Papers by J.A. Grootegoed
Current topics in molecular endocrinology, 1975
Molecular and cellular endocrinology, 1992
In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of i... more In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of beta B-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the beta B-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of beta B-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of inhibin changed markedly. The meaning of this changing ratio between beta B-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The tot...
Journal of andrology
The in vitro response of Sertoli cells isolated from adult rat testes to testosterone (T) and fol... more The in vitro response of Sertoli cells isolated from adult rat testes to testosterone (T) and follicle-stimulating hormone (FSH) treatment was investigated. Sertoli cells from >70-day-old Sprague-Dawley rats were isolated by a combined enzymatic treatment followed by the removal of the majority of contaminating germ cells with immobilized peanut agglutinin lectin. Sertoli cells were then cultured for 6-10 days, forming a confluent layer with a cell viability of >83% and 74-77% purity. The contaminating cells were peritubular cells (4-6%), pachytene spermatocytes (4-5%), round spermatids (<2%), elongated spermatids (<1%), and degenerating germ cells (14.8%). The proportion of degenerating germ cells decreased from 14.8% to 8.6% between days 6 and 10 in culture. After a prestimulation culture period of 4 days, FSH treatment over a 2-day period resulted in a dose-related increase of inhibin with a median effective dose (ED50) value of 36.7+/-20.4 ng/ml in comparison with an...
Journal of andrology
We have studied the ability of the synthetic androgen methyltrienolone (R1881) to maintain testis... more We have studied the ability of the synthetic androgen methyltrienolone (R1881) to maintain testis and accessory organ weights, as compared to the effect of testosterone propionate (TP). In contrast to TP, R1881 is not metabolized and does not significantly bind to androgen-binding protein (ABP). Thirty-six rats were treated with ethane dimethane sulphonate (EDS) and GnRH antagonist (Org30267) to abolish all testicular androgen production, and recombinant human FSH (rec-hFSH, Org32489) was administered to ensure adequate FSH levels. Of these rats, five groups of four rats were treated daily with 0-, 50-, 100-, 200-, and 400-microgram TP, s.c., and four groups of four rats were treated daily with 150-, 300-, 600-, and 1200-microgram R1881, s.c. One control group of four rats received vehicle injections only. EDS treatment, followed by GnRH antagonist and rec-hFSH treatment for 17 days, significantly reduced testis, prostate, and seminal vesicle weights (P < 0.001, P < 0.01, P &l...
The Biochemical journal, 1993
Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by a... more Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P(i) to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both nontransformed and transformed and androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110 kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (>...
Journal of reproduction and fertility, 1982
During incubation of fragments of seminiferous tubules in the absence of glucose, pachytene sperm... more During incubation of fragments of seminiferous tubules in the absence of glucose, pachytene spermatocytes and round spermatids died within 24 h, while Sertoli cells were still viable. The germ cells survived for at least 72 h in seminiferous tubule fragments which were incubated in the presence of glucose. Lactate rather than glucose is essential for [3H]uridine incorporation and survival of isolated pachytene spermatocytes. However, if the spermatocytes were incubated in the presence of Sertoli cells, glucose maintained the incorporation of [3H]uridine into the germ cells. Sertoli cells secreted lactate in the presence of glucose and the lactate secretion was stimulated 2--4-fold by FSH. It is concluded that the activity and survival of pachytene spermatocytes in vitro can be regulated by the supply of lactate from Sertoli cells.
The Biochemical journal, Jan 15, 1985
A unique intercellular pathway of leucine catabolism was observed in vitro in rat spermatogenic e... more A unique intercellular pathway of leucine catabolism was observed in vitro in rat spermatogenic epithelium. Sertoli cells convert leucine via transmination into 4-methyl-2-oxovalerate, and spermatocytes and spermatids reduce exogenous 4-methyl-2-oxovalerate to 2-hydroxy-4-methylvalerate, which is then released by the spermatogenic cells. The NADH-dependent reduction of 4-methyl-2-oxovalerate could be catalysed by the male-germ-cell-specific lactate dehydrogenase isoenzyme LDH-C4 in the cytosol of the spermatogenic cells, concomitant with the NAD+-dependent conversion of exogenous lactate into pyruvate.
The Journal of experimental zoology, 1985
Isolated rat pachytene spermatocytes were incubated in chemically defined medium supplemented wit... more Isolated rat pachytene spermatocytes were incubated in chemically defined medium supplemented with pyruvate and lactate, which are known to be essential energy substrates for these germ cells. Protein synthesis by the isolated cells was investigated by means of two-dimensional polyacrylamide gel electrophoresis. The electrophoretic patterns of (35S)-labeled proteins, synthesized by the pachytene spermatocytes during incubation in the presence of (35S)methionine either from 0-2 h or from 24-26 h after isolation, were almost completely identical. The patterns of newly synthesized proteins of freshly isolated spermatocytes and spermatids, however, showed several stage-specific proteins in addition to many proteins common to both spermatogenic cell types. Hence, it was concluded that a stage-specific pattern of protein synthesis can be maintained by pachytene spermatocytes during incubation for a period of 24 h in the absence of Sertoli cells but in the presence of a proper energy source.
