Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

Effect of luteinizing hormone on RNA synthesis in Leydig cells of immature rats

Luteotrophic hormone acts on testicular interstitial cells, promoting the activation of several cellular events that culminate in steroids synthesis. Since the interstitial tissue include several cell types, purified Leydig cells were used in this work. Isolated interstitial cells from immature rats were purified through a 0-40% metrizamide gradient. Either LH, HCG or Bt2-cAMP significantly stimulated the incorporation of [3H]uridine into RNA, when compared to control. The effect of HCG on RNA synthesis was developed within 30 min after the addition of the hormone and was dose-dependent. The maximum effect was attained with 10 mIU/ml of HCG. These results indicate that HCG/LH or Bt2-cAMP but not FSH, promote an acute stimulation of RNA synthesis by Leydig cells from immature rats.     

Hormonal studies of uridine utilization in an insect cell line CP-1268 derived from the codling moth Laspeyresia pomonella.

Ecdysterone decreased cellular growth and the incorporation of uridine into RNA following 4 days of hormone exposure. This hormone did not affect uridine incorporation following short-term exposure up to 25 hours. Juvenile hormone and farnesol both significantly decreased uridine uptake and incorporation into RNA; however, uridine uptake was inhibited to a greater extent than uridine incorporation. Cyclic AMP increased the incorporation of uridine into RNA but had no demonstrable effect on the uptake process. This stimulation was not the result of cAMP degradation products. Cyclic AMP and ecdysterone together produced a significant increase in uridine incorporation into RNA. These studies demonstrate the potential utilization of insect cell lines for studying the mode of action of insect developmental hormones.     

Hormonal studies of uridine utilization in an insect cell line CP-1268 derived from the codling mothLaspeyresia pomonella

Summary Ecdysterone decreased cellular growth and the incorporation of uridine into RNA following 4 days of hormone exposure. This hormone did not affect uridine incorporation following short-term exposure up to 25 hours. Juvenile hormone and farnesol both significantly decreased uridine uptake and incorporation into RNA; however, uridine uptake was inhibited to a greater extent than uridine incorporation. Cyclic AMP increased the incorporation of uridine into RNA but had no demonstrable effect on the uptake process. This stimulation was not the result of cAMP degradation products. Cyclic AMP and ecdysterone together produced a significant increase in urdine incorporation into RNA. These studies demonstrate the potential utilization of insect cell lines for studying the mode of action of insect developmental hormones.     

Effects of inhibin on the in vitro synthesis of desoxyribonucleic acid in the rat testis

Three preparations of inhibin extracted from ram rete testis fluid (RTF) and from human seminal plasma (HSP) reduce tritiated thymidine incorporation into testicular desoxyribonucleic acids (DNA) in vitro. Effect of low molecular inhibin from RTF is dose-dependent. Castrated ram serum does not modify testicular DNA synthesis in vitro. Besides their suppressive action on follicle stimulating hormone (FSH) secretion in vivo and in vitro, these inhibin preparation display a direct inhibiting effect on testicular DNA synthesis and, thus, on mitotic activity. Identity between inhibin and testicular chalones are discussed.     

Effects of prostaglandin E1 on the metabolism in rat parathyroid gland in vitro

We investigated some effects of prostaglandin E₁ on the metabolism of rat parathyroid glands using a culture system containing basal Eagle's medium supplemented with 5–10% heat-inactivated rat serum. Rat parathyroid glands incorporate [³H]fucose and ¹⁴C-labeled amino acids into cellular glycoproteins and secrete some of these into the culture medium. Gel filtration chromatography separates these glycoproteins into three classes, the smallest of which (peak 3) is secreted with immunoreactive parathyroid hormone. In cultures of 48 h, prostaglandin E₁ (1 μg/ml) specifically inhibits the secretion of peak 3 and of parathyroid hormone but has no effect on the incorporation of [³H]-fucose, ¹⁴C-labeled amino acids, or [³H]uridine into parathyroid glands. Cytochalasin B inhibits the secretion of parathyroid hormone and the incroporation of isotopic fucose and amino acids. Cortisol stimulates incorporation of [³H]fucose and the secretion of parathyroid hormone even in the presence of inhibitory doses of prostaglandin E₁. It is concluded that, in organ culture, prostaglandin E₁ inhibits the secretion of parathyroid hormone and of a specific glycoprotein the function of which may be related to the secretion of the hormone.     

