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Biomedical subjects

R M Sharpe

Publications and source records attributed to R M Sharpe.

At least 109 records · Page 6Linked to original sources
PubMed1989

Macrophage activation enhances the human chorionic gonadotrophin-induced disruption of spermatogenesis in the rat.

The objective of this study was to examine the role of intertubular macrophages in modifying the response of the rat testis to stimulation by human chorionic gonadotrophin (hCG). The phagocytic activity of macrophages was stimulated by a unilateral intratesticular injection of polystyrene latex beads. Latex beads were engulfed by the resident macrophages and retained within their cytoplasm. Contralateral testes received injection of vehicle alone. A group of control rats was killed 3 days later; other groups received 100 IU hCG s.c. and the morphological and functional responses of the testes were examined 12, 24 and 48 h later. Spermatogenesis was unaffected in control rats, whereas in the testes of all hCG-treated rats leukocytes infiltrated into the intertubular tissue and the seminiferous tubules exhibited focal disruptions of spermatogenesis which were more severe in testes containing activated macrophages. Spermatogenic disruption was dependent upon the stage of the spermatogenic cycle, with the maximum tubule degeneration occurring at or near stages III and IX-XI. However these changes were not a consequence of androgen deprivation, since no consistent correlation was demonstrated between alterations in testosterone levels in testicular interstitial fluid and the accompanying damage to germ cells. It is concluded that hCG alone or in combination with activated macrophages induces an inflammatory-type response of the intertubular tissue and localized degeneration of the seminiferous epithelium. The antispermatogenic effects of hCG may have important implications for in-vivo investigations of Leydig cell function in laboratory animals and for the efficacy of hCG administration used in the clinical treatment of male hypogonadism.

Animals↗
PubMed1989

Dynamics of testosterone secretion by the rat testis: implications for measurement of the intratesticular levels of testosterone.

Testosterone concentrations have been measured in testicular interstitial fluid (IF), and in blood plasma sampled from various parts of the rat testis and spermatic cord, to assess (1) the most accurate method for determination of the intratesticular levels of testosterone, and (2) the route of secretion of testosterone from the testis. In untreated adult rats, testosterone concentrations were highest in blood collected from veins on the surface of the testis (269.50 +/- 30.63 (S.E.M.) nmol/l), but were reduced by 56% on average in blood collected from veins at the proximal end of the spermatic cord (123.06 +/- 24.75 nmol/l), and were reduced considerably in peripheral venous blood (4.55 +/- 0.55 nmol/l). Similar changes occurred in adult rats in which steroidogenesis was either stimulated (by treatment with human chorionic gonadotrophin; hCG) or inhibited (by treatment with aminoglutethimide; AMG), and in rats of various ages during sexual maturation. The reduction in testosterone levels during passage of blood from the testis up the spermatic cord is probably due mainly to dilution by incoming arterial blood which transfers to venous blood via anastomoses in the spermatic cord. Venous-arterial transfer of testosterone in the cord contributed to this in only a minor way. Concentrations of testosterone in testicular IF were always greater than testicular venous concentrations in control, developing and hCG-stimulated rats, but were comparable in rats treated with AMG to suppress Leydig cell steroidogenesis. These and other results demonstrate that the method of drip-collection of IF results in over-estimation of the actual intratesticular levels of testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Evidence for a role of the Leydig cells in control of the intratesticular secretion of inhibin.

Injection of adult male rats with human chorionic gonadotrophin (hCG) caused a dose- and time-dependent increase in the levels of immunoactive inhibin in testicular interstitial fluid (IF), which differed from the pattern of change in testosterone levels. Blockage of the hCG-induced increase in IF levels of testosterone, by administration of aminoglutethimide, only partially attenuated the increase in levels of inhibin. Inhibin levels in IF were only increased by doses of hCG which cause inflammatory changes and focal seminiferous tubule damage, but there was no association between the degree of tubule damage and inhibin levels. It is concluded that one or more luteinizing hormone (LH)-regulated, non-steroidogenic Leydig cell products may be involved in the paracrine control of inhibin secretion. These data are of clinical relevance and of potential physiological significance.

Aminoglutethimide↗
PubMed1988

Assessment of the role of Leydig cell products other than testosterone in spermatogenesis and fertility in adult rats.

Adult male rats were treated with ethane dimethanesulphonate (EDS) to destroy the Leydig cells and were then supplemented for 3-10 weeks with testosterone esters (TE) by injection every 3 days. The latter treatment prevented Leydig cell regeneration but maintained quantitatively the androgen-dependent aspects of spermatogenesis, as judged by germ cell counts at stage VII of the spermatogenic cycle. Other than the absence of Leydig cells, the testes of EDS-treated, TE-supplemented rats showed only two morphological changes, (1) the appearance of mast cells throughout the interstitium, and (2) a 3- to 4-fold increase in the number of degenerating germ cells (secondary spermatocytes) at stages XIV-I; this was reflected in a significant decrease in the ratio of spermatids to pachytene spermatocytes at stage VII. These changes were not observed in either oil-treated or TE-treated control rats although similar, but less marked, changes in cell degeneration at stages XIV-I were observed in rats actively immunized against oxytocin. Epididymal sperm number was reduced marginally (approximately 15%) in EDS-treated, TE-supplemented rats while sperm motility was affected even less. In a serial mating trial, some of these treated rats showed evidence of subfertility/infertility, but this was mostly transient and may have been the result of epididymal effects of EDS. These results suggest that Leydig cell products other than testosterone are not essential for maintenance of spermatogenesis and fertility in rats, although because of increased germ cell degeneration during the final stages of meiosis (perhaps as the result of oxytocin withdrawal), a small reduction in sperm count may occur.

