scholarly journals Characterization and hormonal modulation of immunoreactive thiamin carrier protein secreted by adult rat Leydig cells in vitro

1999 ◽  
Vol 162 (1) ◽  
pp. 49-56
Author(s):  
S Subramanian ◽  
PR Adiga
Keyword(s):  
Leydig Cells ◽  
Polyclonal Antibodies ◽  
Carrier Protein ◽  
Adult Rats ◽  
Adult Rat ◽  
Sequence Of Events ◽  
Specific Radioimmunoassay ◽  
Hormonal Modulation ◽  
Lh Secretion

Leydig cells isolated from adult rats and maintained under defined conditions in culture secrete a protein of molecular weight (Mr) 70 000 which is immunologically similar to chicken thiamin carrier protein (TCP). Synthesis of immunoreactive TCP by these cells is demonstrated by immunoprecipitation of [35S]methionine incorporated, newly synthesized proteins with monoclonal and polyclonal antibodies to chicken TCP. The amount of immunoreactive TCP secreted into the culture supernatant is quantitated by using a specific radioimmunoassay. Under the influence of LH, secretion of immunoreactive TCP is enhanced 3-fold and can be inhibited by up to 70% with aromatase inhibitor (1,4,6-androstatrien-3,17-dione). Cyclic AMP acts as a second messenger in the sequence of events involved in LH-induced elevation of immunoreactive TCP in Leydig cells. The effects of exogenous estradiol-17beta and diethylstilbestrol are comparable in terms of stimulation of secretion of immunoreactive TCP by these cells. Tamoxifen brought about a 70% decrease in the elevated levels of immunoreactive TCP. These results suggest that estrogen mediates immunoreactive TCP induction in hormonally stimulated adult rat Leydig cells.

Ethan-1,2-dimethanesulphonate reduces testicular oxytocin content and seminiferous tubule movements in the rat

1987 ◽  
Vol 112 (2) ◽  
pp. 311-NP ◽  
Author(s):  
H. D. Nicholson ◽  
R. T. S. Worley ◽  
S. E. F. Guldenaar ◽  
B. T. Pickering
Keyword(s):  
Leydig Cell ◽  
Seminiferous Tubule ◽  
Leydig Cells ◽  
Contractile Activity ◽  
Histological Study ◽  
Interstitial Cells ◽  
Seminiferous Tubules ◽  
Adult Rats ◽  
Adult Rat

ABSTRACT An oxytocin-like peptide is present in the interstitial cells of the testis, and testicular concentrations of oxytocin have been shown to increase seminiferous tubule movements in vitro. We have used the drug ethan-1,2-dimethanesulphonate (EDS), which depletes the Leydig cell population of the adult rat testis, to examine further the relationships between the Leydig cell, testicular oxytocin and tubular movements. Adult rats were injected i.p. with a single dose of EDS (75 mg/kg) or of vehicle (25% dimethyl sulphoxide). Histological study 3 and 10 days after treatment with EDS showed a reduction in the number of interstitial cells, and levels of oxytocin immunoreactivity were undetectable by radioimmunoassay. Immunostaining revealed very few oxytocin-reactive cells. Spontaneous contractile activity of the seminiferous tubules in vitro was also dramatically reduced, but could be restored by the addition of oxytocin to the medium. Four weeks after EDS treatment, the interstitial cells were similar to those in the control animals both in number and in immunostaining; immunoassayable oxytocin was present and tubular movements were normal. The EDS effect, seen at 3 and 10 days, was not altered by daily treatment with testosterone. However, repopulation of the testes with oxytocin-immunoreactive cells was not seen until 6 weeks in the testosterone-treated animals. We suggest that the Leydig cells are the main source of oxytocin immunoreactivity in the testis and that this oxytocin is involved in modulating seminiferous tubule movements and the resultant sperm transport. The results also imply that testosterone does not play a major role in controlling tubular activity in the mature rat. J. Endocr. (1987) 112, 311–316

Reversal of long-term LH deprivation on testosterone secretion and Leydig cell volume, number and proliferation in adult rats

