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R M Sharpe

Publications and source records attributed to R M Sharpe.

At least 145 records · Page 8Linked to original sources
PubMed1983

Testicular interstitial fluid as a monitor for changes in the intratesticular environment in the rat.

The use of testicular interstitial fluid (IF) collected from the rat testis has been validated as (1) an index of the total extracellular extratubular fluid volume of the testis (which reflects the permeability of testicular capillaries) and (2) as a means of measuring changes in the interstitial hormonal environment. The former was tested by comparing the albumin 'space' with the volume of recovered IF in the same testes from control and bilaterally cryptorchid rats sampled at 0-40 h after injection of hCG. Although the volume of IF recovered was on average only 50% of the albumin 'space', both measures increased in parallel after hCG injection and were always closely correlated (P less than 0.001) over a 4- to 5-fold range. The volume of recovered IF increased with age in parallel with increase in testicular weight, and the testosterone concentration in IF paralleled changes in peripheral serum, increasing from 45 to 80 days of age and then declining. After injection of 25 micrograms bovine LH, testosterone levels in IF, spermatic venous (SV) and peripheral venous (PV) blood increased up to 10-fold by 1 h and returned to control levels over the next 11 h. Testosterone levels in IF were always considerably higher than those in SV blood, but this difference was not constant. Subcutaneous injection of rats with an LH-RH agonist resulted in parallel increases in the serum levels of LH and in the IF and PV levels of testosterone. However, at 6 h there was an 'LH-independent' secondary increase in testosterone levels which was associated with an increase in IF volume, reflecting an increase in capillary wall permeability and hence increased transport of LH into the testis.

Age Factors↗
PubMed1982

Stimulatory effect of LHRH and its agonists on Leydig cell steroidogenesis in vitro.

Short-term (4 h) incubation of collagenase-dispersed Leydig cells from adult rats in the presence of an LHRH agonist caused a 2-3-fold stimulation (P less than 0.001) of testosterone production. This effect was dose-dependent and as little as 5 x 10(-11) M LHRH agonist caused significant stimulation whilst maximal effects were achieved with 10(-9) M concentrations. Stimulation of steroidogenesis by LHRH agonist was prevented by addition of an antiserum specific for the peptide, but was exaggerated in the presence of the phosphodiesterase inhibitor MIX, suggesting the involvement of cyclic AMP in the response of the Leydig cells to the agonist. Native LHRH caused a similar degree of stimulation of testosterone secretion to LHRH agonist but concentrations 1000 times greater than those of the agonist were required to achieve this, a finding consistent with the known affinities of these 2 peptides for the Leydig cell LHRH-receptor. The addition of LHRH agonist also enhanced (P less than 0.001) testosterone secretion by adult rat Leydig cells in response to hCG or dibutyryl cyclic AMP, and this effect was still evident in the presence of maximally-stimulating concentrations of these factors. LHRH agonist also stimulated testosterone secretion by Leydig cells from immature rats, but this effect differed from that in the adult in being of smaller magnitude and being restricted to effects on basal secretion or secretion elicited by low concentrations of hCG. These results show for the first time (a) that LHRH and its agonists can exert effects on Leydig cell steroidogenesis during short-term incubation, and (b) that these effects are stimulatory, which contrasts with the inhibitory effects reported after long-term (2-3 days) exposure of Leydig cells to LHRH agonists in vivo and in vitro. The availability of this simple and rapid measure of a biological action of LHRH on the Leydig cell should enable its precise mode of action to be determined, and should throw light on the physiological role of endogenously produced testicular 'LHRH'.

1-Methyl-3-isobutylxanthine↗
PubMed1982

The mode of action of LHRH agonists on the rat Leydig cell.