Effect of hypophysectomy and replacement therapy on fatty acid metabolism in the rat testis

1. The influence of pituitary gonadotrophins and of testosterone on the conversion of linoleic acid into other polyunsaturated fatty acids by rat testicular tissue was studied. 2. In immature hypophysectomized rats, follicle-stimulating hormone caused a threefold increase in the incorporation of radioactivity from [1-(14)C]linoleic acid into testicular lipids; the distribution of (14)C in the polyunsaturated fatty acids, however, was not significantly affected. 3. In mature hypophysectomized rats, the hormonal treatments had less pronounced effects on (14)C incorporation into testicular lipids, but caused a significant increase in the percentage of (14)C incorporated into polyunsaturated fatty acids of the omega-6 series, luteinizing hormone and testosterone having the more pronounced influences. 4. A time-course study of the appearance of radioactivity in the ejaculated spermatozoa of rabbits, after they had been given a tracer dose of [1-(14)C]linoleic acid, indicated that incorporation of radioactivity into spermatozoa occurred during all stages of spermatogenesis.     

Macrophage regulation of DNA synthesis in lymphoid cells: effects of a soluble factor from macrophages

Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [³H]uridine and [¹⁴C] amino acids by spleen cells was also moderately suppressed. Based on ⁵¹Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [³H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.     

Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: Possible role of uncharged tRNA in regulating metabolism

Previous studies (J. Biol. Chem, 253: 99–105, 1978) showed that thyrotropin-releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH₄C₁) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid-depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time-course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid-depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino-acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximde can be attributed solely to inhibition of the synthesis of proteins.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

Effects of photoperiod, hypophysectomy, and follicle-stimulating hormone on testicular follicle-stimulating hormone binding sites in golden hamsters

Experiments were conducted to partially characterize and to examine the regulation of unoccupied testicular follicle-stimulating hormone (FSH) binding sites in adult golden hamsters. Testicular FSH binding sites were measured in the 1800 X gav fraction of whole testicular homogenates using iodinated bovine FSH. Binding of FSH was highly specific for FSH, located primarily in the testes, was time- and temperature-dependent, initially reversible, saturable, and consistent with a model consisting of a single class of high-affinity binding sites (range of equilibrium association constants (Ka) 2-12 X 10(10) M-1). Exposure of hamsters to a short photoperiod consisting of 5L:19D was associated with an increase in concentration (fmol/mg protein), but a reduction in total content (fmol/testes) of testicular FSH binding sites. There was no appreciable 5L:19D-associated alteration in receptor affinity (average Ka = 7.83 X 10(10) M-1). Injections of ovine prolactin (oPRL), ovine luteinizing hormone (oLH), or ovine FSH (oFSH) for 3 days into hamsters housed in 5L:19D for 12 wk had no effect on photoperiod-induced changes in testicular FSH binding sites. On Days 5 and 6 post hypophysectomy, a dramatic increase in FSH binding site concentration occurred, with but marginal effects on binding site affinity. Injections of 5 micrograms oFSH on Days 2, 3, and 4 after hypophysectomy prevented the increase in binding site concentrations measured on Day 5. Injection of a combination of 5 micrograms oFSH, 50 micrograms oPRL, and 25 micrograms oLH also reduced testicular FSH binding site concentrations in hypophysectomized hamsters, but oPRL or oLH by themselves were ineffective. The data indicate a homologous down-regulation of testicular FSH binding sites, but do not exclude the involvement of other hormones.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Follicle-stimulating hormone interacts with exoloop 3 of the receptor

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.     

Amino acid dependent and independent insulin stimulation of cartilage metabolism

The effects of insulin on embryonic chicken cartilage in organ culture and the dependence of these effects on essential amino acids have been studied. In the presence of all essential amino acids, insulin: (1) increases 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake; (2) increases [5(-3H] uridine flux into uridine metabolites and the intracellular UTP pool; (3) expands the size of the intracellular UTP pool; (4) does not change the specific activity of the UTP pool; and (5) stimulates RNA, proteoglycan, and total protein synthesis. In lysine (or other essential amino acid)-deficient medium, the effects of insulin are different. While insulin stimulates incorporation of [5(-3)H] uridine into RNA, it does so by increasing the specific activity of the UTP pool without increasing RNA synthesis. Insulin stimulates 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake but no longer stimulates proteoglycan, total protein, or RNA synthesis or expands the size of the UTP pool. These data indicate that there are amino acid dependent and independent effects of insulin on cartilage. Transport processes are amino acid independent, while synthetic processes are amino acid dependent.     