1990 ◽  
Vol 127 (1) ◽  
pp. 47-NP ◽  
Author(s):  
D. S. Keeney ◽  
R. L. Sprando ◽  
B. Robaire ◽  
B. R. Zirkin ◽  
L. L. Ewing
Keyword(s):  
Cell Volume ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Number ◽  
Implant Removal ◽  
Adult Rats ◽  
Testosterone Secretion ◽  
Lh Secretion

ABSTRACT The purpose of this study was to determine whether Leydig cell volume and function could recover fully from long-term LH deprivation upon restoration of endogenous LH secretion, and whether the restoration of LH would elicit a mitogenic response, i.e. stimulate Leydig cell proliferation or affect Leydig cell number per testis. LH secretion was inhibited by treating adult rats with testosterone and oestradiol-filled (TO) silicone elastomer implants (16 weeks), and was restored by removing the implants. Changes in serum concentrations of LH and FSH, LH-stimulated testosterone secretion by testes perfused in vitro, Leydig cell volume and number per testis, average Leydig cell volume and Leydig cell [3H]thymidine incorporation were measured at weekly intervals following implant removal. The TO implants inhibited (P < 0·01) LH secretion, but serum concentrations of FSH were not significantly different (P > 0·10) from control values. After implant removal, serum LH returned to control values within 1 week, whereas serum FSH increased twofold (P < 0·01) and returned to control values at 4 weeks. LH-stimulated in-vitro testosterone secretion was inhibited by more than 99% in TO-implanted rats, but increased (P < 0·01) to 80% of control values by 8 weeks after implant removal. The total volume of Leydig cells per testis and the volume of an average Leydig cell were 14 and 19% of control values respectively, after 16 weeks of TO implantation (P < 0·01), but returned to 83 and 86% of controls (P > 0·10) respectively, by 6 weeks after implant removal. Leydig cell proliferation ([3H]thymidine labelling index) was low (< 0·1%) in both control and TO-implanted rats, increased (P < 0·01) fivefold from 1 to 4 weeks after implant removal and then declined to control values at 6 weeks. The increase in Leydig cell [3H]thymidine incorporation was mimicked by treating TO-implanted rats with exogenous LH, but not FSH. Leydig cells were identified in both the interstitium and the lamina propria of the seminiferous epithelium. The proportion of Leydig cell nuclei in the lamina propria was 30-fold greater (P < 0·01) at 1 and 3 weeks after implant removal (3%) compared with that for control and TO-implanted rats (0·1%). Total Leydig cell number per testis was marginally but not significantly (P = 0·06) decreased in rats treated with TO implants for 16 weeks when compared with controls (18·4±2·2 vs 25·4±1·2 × 106). Three weeks after implant removal, the numbers of Leydig cells per testis were identical (26·8±2·8 × 106) to those in control animals. These results not only demonstrate dramatic morphogenic effects of LH on mature rat Leydig cells, but also suggest that endogenous LH might be mitogenic at least to a subpopulation of Leydig cells. Journal of Endocrinology (1990) 127,47–58

Comparison of the effects on purified Leydig cells of four hormones (oxytocin, vasopressin, opiates and LHRH) with suggested paracrine roles in the testis

1987 ◽  
Vol 113 (1) ◽  
pp. 89-96 ◽  
Author(s):  
R. M. Sharpe ◽  
I. Cooper
Keyword(s):  
Leydig Cells ◽  
Interstitial Fluid ◽  
Adult Rats ◽  
Adult Rat ◽  
Clear Cut ◽  
Testosterone Production ◽  
Time Period ◽  
High Doses