The relationship between LHRH agonist-receptor interaction and subsequent stimulation of steroidogenesis has been investigated using dispersed adult rat Leydig cells. Binding of 125I-labelled LHRH agonist to these cells was rapid and was readily and completely reversible, which contrasted with the binding of 125I-labelled hCG which was never completely reversible and which became progressively less reversible with increase in time. Following 125I-LHRH agonist-receptor interaction for 5 min at 21 degrees C, over 80% of the bound hormone dissociated at a fast rate (t 1/2 1.5 min) and the remainder at a slower rate (t 1/2 20 min). Following LHRH agonist-receptor interaction for a longer period (45 min) at 21 degrees C, only the slower of these 2 components of dissociation was evident, although binding was still completely reversible. Evidence was also obtained that following preincubation of Leydig cells for 2 hr at 34 degrees C with a near-saturating concentration (2000 pg/ml) of unlabelled LHRH agonist, all or most of the bound hormone dissociated during subsequent incubation at 21 degrees C. Incubation of Leydig cells at 34 degrees C in the continuous presence of either a low dose (1 pM) of hCG or a high dose (2000 pg/ml) of LHRH agonist resulted in similar percentage stimulation of testosterone secretion with a similar time lag (2 h) before this increase was evident. Preincubation with this dose of hCG for as little as 15 or 30 min still led to increased testosterone secretion following removal of the free hormone. In contrast, preincubation with LHRH agonist for up to 2 h was without subsequent stimulatory effect, presumably due to dissociation of the bound hormone from its receptors. Therefore, for LHRH agonist to stimulate Leydig cell steroidogenesis in vitro, it must be present continuously in the incubation medium, and the implications of this with respect to the secretion and mode of action of 'testicular LHRH' in vivo are discussed.

Animals↗
PubMed1982

The secretion, measurement, and function of a testicular LHRH-like factor.

In this paper we have demonstrated the presence and secretion within the testis of a factor that is immunologically distinct from LHRH, but that has receptor-binding and bioactive features similar to LHRH. The major source of this LHRH-like factor is probably the Sertoli cell and, as its site of action appears to be restricted to the Leydig cells, it is possible that it is one of the means by which the Sertoli cell has been postulated to regulate the Leydig cell. All of the available evidence suggests that the action of the LHRH-like factor on the Leydig cell is inhibitory, and it may mediate some or all of the negative effects on the Leydig cell of LH and hCG. The precise role of the LHRH-like factor, particularly under physiological conditions (i.e., low level stimulation with LH), remains to be determined, but the availability of methods for its extraction, measurement, and inactivation should enable this problem to be resolved within the near future. Moreover, the recognition that Leydig cell function may be regulated by local factors within the testis should open up new avenues of research that may lead to improved methods for the regulation of fertility in the male.

Aging↗
PubMed1982

Variation in the steroidogenic responsiveness of isolated rat Leydig cells.

Isolated Leydig cells were prepared from adult rat testes by (1) mechanical dissection, (2) collagenase dispersion or (3) mechanical dissection followed by collagenase dispersion, and their functional characteristics were assessed. Compared with Methods 2 and 3, mechanical isolation alone resulted in the purest preparation of Leydig cells but the lowest yield. Leydig cells isolated by any of the 3 methods had similar numbers of LH- and LH-RH receptors, but cells isolated by Methods 1 and 3 showed poor testosterone responsiveness compared to cells isolated by Method 2. This reduced response was evident following stimulation with hCG, dibutyryl cyclic AMP or an LH-RH agonist, and could not be accounted for simply on the grounds of diminished cell viability. It is concluded that Leydig cells in the rat testis are particularly sensitive to mechanical intrusion, and this is an important factor to bear in mind when preparing Leydig cells or when comparing results between laboratories.

Animals↗
PubMed1981

Sertoli-Leydig cell communication via an LHRH-like factor.

The primary function of the testosterone secreted by Leydig cells is the maintenance of spermatogenesis and hence fertility. This action of testosterone is mediated by the Sertoli cells which nourish and support the developing spermatozoa. As normal Sertoli cell function is so critically dependent on normal Leydig cell function, a regulatory influence of the Sertoli cells on the Leydig cells has been suggested. Indeed, follicle-stimulating hormone (FSH), which acts only on Sertoli cells, can also cause profound changes in Leydig cell function, although how this is effected is unknown. We recently hypothesized that the Sertoli cell might be the source of a luteinizing hormone releasing hormone (LHRH)-like factor which we detected in the interstitial fluid surrounding the Leydig cells. As injected LHRH agonists cause impairment of gonadal function and directly inhibit FSH-induced changes in Leydig cell function through specific membrane receptors, this 'LHRH-like' factor has all the correct credentials for the postulated messenger between the Sertoli and Leydig cells. Here, we strengthen this case by demonstrating that seminiferous tubules from both the rat and the stumptailed macaque (Macaca arctoides) contain a factor which has LHRH-like receptor-binding and biological activity in vitro, but which is immunologically distinct from native LHRH. We have also shown that this factor is secreted in vitro by cultured rat Sertoli cells.

