution of the cellular GSH pool, however, did not affect the ATP content and viability of the cells. The present results indicate that spermatids can utilize GSH-dependent defence mechanisms against a number of model compounds.

Adenosine Triphosphate↗
PubMed1990

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids.

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.

Adenosine Triphosphate↗
PubMed1989

Differential effects of follicle-stimulating hormone, insulin, and insulin-like growth factor I on hexose uptake and lactate production by rat Sertoli cells.

The stimulatory effects of follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor I (IGF-I) on lactate production and hexose uptake by Sertoli cells from immature rats were studied. The time-courses and the maximal stimulatory effects of FSH, insulin, and IGF-I on lactate production were virtually identical. When Sertoli cells were incubated in the presence of FSH in combination with insulin or IGF-I (submaximal doses), additive but no pronounced synergistic effects were observed. The stimulatory effects of FSH and insulin were not dependent on the presence of extracellular calcium. 2-Deoxy-D-glucose (2-DOG), an analogue of D-glucose, was used to investigate the hexose transport system of Sertoli cells. Uptake of 2-DOG was linear in time and virtually all of the intracellular 2-DOG was phosphorylated up to 30 min of incubation; 2-DOG uptake was inhibited by cytochalasin B, but not by cytochalasin E. D-glucose, but not D-galactose, appeared to be an effective competitor of 2-DOG uptake. The Km of 2-DOG uptake was not influenced by FSH, insulin, and IGF-I. FSH had no effect on the Vmax of 2-DOG uptake, whereas insulin and IGF-I caused a 30% stimulation of the Vmax. It is concluded that FSH, insulin, and IGF-I stimulate lactate production by cultured Sertoli cells, but that only insulin and IGF-I stimulate hexose transport. The insulin-like effect of FSH on Sertoli cells may principally involve stimulation of glycolytic enzyme activities.

Animals↗
PubMed1989

Follicle-stimulating hormone regulates androgen receptor mRNA in Sertoli cells.

Follicle-stimulating hormone (FSH) and testosterone stimulate the production of a variety of proteins by immature Sertoli cells. A highly purified Sertoli cell preparation was incubated for 3 days with FSH and testosterone. Both androgen receptor protein and mRNA concentrations were markedly increased by FSH. Testosterone also increased the androgen receptor protein concentration, but did not increase the expression of the androgen receptor mRNA. It is concluded that FSH plays a role in the responsiveness of Sertoli cells to testosterone.

Animals↗
PubMed1989

Quantitative evaluation of the maintenance and development of spermatocytes and round spermatids in cultured tubule fragments from immature rat testis.

Maintenance and development of spermatocytes and round spermatids was studied in an in-vitro incubation system. This system consisted of open tubule fragments from 26-day-old rat testes, obtained after collagenase treatment. The tubule fragments contained Sertoli cells and spermatogenic cells up to and including a small number of early round spermatids. The number of primary spermatocytes and round spermatids in the tubule fragments was estimated using flow-cytometric analysis, immediately after isolation and after 72 h of incubation. In addition, the activity of LDH-C4 in the tubule fragments was measured. After 72 h of incubation, the percentage of spermatocytes was reduced by 70-80%, but the percentage of spermatids was doubled. The total LDH-C4 activity per well was increased 2-3-fold during 72 h of incubation of the fragments. A modest improvement of the culture results was observed when a combination of FSH, insulin, retinol and testosterone was added to the medium. LDH-C4 activity was investigated to see whether it could be used as a quantitative marker of isolated and cultured spermatocytes and spermatids. It was observed that LDH-C4 activity per cell was decreased when spermatocytes and spermatids were isolated and/or incubated at 4 degrees C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32 degrees C, either in the absence or presence of a protein synthesis inhibitor. Cellular LDH-C4 activity may be influenced not only by temperature, but possibly also by other cell isolation conditions. It is concluded that LDH-C4 activity may not be a reliable quantitative marker for the presence of spermatocytes and spermatids in culture, but should be used in combination with other analytical methods such as DNA estimation and DNA flow cytometry.

Adenosine Triphosphate↗
PubMed1989

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters.

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.

Animals↗
PubMed1989

Effects of FSH and testosterone on highly purified rat Sertoli cells: inhibin alpha-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone.

The effects of FSH and testosterone on inhibin mRNA expression and inhibin production by highly purified Sertoli cell preparations were examined. Sertoli cells were isolated from testes of 22-day-old rats by sequential trypsin, collagenase and hyaluronidase treatments, with subsequent osmotic shock treatment on day 3 of culture. Contamination by peritubular and germ cells was less than 0.5 and 1-3% respectively. Intracellular and secreted inhibin levels were measured by radioimmunoassay, using Sertoli cells which were incubated for 24 h in the absence or presence of FSH and testosterone from days 4 to 5 of culture. FSH stimulated the cellular inhibin content and the secreted inhibin level by four- and sevenfold respectively, with a half-maximal effective dose of 5-50 ng/ml. Under the present incubation conditions, testosterone (1 mumol/l) had no effect on immunoreactive inhibin levels in either the presence or absence of FSH. Similarly, the expression of inhibin alpha-subunit mRNA was increased following FSH stimulation, whereas testosterone had no effect. The expression of inhibin beta B-subunit mRNAs was not influenced by FSH or testosterone. It is concluded that highly purified Sertoli cell preparations, with a very low number of peritubular or germ cells, are fully responsive to FSH with respect to inhibin mRNA expression and inhibin production.