The Journal of biological chemistry, Jan 15, 1991
A full length human androgen receptor (hAR) cDNA was constructed from cDNA and genomic clones. St... more A full length human androgen receptor (hAR) cDNA was constructed from cDNA and genomic clones. Structurally the 10.6-kilobase (kb) hAR cDNA consists of a long 5'-untranslated region (5'-UTR, 1.1 kb), a previously described open reading frame (ORF, 2.7 kb) (Trapman, J., Klaassen, P., Kuiper, G. G. J. M., van der Korput, J. A. G. M., Faber, P. W., van Rooij, H. C. J., Geurts van Kessel, A., Voorhorst, M. M., Mulder, E., and Brinkmann, A. O. (1988) Biochem. Biophys. Res. Commun. 153, 241-248; Faber, P. W., Kuiper, G. G. J. M., van Rooij, H. C. J., van der Korput, J. A. G. M., Brinkmann, A. O., and Trapman, J. (1989) Mol. Cell. Endocrinol. 61, 257-262), and a very long 3'-untranslated region (3'-UTR, 6.8 kb). The complete 5'- and 3'-UTRs were found to be encoded by the previously reported first and eight protein coding exons of the hAR gene, respectively (Kuiper, G. G. J. M., Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Ris-Stalpers, C., Klaass...
The Biochemical journal, Jan 15, 1977
The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H... more The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolate...
Cell Differentiation, 1973
Reproduction, 1979
Fragments of seminiferous epithelium were prepared from 3-week-old rats. Although the Sertoli cel... more Fragments of seminiferous epithelium were prepared from 3-week-old rats. Although the Sertoli cells formed a monolayer, germ cells (spermatogonia and early spermatocytes) remained in association with them and were of normal ultrastructural appearance. Germ cells became completely separated from Sertoli cells after 3 weeks of culture in a chemically defined medium. The contact areas between Sertoli and germ cells were characterized by desmosome-like junctions while those between germ cells appeared to be pentalaminar.
Reproduction, 1983
Sertoli cells were obtained from 3\p=n-\6-week-oldrats, which were sterile after prenatal irradia... more Sertoli cells were obtained from 3\p=n-\6-week-oldrats, which were sterile after prenatal irradiation. The production of lactate by these Sertoli cells, measured 24\p=n-\48h after isolation during incubation in the absence of hormones, increased with age of the rats from 3 to 6 weeks. At all ages investigated, the production of lactate was enhanced in the presence of FSH plus testosterone, but the stimulation was most pronounced at 4 weeks of age. Lactate production was increased by FSH alone but testosterone had no effect in the presence or absence of FSH. Sertoli cells from 4-week-old rats produced both pyruvate and lactate, which accumulated in the incubation medium in a ratio of 1:4. The stimulation of the production of pyruvate and lactate by FSH was dose\x=req-\ dependent (ED50 at~10 ng NIH-FSH-S13/ml). The production of pyruvate and lactate was stimulated 2-fold by insulin, 4-fold by FSH and > 6-fold by dibutyryl cyclic AMP (in the presence of 3-isobutyl-1-methylxanthine). The effects of FSH (0\m=.\5\ g=m\ g NIH-FSH-S13/ml) and insulin (5 \g=m\g/ml) were not additive. Leucine incorporation into isolated pachytene spermatocytes and round spermatids was stimulated by exogenous pyruvate and lactate in a dose-dependent way : maximal incorporation was obtained with 0\m=.\2 mM-pyruvate or 2 mM L-lactate. Spent medium from incubated Sertoli cells (from 4-week-old rats) stimulated the leucine incorporation into isolated pachytene spermatocytes and round spermatids 4\p=n-\8-fold.This effect could be explained by the amounts of pyruvate and lactate present in the spent medium. It is suggested that pyruvate and lactate are major products from Sertoli cells which can support synthetic activities in germ cells, and the present results indicate that pyruvate and lactate may play a role in the hormonal regulation of spermatogenesis.
Reproduction, 1983
An unusual enolase isoenzyme, ENO S, was found in human, ram and mouse spermatozoa. This isoenzym... more An unusual enolase isoenzyme, ENO S, was found in human, ram and mouse spermatozoa. This isoenzyme is unique to spermatozoa and distinguished from the somatic enolases ENO 1, ENO 2 and ENO 3 by electrophoretic mobility, high thermostability and ability to undergo structural alteration at high temperatures. The pattern of expression of ENO S during sperm differentiation suggests that this isoenzyme is synthesized relatively late in the presence of a haploid genome.
Reproduction, 1986
Round spermatids were isolated from rat testes and the effects of different energy-yielding subst... more Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6\p=n-\8nmol/106 cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect \g=b\-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6\p=n-\7nmol/106 cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.
Reproduction, 1986
The rates of metabolism in vitro of 3Hor 14C-labelled glucose, pyruvate, glutamine and leucine by... more The rates of metabolism in vitro of 3Hor 14C-labelled glucose, pyruvate, glutamine and leucine by Sertoli cells from immature rats were estimated. The overall rate of glucose utilization exceeded by far the rates of oxidation of pyruvate (derived from glucose) via the citric acid cycle and glucose metabolism via the oxidative branch of the pentose phosphate pathway. This pattern of glucose metabolism was not markedly altered after stimulation of glucose metabolism by FSH. The rate of oxidation of exogenous pyruvate indicated that the energy yield from glucose metabolism by Sertoli cells could be dependent on the extracellular concentrations of pyruvate and lactate. There is no evidence that a high rate of aerobic glycolysis is of vital importance for Sertoli cells. In medium containing glucose and all amino acids, 14C-labelled glutamine and leucine were converted to 14CO2 at considerable rates. It was calculated that the oxidation of glutamine and leucine in addition to glucose and fatty acids can yield much of the required energy of Sertoli cells.
Reproduction, 1988
The ATP content of round spermatids isolated from hamsters was decreased 90% after 18 h of incuba... more 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.
Proceedings of the National Academy of Sciences, 1992
Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotei... more Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated a and 1A. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10-4 to 10-5 M) relative to the hormone itself (10-8 to 10-11 M). This suggests that the
Nucleic Acids Research, 2004
GermOnline provides information and microarray expression data for genes involved in mitosis and ... more GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on~700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subjectoriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.
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Papers by J.A. Grootegoed