Animals↗
PubMed1981

The importance of testicular interstitial fluid in the transport of injected hCG to the Leydig cells.

High levels of hCG occur in testicular interstitial fluid (IF) following injection of the hormone, and the proportion that is taken up by the testis has been assessed by comparing the concentrations in IF of [125I]hCG and [125I]BSA (i.e. an inert protein) in rats treated with the same total dose of hCG. Adult male rats were injected with a total dose of either 0.5 or 45.5 IU hCG and which included either [125I]hCG or [125I]BSA, and were killed between 2 and 40 h later when the concentrations of the labelled proteins in plasma IF and testicular tissue were assessed. Treatment with either dose of hCG increased IF levels in the testis at 4-24 h after injection although this increase was much greater (350% v 61%) with the higher dose of hCG. In rats injected with [125I]hCG, the ratio of radioactivity in testicular tissue to that in IF (T/IF) varied between 0.3 and 2.1 depending on the total injected dose of hCG and the time after injection, whilst in rats injected with the same amounts of unlabelled hCG together with [125I]BSA, the T/IF ratio never exceeded 0.09 and remained constant with time. In the latter group, the IF/plasma ratio of [125I]BSA was always significantly higher than in rats injected with [125I]hCG, irrespective of the total dose of hCG injected. The difference between the IF/plasma ratios for [125I]BSA and [125I]hCG gives a measure of how much of the hCG has been removed by testicular uptake. The results show that following injection of either 0.5 or 45.5 IU hCG, the temporal pattern of testicular hCG-uptake from IF is similar, although proportionately more (50-59%) of the hCG in IF has been taken up within 24 h of injection of the low dose of hCG than with the high dose (32-52%). It is concluded that testicular IF is an important medium for the transport of injected hCG from the blood to the Leydig cells.

Animals↗
PubMed1981

Effect of injected human chorionic gonadotrophin on capillary permeability, extracellular fluid volume and the flow of lymph and blood in the testes of rats.

The subcutaneous injection of human chorionic gonadotrophin (HCG) into adult male rats caused appreciable rises in capillary permeability and lymph flow in the testis, accompanied by smaller rises in the volume of extratubular, extracellular fluid. Most of these changes were already apparent 8 h after injection, but became progressively greater during the next 12 h. Testicular blood flow was unchanged at 12 h but increased slightly between 12 and 6 h after injection. The primary effect is probably the increase in capillary permeability. The timing of these changes suggests that HCG does not affect the capillaries directly, but it would seem that the changes are due to some substances secreted by the testis in response to the HCG. It is clear that these changes will have important influences both on the access to the testicular cells of peptide hormones in the blood and also on the passage into the venous blood of hormones secreted by the testis.

Animals↗
PubMed1980

HCG stimulation of testicular LHRH-like activity.

It is well established that the administration of high doses of luteinizing hormone (LH) or human chorionic gonadotropin (HCG) to animals can impair gonadal function in both the male and female. These effects can be duplicated by the administration of LH releasing hormone (LHRH) or its agonists to intact animals, including man. Somewhat surprisingly, recent results have demonstrated that similar impairment of gonadal function by LHRH and its agonists can be achieved in the absence of the pituitary (for review see ref. 7), and the demonstration of specific, high-affinity binding sites for LHRH and its agonists on ovarian luteal cells and on testicular Leydig cells, could mean that LHRH-like factors are involved in the local regulation of the gonads. A further intriguing finding is the striking similarity between the direct, inhibitory effects of LHRH and LH/HCG on the gonads; for example, both treatments reduce LH receptor numbers and the steroidogenic responsiveness of rat Leydig cells. We now offer a possible explanation for this similarity by demonstrating that HCG treatment stimulates the testicular production of a biologically active LHRH-like factor, suggesting that the inhibitory actions of HCG on the Leydig cell may be mediated by the local production of this factor.