Some biochemical effects of triamcinolone acetonide on rat liver and muscle

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [(3)H]uridine and [(32)P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4-5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of alpha-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with (14)C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of (14)C-labelled amino acids into protein, (b) [(3)H]uridine and [(32)P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.     

Action of insulin and growth hormone on protein synthesis in muscle from non-hypophysectomized rabbits

The separate effects of insulin and growth hormone on the uptake and incorporation of five amino acids into diaphragm muscle from non-hypophysectomized rabbits has been examined. Both growth hormone and insulin, when present in the medium separately, stimulated the incorporation into protein of the amino acids, leucine, arginine, valine, lysine and histidine. Insulin also stimulated amino acid uptake, but growth hormone did not. When insulin and growth hormone were present in the incubation medium together, the uptake and incorporation of valine, the only amino acid studied under these conditions, tended to be greater than the sum of the separate effects of the two hormones.     

A direct effect of growth hormone on the incorporation of precursors into proteins and nucleic acids of perfused rat liver

1. The livers of rats were perfused in situ. When the amino acid concentration in the perfusing medium was that present in rat plasma, the addition of growth hormone to the medium stimulated the incorporation of labelled amino acids into liver protein only marginally and not to a statistically significant extent. When, however, the amino acid concentration was raised to three times that present in rat plasma, growth hormone significantly and substantially stimulated amino acid incorporation into protein within 30min. of perfusion of normal rat liver. 2. A significant effect of growth hormone on labelling of normal rat-liver protein was seen with concentrations not much greater than those reported to be present in rat plasma. 3. The labelling of nucleic acids of normal and hypophysectomized rat liver by [(3)H]orotic acid was enhanced by addition of growth hormone to the perfusing medium when normal concentrations of amino acids were used. 4. At elevated concentrations of amino acids, growth hormone stimulated labelling of nucleic acids of hypophysectomized rat liver at 30 and 60min. of perfusion. Under these conditions, nucleic acids of normal rats were labelled to about the same extent in control and hormone-treated livers at 30min. and, because of a fall in the radioactivity of the control livers, there was more labelled nucleic acids in growth-hormone-treated livers at 60min. than in the control livers. 5. Growth hormone, unlike insulin, had no inhibitory effect on the release of glucose by the perfused liver. 6. It is concluded that growth hormone can stimulate the incorporation of precursor into proteins and nucleic acids of liver directly and without the mediation of other organs or of insulin.     

Stimulation of protein synthesis in vivo in immature mouse testis by FSH.

Human pituitary FSH was found to increase the incorporation of tritiated lysine into testicular protein in prepubertal mice in vivo. Radioactivity was measured in washed trichloracetic acid precipitates prepared from crude testicular homogenates. The time of maximum response was 8 to 16 hr after subcutaneous injection of the hormone. This was considerably later than the maximum response in vitro reported by other workers. Neither HCG nor dibutyryl cyclic 3',5'-adenosine monophosphate had a significant effect on the incorporation of lysine.     

Excitatory amino acid regulation of gonadotropin secretion: modulation by steroid hormones

Excitatory amino acids (EAAs) can potently modulate gonadotropin secretion in the male rat and monkey. In the present study we examined of EAAs on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the female rat under low estrogen (ovariectomized) and high estrogen (proestrus) backgrounds. In ovariectomized immature female rats (NMDA) inhibited LH but not FSH secretion at 30 min post-injection. In contrast, NMDA potently stimulated LH but not FSH secretion when administered on proestrus to adult female rats. Both glutamate and kainate were also found to stimulate LH but not FSH secretion in estrogen-treated ovariectomized immature rats. This study suggests that EAA neurotransmission may be an important component in the expression of gonadotropin surges and that EAA effects appear to be subject to gonadal steroid regulation.