ABSTRACT Four hormones have been identified by various authors as possible paracrine regulators of testicular Leydig cells. The aim of this study was to evaluate their effects on purified adult rat Leydig cells under various conditions in vitro, and then to assess whether comparable effects occurred in vivo. In agreement with previous findings, an LHRH agonist (LHRH-A) exerted clear-cut effects on testosterone secretion by Leydig cells both in vitro and in vivo. On its own, LHRH-A stimulated testosterone production by Leydig cells for up to 24 h in culture but inhibited testosterone production stimulated by human chorionic gonadotrophin (hCG) between 24 and 72 h of culture. In-vivo, unilateral intratesticular injection of adult rats with 1 ng LHRH-A resulted 5 h later in a significant increase in testosterone concentrations in testicular interstitial fluid (IF). Vasopressin exerted effects in vitro which were similar to those of LHRH-A. On its own, vasopressin stimulated testosterone production for up to 5 h of culture, but not thereafter, while in the presence of hCG, vasopressin inhibited testosterone production beyond 24 h of culture. The initial stimulatory effect of vasopressin on testosterone production occurred with concentrations of 1 nmol/l and higher, but the magnitude of stimulation (threefold or less) was considerably less than that induced by LHRH-A (ninefold) over the same time period. In contrast to LHRH-A, unilateral intratesticular injection of vasopressin in high doses (20 and 2 ng) had no effect on IF testosterone levels 5 h later. When Leydig cells were cultured in the presence of testicular IF, to approximate in-vivo conditions, there was marked stimulation of testosterone production, but the effects of vasopressin and LHRH-A in the presence of IF were comparable to those observed in its absence. Neither morphine nor oxytocin at concentrations of 0·1 μmol/l had any effect on testosterone production under any of the conditions of culture, and unilateral intratesticular injection of oxytocin, morphine or naloxone was without effect on the IF levels of testosterone. It is concluded that opiates and oxytocin are probably not involved in the paracrine regulation of Leydig cells, whereas vasopressin may play such a role. However, as the stimulatory effects of vasopressin were small in relation to those of LHRH-A and were not evident in vivo, the physiological significance of the effects of vasopressin are uncertain. J. Endocr. (1987) 113, 89–96

Effects of hypophysectomy and subsequent FSH and testosterone treatment on inhibin production by adult rat testes

1985 ◽  
Vol 105 (1) ◽  
pp. 1-6 ◽  
Author(s):  
C. L. Au ◽  
D. M. Robertson ◽  
D. M. de Kretser
Keyword(s):  
Seminiferous Tubule ◽  
Hormonal Control ◽  
Human Chorionic Gonadotrophin ◽  
Experimental Conditions ◽  
Adult Rats ◽  
Adult Rat ◽  
Fluid Production ◽  
Tubule Fluid

ABSTRACT The hormonal control of inhibin production by adult rat testes was investigated using an in-vitro inhibin bioassay validated for the measurement of inhibin activity in charcoal-treated rat testicular extracts. The effect of hypophysectomy examined at 16 h, 3, 7 and 42 days after surgery showed a decrease in testicular inhibin content and seminiferous tubule fluid production by 7 days and a decrease in inhibin production by 42 days. Serum FSH and LH were suppressed 3 days after surgery. In 30-day chronically hypophysectomized adult rats treated for 3 days with twice daily s.c. injections of (a) human FSH (hFSH, 22 i.u./rat per day), (b) testosterone (5 mg/rat per day), (c) hFSH + testosterone (same doses as a and b), or (d) human chorionic gonadotrophin (hCG, 12 i.u./rat per day), hFSH or hFSH and testosterone stimulated an increase in testicular inhibin content but not in inhibin production or tubule fluid production. Testosterone and hCG had no effect on these parameters. It is concluded that in vivo, FSH alone stimulates an increase in testicular inhibin content. The failure to observe an increase in inhibin production in vivo is attributed to the suppression of seminiferous tubule fluid production under the same experimental conditions. J. Endocr. (1985) 105, 1–6

Selective Modulation of Tonic and Phasic Inhibitions in Dentate Gyrus Granule Cells

2002 ◽  
Vol 87 (5) ◽  
pp. 2624-2628 ◽  
Author(s):  
Zoltan Nusser ◽  
Istvan Mody
Keyword(s):  
Dentate Gyrus ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Hippocampal Slices ◽  
Tonic Inhibition ◽  
Adult Rats ◽  
Adult Rat ◽  
Phasic Inhibition ◽  
The Mean