Animals↗
PubMed1980

Differences between dispersed Leydig cells and intact testes in their sensitivity to gonadotrophin-stimulation in vitro after alteration of LH-receptor numbers.

The sensitivity to hCG-stimulation in vitro of intact hemi-testes and collagenase-dispersed Leydig cells has been compared directly following either an hCG-induced loss of LH-receptors or after a hyperprolactinaemia-induced increase in LH-receptors. Injection of hCG 65 h previously, reduced hCG-binding to dispersed Leydig cells by over 84%. The sensitivity of the steroidogenic response of these cells to hCG-stimulation in vitro was reduced 22-fold whereas intact testes from the same animals showed only a 3-fold reduction in sensitivity to hCG. Dispersed Leydig cells from control rats were 8 times more sensitive to hCG-stimulation than intact testes from the same rats, a difference not evident with hCG-injected rats. In contrast, there was no difference between intact testes and dispersed Leydig cells from control and hCG-injected rats in their sensitivity to stimulation with dibutyryl cyclic AMP in vitro. Induction of hyperprolactinaemia increased hCG-binding to dispersed Leydig cells by 55%. The sensitivity of these cells to hCG-stimulation in vitro was increased by a factor of 4.5, a difference not found with intact testes from the same animals. These results show that experimental manipulation of LH-receptor numbers alters the sensitivity of dispersed Leydig cells, but not of the intact testis, to hCG-stimulation in vitro, a difference which appears to reside at the receptor level. Possible explanations for these findings are discussed together with their implications with respect to the distribution of LH-receptors over the Leydig cell surface.

Animals↗
PubMed1980

Inhibition of maturational changes in Leydig cell function by treatment of rats with an agonist of LH-RH.

Daily treatment of immature (30-day-old) male rats for 40 days with 50 ng of an agonist of LH-RH impaired normal development of Leydig cell function. This treatment partly or completely inhibited maturational increases in (1) the serum levels of testosterone, (2) seminal vesicle weight, (3) the in-vitro steroidogenic responsiveness of the testis, and (4) the in-vitro testicular binding of 125I-labelled hCG. In contrast, twice-weekly treatment of immature or adult rats with 50 ng LH-RH agonist had only minor effects on Leydig cell function, although hCG-binding was always significantly reduced. Testicular growth in immature rats was unaffected by daily injection of LH-RH agonist whereas twice-weekly treatment caused a small reduction in weight. None of the treatments had any major consistent effect on the pituitary or serum levels of gonadotrophins and prolactin, although daily treatment with the LH-RH agonist clearly reduced the responsiveness of the pituitary to the agonist, in terms of the amounts of LH and FSH released.

Animals↗
PubMed1980

Effect of adrenalectomy or castration on the inhibition of gonadotrophin secretion induced by hyperprolactinaemia in the adult male rat.

To investigate the role of adrenal and gonadal steroids in the long-term suppression of gonadotrophin secretion induced by prolactin the effects of adrenalectomy or castration on the serum and pituitary levels of LH, FSH and prolactin and the hypothalamic content of LH releasing hormone (LH-RH) have been studied in adult male rats with hyperprolactinaemia produced by the transplantation of pituitary glands under the kidney capsule. Levels of LH and FSH in serum were significantly suppressed in al intact pituitary-grafted rats. Adrenalectomy on the day of pituitary implantation or 20 days later did not affect the suppression. However, castration on days 0, 28 or 49 after pituitary grafting resulted in a rise in levels of FSH in serum indistinguishable from that in control rats. While the rise in levels of LH after castration on day 0 was the same as the controls, this increase was significantly reduced 2 days after castration on days 28 and 49 after pituitary grafting. Castration resulted in an increase in the pituitary content of LH and a reduction in the hypothalamic content of LH-RH but no change in the pituitary content of FSH. Hyperprolactinaemia did not appear to affect these response. The present results showed clearly that the gonad but not the adrenal must be present for prolactin to exert an inhibitory effect on gonadotrophin secretion.

Adrenalectomy↗