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J A Grootegoed

Publications and source records attributed to J A Grootegoed.

At least 91 records · Page 5Linked to original sources
PubMed1987

Identification of insulin receptors on rat Sertoli cells.

The binding of insulin to rat Sertoli cells was investigated to establish if effects of insulin on Sertoli cells can be mediated via insulin receptors. Sertoli cells were isolated from the testes of 3-week-old rats, and preincubated for 3 days in the absence of hormones. Binding of 125I-porcine insulin to the Sertoli cells was 75-80% specific and this binding was time- and pH-dependent and reversible. Scatchard analysis of the binding data resulted in curvilinear plots with a high affinity binding of Kd = 1.8 X 10(-9) M. Porcine and bovine insulin competed equally well for 125I-porcine insulin binding. Porcine proinsulin was 10-50 times less potent, corresponding to its lower biological activity. Insulin-like growth factor-I (IGF-I) was 30-40 times less potent, indicating low affinity binding of IGF-I to the insulin receptor. Lutropin which was used as a control gave no competition with the 125I-insulin binding. Affinity labelling of Sertoli cell membrane proteins with 125I-insulin using the cross-linking agent disuccinimidylsuberate revealed binding of insulin to (a) protein(s) of Mr greater than 300,000 or Mr = 130,000 after electrophoresis under non-reducing or reducing conditions, respectively. Affinity labelling with 125I-insulin was largely prevented by unlabelled insulin. It is concluded that the protein of Mr 130,000 may represent the alpha-subunit of the insulin receptor. The presence of insulin receptors as well as IGF-I receptors on cultured rat Sertoli cells may suggest that insulin and IGF-I have specific functions in regulating the maturation and activities of Sertoli cells during the initiation and maintenance of spermatogenesis.

Affinity Labels↗
PubMed1987

Transport of transferrin-bound iron into rat Sertoli cells and spermatids.

Transferrin (Tf), a major secretory protein of Sertoli cells, may transport iron to spermatogenic cells. This was assessed by measuring the uptake of Fe from 59Fe-125I-labelled rat Tf by Sertoli cells and round spermatids in vitro. Uptake of Fe from labelled Tf by Sertoli cells after a 72-h pre-incubation period was linear for 20 h (approximately 18 pmol/10(6) cells/20 h), whereas the uptake of Fe from labelled Tf by round spermatids after a 16-h pre-incubation period reached a plateau by 2 h (approximately 5 pmol/10(6) cells/2 h). The corresponding net uptake of Tf by both cell types was less than 0.1 pmol. High speed supernatants prepared from Sertoli cells and spermatids labelled with 59Fe-125I-Tf were fractionated by gel permeation chromatography. Separate peaks of protein-bound 59Fe and 125I-Tf were observed. Protein bound 59Fe could be precipitated with an antiserum to rat ferritin. It is concluded that iron from exogenous Tf is transported into Sertoli cells and round spermatids in vitro, and is complexed to intracellular ferritin. However, the present results do not exclude the possibility that Sertoli cell Tf may serve purposes other than iron transport.

Animals↗
PubMed1986

Effect of glucose on ATP dephosphorylation in rat spermatids.

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 (6-8 nmol/10(6) 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 beta-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 (6-7 nmol/10(6) 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.

Adenosine Triphosphate↗
PubMed1986

Metabolism of radiolabelled energy-yielding substrates by rat Sertoli cells.

The rates of metabolism in vitro of 3H- or 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.

Animals↗
PubMed1985

Intercellular pathway of leucine catabolism in rat spermatogenic epithelium.

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.

Animals↗
PubMed1985

Protein synthesis by isolated pachytene spermatocytes in the absence of Sertoli cells.

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.

Animals↗
PubMed1985

Comparison of the effects of insulin and follitropin on glucose metabolism by Sertoli cells from immature rats.

Sertoli cells were isolated from the testes of 3-week-old sterile rats (prenatally irradiated) and incubated for 3 days in the absence of added hormones. Subsequently the effects of follitropin and insulin on glucose metabolism were investigated using this in vitro system. A marked stimulation of net lactate production by either follitropin or insulin was observed within 3 h after addition of the hormones. This response was not inhibited in the presence of the protein synthesis inhibitor cycloheximide. Production of cAMP by the Sertoli cells was markedly enhanced by follitropin, but not at all by insulin. The addition of 0.5 mM dibutyryl cAMP to the incubation medium also resulted in a rapid increase of the rate of lactate production by the Sertoli cells. The stimulation of lactate production by follitropin and insulin was dose-dependent (ED50 of approx. 10 ng NIH-FSH-S13/ml and of approx. 50 ng insulin/ml). It is suggested that the observed effects of insulin on Sertoli cells are mediated via insulin receptors, rather than via receptors for insulin-like growth factors. Within 18 h after addition of either follitropin or insulin the cells became refractory with respect to lactate production to the homologous hormone, whereas the cells could still respond to the heterologous hormone. It is concluded, that follitropin and insulin, acting via different mechanisms, exert similar rapid stimulatory effects on glucose metabolism by Sertoli cells from immature rats in vitro. These effects are not dependent on de novo protein synthesis and may differ from long-term trophic effects of follitropin, insulin, and/or insulin-like growth factors.

Age Factors↗
PubMed1984

The role of glucose, pyruvate and lactate in ATP production by rat spermatocytes and spermatids.

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous L-lactate (3-6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.

Adenosine Triphosphate↗
PubMed1983

A sperm-specific enolase.

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.

Animals↗
PubMed1982

Regulation of survival of rat pachytene spermatocytes by lactate supply from Sertoli cells.

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.

Animals↗
PubMed1981

Exogenous lactate is essential for metabolic activities in isolated rat spermatocytes and spermatids.

Spermatocytes and round spermatids were isolated from rat testis and the effects of addition of 3.3 mM-glucose and 6 mM-DL-lactate to the incubation medium on the morphology, oxygen consumption and incorporation of uridine and amino acids of these cells were investigated. Addition of lactate to isolated germ cells increased O2 consumption 1.8-fold and incorporation of precursors of RNA and protein by at least 5-fold. The amino acid incorporation into spermatocytes and spermatids was irreversibly decreased after a preincubation for at least 5 h or 70 min respectively in the absence of lactate, indicating degeneration of the germ cells. In the presence of lactate, however, spermatocytes maintained their morphological integrity for at least 24 h. Addition of glucose to isolated germ cells had no effect on any of the parameters investigated. It is concluded that isolated spermatocytes and round spermatids may utilize lactate, perhaps secreted by Sertoli cells, as the main source of energy.

Animals↗
PubMed1979

Reversible interaction between androgen binding protein and testicular macromolecules causing inhibition of androgen binding activity.

Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.

Androgens↗