In some nerve cells, activation of GABAA receptors by GABA results in phasic and tonic conductances. Transient activation of synaptic receptors generates phasic inhibition, whereas tonic inhibition originates from GABA acting on extrasynaptic receptors, like in cerebellar granule cells, where it is thought to result from the activation of extrasynaptic GABAA receptors with a specific subunit composition (α6βxδ). Here we show that in adult rat hippocampal slices, extracellular GABA levels are sufficiently high to generate a powerful tonic inhibition in δ subunit–expressing dentate gyrus granule cells. In these cells, the mean tonic current is approximately four times larger than that produced by spontaneous synaptic currents occurring at a frequency of ∼10 Hz. Antagonizing the GABA transporter GAT-1 with NO-711 (2.5 μM) selectively enhanced tonic inhibition by 330% without affecting the phasic component. In contrast, by prolonging the decay of inhibitory postsynaptic currents (IPSCs), the benzodiazepine agonist zolpidem (0.5 μM) augmented phasic inhibition by 66%, while leaving the mean tonic conductance unchanged. These results demonstrate that a tonic GABAA receptor–mediated conductance can be recorded from dentate gyrus granule cells of adult rats in in vitro slice preparations. Furthermore, we have identified distinct pharmacological tools to selectively modify tonic and phasic inhibitions, allowing future studies to investigate their specific roles in neuronal function.

scholarly journals Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes.

1989 ◽  
Vol 108 (4) ◽  
pp. 1353-1361 ◽  
Author(s):  
G A Keller ◽  
T J Scallen ◽  
D Clarke ◽  
P A Maher ◽  
S K Krisans ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Rat Liver ◽  
Polyclonal Antibodies ◽  
Rat Hepatocytes ◽  
Carrier Protein ◽  
Sterol Carrier Protein ◽  
Rat Liver Cells ◽  
Ultrathin Frozen Sections ◽  
Sterol Carrier Protein 2

Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

Distribution of α1-adrenoceptor subtype proteins in different tissues of neonatal and adult rats

2000 ◽  
Vol 78 (3) ◽  
pp. 237-243 ◽  
Author(s):  
Hao Shen ◽  
Krishna G Peri ◽  
Xing-Fei Deng ◽  
Sylvain Chemtob ◽  
Daya R Varma
Keyword(s):  
Vas Deferens ◽  
Polyclonal Antibodies ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Rat Tissues ◽  
Adult Rats ◽  
Adult Rat ◽  
Old Rats ◽  
Kidney Liver ◽  
The Brain

Distribution of α1-adrenoceptor (α1AR) subtype (α1A, α1B, α1D) proteins in brain, heart, kidney, and liver of 1-week-old rats and in brain, heart, aorta, kidney, liver, vas deferens, prostate, and adrenal glands of adult rats was investigated by Western analysis, using receptor subtype specific polyclonal antibodies. High levels of immunoreactive α1AAR and α1DAR in brain and heart and of α1BAR in liver and heart of neonatal rats were detected. In adult rat tissues, the abundance of α1AAR protein was most marked in the brain, intermediate in heart, aorta, liver, vas deferens, and adrenals, and minimal in the kidney and prostate; relative to other tissues, the expression of α1BAR was higher in brain and heart and that of α1DAR in brain. All the three receptor subtypes increased with age in the brain cortex, whereas the abundance of α1BAR increased in the heart but decreased in the liver; α1AAR and α1DAR in liver, kidney, and heart were not affected by age. It is concluded that α1AR subtypes are widely expressed in different neonatal and adult rat tissues.Key words: α1A-adrenoceptors, α1B-adrenoceptors, α1D-adrenoceptors, α1-adrenoceptor proteins.

scholarly journals Seizure-Like Events in Disinhibited Ventral Slices of Adult Rat Hippocampus

1999 ◽  
Vol 82 (5) ◽  
pp. 2130-2142 ◽  
Author(s):  
Cornelius Borck ◽  
John G. R. Jefferys
Keyword(s):  
Hippocampal Neurons ◽  
Peak Frequency ◽  
Ventral Hippocampus ◽  
Synaptic Activity ◽  
Receptor Antagonists ◽  
Adult Rats ◽  
Adult Rat ◽  
Key Factor ◽  
Interictal Discharges