Animals↗
PubMed1988

Insulin-like growth factor I (IGF-I) receptors on Sertoli cells from immature rats and age-dependent testicular binding of IGF-I and insulin.

Insulin-like growth factor I (IGF-I) binding to cultured Sertoli cells from immature rats was quantitatively evaluated. The binding of 125I-IGF-I to the Sertoli cells was specific, time- and pH-dependent and reversible. Scatchard analysis yielded a Kd of 3.5 X 10(-9) M and a binding capacity of 2080 fmol/mg protein. Competition with IGF-I resulted in a half-maximal displacement by 2 nM IGF-I, whereas insulin up to a concentration of 100 nM gave virtually no displacement of IGF-I binding. Similarly, the gonadotropic hormones follitropin and lutropin did not compete with 125I-IGF-I binding. In previous studies, it was shown that cultured Sertoli cells from immature rats bind insulin with a Kd of 1.8 X 10(-9) M and a binding capacity of 8.5 fmol/mg protein. The binding of IGF-I and insulin to a total testis membrane fraction was studied using testes from immature and adult rats. In testis from 21-day-old rats, the maximal specific binding was relatively high for IGF-I (871 +/- 50 fmol/g wet weight) and relatively low for insulin (118 +/- 11 fmol/g wet weight). In adult testis, the maximal specific binding of IGF-I was 324 +/- 40 fmol/g wet weight and that of insulin was 330 +/- 17 fmol/g wet weight. The binding of IGF-I and insulin expressed as fmol bound per testis was increased 6-fold and 45-fold, respectively, between the age of 21 days and adult age. It is discussed that the numbers of receptors for IGF-I and insulin in testis may be developmentally regulated, and that IGF-I may be more important than insulin with respect to testis development and Sertoli cell maturation in the immature rat.

Aging↗
PubMed1988

Effects of FSH and IGF-I on immature rat Sertoli cells: inhibin alpha- and beta-subunit mRNA levels and inhibin secretion.

Effects of follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) on inhibin production by cultured Sertoli cells from 21- to 23-day-old rats were studied. The expression of inhibin alpha- and beta-subunit mRNAs, and inhibin immunoreactivity and in vitro bioactivity were estimated. Using a cDNA probe corresponding to the alpha-subunit of bovine inhibin, specific hybridization with a 1.5-1.7 kilobase (kb)mRNA species was observed. Addition of FSH to the cultured Sertoli cells for 24 h markedly increased the level of this mRNA in a dose-dependent way. IGF-I had no effect on the intensity of the hybridization. Using a cDNA probe corresponding to the beta B-subunit of human inhibin, 3.5 and 4.2 kb mRNA species were detected. FSH and IGF-I had no effect on the hybridization signal. No hybridization was observed with a cDNA probe corresponding to the beta A bovine inhibin subunit. Inhibin activity was detected in cells and medium by immunoassay, and in the medium by in vitro bioassay. FSH stimulated both immunoreactivity and in vitro bioactivity, whereas IGF-I had no effect at all. The present effect of FSH on inhibin alpha-subunit mRNA expression in cultured Sertoli cells indicates that regulation of inhibin production by FSH includes an effect at the transcriptional level. However, this does not exclude additional translational and posttranslational effects.

Animals↗
PubMed1988

Mechanism of action of (-)gossypol on ATP production in isolated hamster spermatids.

The ATP content of round spermatids isolated from hamsters was decreased 90% after 18 h of incubation in the presence of 4 microM-(-)gossypol and 0.10% bovine serum albumin (BSA). The (+)-enantiomer had no effect under these incubation conditions. The Michaelis-Menten constant Km and the maximal initial velocity Vmax of cellular LDH-C4 were not significantly altered after 18 h of incubation of the spermatids with (-)gossypol. Furthermore, there was no effect of (-)gossypol on the production of 14CO2 from L-[U-14C]lactate. It is concluded that (-)gossypol does not inhibit ATP production in spermatids by an effect on the sperm-specific LDH-C4 enzyme or on the mitochondrial oxidation of pyruvate. Rather, (-)gossypol may have an effect on the coupling between electron transport and ATP synthesis in the mitochondria. This action of (-)gossypol may not involve the H+-conducting activity of gossypol, but could be produced through binding of (-)gossypol to specific mitochondrial proteins.

Adenosine Triphosphate↗
PubMed1988

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro.

Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.

Animals↗