Epileptic discharges lasting 2–90 s, were studied in vitro in slices from the ventral hippocampus of adult rats, in which inhibition was blocked acutely with bicuculline methiodide (BMI, 5–30 μM) and potassium ([K+]o) raised to 5 mM. These seizure-like events (SLEs) comprised three distinct phases, called here primary, secondary, and tertiary bursts. Primary bursts lasted 90–150 ms. Secondary bursts lasted a further 70–250 ms, comprising a short series of afterdischarges riding on the same depolarization as the primary burst. Finally a train of tertiary bursts started with a peak frequency of 5–10 Hz and could last >1 min. Slices from the ventral hippocampus showed significantly higher susceptibility to SLEs than did dorsal slices. SLEs proved sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists. They were insensitive to N-methyl-d-aspartate (NMDA) receptor antagonists; 50 μl d-2-amino-5-phosphonopentanoic acid (d-AP5) did block the transient secondary bursts selectively. SLEs were restricted to the hippocampus proper even if the entorhinal cortex was present. Entorhinal bursts could last <2 s and were only coupled with hippocampal bursts in a minority of slices. Reentry of epileptic bursts occasionally occurred during interictal discharges, but not during the later stages of SLEs. Full-length SLEs always started in CA3 region and could be recorded in minislices containing CA3 plus dentate hilus. Ion-sensitive microelectrodes revealed that interictal discharges were followed by short (2–3 s) [K+]o waves, peaking at ∼7.5 mM. SLEs were always accompanied by increases in [K+]oreaching ∼8.5 mM at the start of tertiary bursts; [K+]o then increased more slowly to a ceiling of 11–12 mM. After the end of each SLE, [K+]o fell back to baseline within 10–15 s. SLEs were accompanied by significant increase in synaptic activity, compared with baseline and/or interictal activity, estimated by the variance of the intracellular signal in the absence of epileptic bursts and action potentials (0.38 mV2, compared with 0.13 mV2, and 0.1 mV2, respectively). No significant increases were observed in the interval preceding spontaneous interictal activity. These studies show that focal assemblies of hippocampal neurons, without long reentrant loops, are sufficient for the generation of SLEs. We propose that a key factor in the transition from interictal activity to SLEs is an increase in axonal and terminal excitability, resulting, at least in part, from elevations in [K+]o.

scholarly journals A novel mechanism for the melatonin inhibition of testosterone secretion by rat Leydig cells: reduction of GnRH-induced increase in cytosolic Ca2+

1999 ◽  
Vol 23 (3) ◽  
pp. 299-306 ◽  
Author(s):  
S Valenti ◽  
S Thellung ◽  
T Florio ◽  
M Giusti ◽  
G Schettini ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Calcium Concentration ◽  
Inhibitory Effect ◽  
Adult Rat ◽  
Testosterone Secretion ◽  
Gi Protein ◽  
Hydroxy Progesterone ◽  
Induced Changes ◽  
Cells Cultured

The site of inhibition, by melatonin, of GnRH-dependent testosterone secretion was investigated in adult rat Leydig cells cultured in vitro. The various effects downstream of the binding of GnRH to its own receptor were isolated and mimicked by specific drugs. Testosterone secretion was then evaluated after 3 h stimulation with GnRH, thapsigargin (1 microM), phorbol-12-myristate-13-acetate (100 nM), arachidonic acid (20 microM), and ionomycin (1 microM) in the presence or absence of melatonin (215 nM). The effect of melatonin on the GnRH-induced changes in cytoplasmic calcium concentration ([Ca(2+)](i)) was also studied, using Fura-2 as fluorescent Ca(2+) indicator. Melatonin attenuated the increase in [Ca(2+)](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin potentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on cells stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibitory effect on GnRH-induced secretion was supressed in cells pretreated with pertussis toxin in a concentration of 180 ng/ml for 20 h. Assay of 17-hydroxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that melatonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca(2+)](i), through impairment of the GnRH-dependent release of Ca(2+) from intracellular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca(2+)](i).

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