Animals↗
PubMed1988

Re-evaluation of the intratesticular level of testosterone required for quantitative maintenance of spermatogenesis in the rat.

The amount of testosterone required for quantitative maintenance of spermatogenesis has been re-evaluated using techniques aimed at minimizing the synthesis of testosterone after removal of the testis. Adult male rats were treated with ethane dimethane-sulphonate (EDS) to destroy the Leydig cells, and were supplemented with 25, 5 or 1 mg testosterone esters by injection every 3 days for 21 days. Serum hormone levels, testicular morphology and spermatogenesis were assessed and the intratesticular levels of testosterone compared in testes either removed under ether anaesthesia and placed in liquid nitrogen (right testis) or removed after collection of blood and placed in ice (left testis). Data for testosterone-supplemented rats were compared with those for control rats and rats treated with EDS alone. All doses of testosterone suppressed LH and FSH levels in serum to within the hypophysectomized range, and Leydig cell regeneration in EDS-treated rats was prevented completely. Treatment of EDS-injected rats with 25 or 5 mg testosterone maintained testicular weight, the number of germ cells and the diameter of seminiferous tubules at stage VII within or above the control range, although there was a significant increase in the number of degenerating pachytene spermatocytes at stage VII with 5 but not 25 mg testosterone; none of these parameters was maintained at control levels by a dose of 1 mg testosterone. Levels of testosterone in testosterone-supplemented rats differed little between testes collected in ice and liquid nitrogen, but in controls and rats treated with EDS alone, testosterone levels were overestimated by 75 and 27% respectively when comparing testes collected in ice with those collected in liquid nitrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Factors affecting the secretion of immunoactive inhibin into testicular interstitial fluid in rats.

Immunoreactive inhibin was measured in testicular interstitial fluid (IF) from rats during sexual maturation or after impairment of spermatogenesis induced by ethane dimethanesulphonate (EDS), unilateral cryptorchidism or local heating (43 degrees C, 30 min) of the testes, to ascertain its usefulness as a marker of changing Sertoli cell function. Cultures of isolated seminiferous tubules were also studied. Inhibin was measured by a radioimmunoassay directed towards the first 26 amino acids of the N-terminus of the alpha-subunit, and the results confirmed for selected pools of IF by in-vitro bioassay using dispersed ovine pituitary cells. During puberty, IF levels of immunoactive inhibin fell by more than 90% (P less than 0.001) between 30 and 60 days of age, a decrease paralleled by the levels of androgen-binding protein (ABP), another Sertoli cell product secreted into IF. These changes also paralleled, but preceded, the fall (60%; P less than 0.001) in serum levels of FSH between 40 and 70 days, while the serum and IF levels of testosterone increased more than two-fold over this period. When adult rats were injected with EDS to destroy the Leydig cells, testosterone levels in IF and serum were undetectable at 3 and 7 days after treatment, were just detectable at 14 days and thereafter returned slowly towards normal by 42 days. The initial androgen withdrawal following EDS treatment caused a progressive reduction in testicular weight up to 21 days and this was accompanied by a significant increase in the serum levels of FSH and a two- to threefold increase in the IF levels of immunoactive inhibin (and also of ABP). Serum FSH and IF levels of immunoactive inhibin returned to within the normal range by 42 days when testosterone levels had normalized. In contrast, in two other experimental situations in which a marked decrease in testicular weight coupled with an increase in IF levels of ABP occurs, different results for the IF levels of immunoactive inhibin were obtained. Thus, in rats exposed to local heating of the testes, IF levels of immunoactive inhibin remained unchanged from control values at 21-40 days after treatment, a finding confirmed by bioassay results. In rats made unilaterally cryptorchid for 10 months, levels of immunoactive inhibin in IF were reduced by 60% (P less than 0.01) in the abdominal compared with the contralateral scrotal testis.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1987

Origin of regenerating Leydig cells in the testis of the adult rat. An ultrastructural, morphometric and hormonal assay study.

Ethane dimethanesulphonate (EDS) was used as a specific cytotoxin to eliminate the Leydig cell population of the adult rat testis. Ultrastructural, morphometric and serum gonadotrophin and testosterone analysis was used to study the response of the intertubular tissue of the testis from 1 day to 10 weeks after EDS treatment. In control animals, the testis contained approximately 28 million Leydig cells and 8 million macrophages. Three to seven days after EDS treatment, Leydig cells were absent and serum testosterone was undetectable. Macrophage numbers increased three-fold by 3 days and returned to pretreatment values thereafter. At 2 and 3 weeks post-EDS, foetal-type Leydig cells (approximately 1-2 million per testis) appeared in proximity to perivascular and peritubular tissues, a feature also observed at 4 weeks when numerous such cells (approximately 15 million per testis) formed prominent clusters in perivascular and peritubular locations. Between 6 and 10 weeks after EDS treatment, the foetal-type Leydig cells were transformed morphologically into adult-type Leydig cells, they occupied central intertubular positions and their numbers were restored to pretreatment values. Regeneration of Leydig cells was reflected by elevated serum testosterone levels which returned towards the normal range. The results demonstrate the regenerative capacity of the testicular intertubular tissue and indicate a dual site of origin of Leydig cells which initially resemble foetal-type Leydig cells prior to establishing the adult-type Leydig cell population. The morphological pattern of Leydig cell regeneration suggests that in addition to gonadotrophic stimulation, local testicular factors from the seminiferous tubules may stimulate Leydig cell growth.

Animals↗
PubMed1987

Relationship between the exposure of Leydig cells to factor(s) present in testicular interstitial fluid and changes in their capacity to secrete testosterone during culture or after hCG-induced desensitization.

This study has evaluated whether the decrease in capacity of Leydig cells to secrete testosterone that occurs during culture or after desensitization with hCG in vivo, is a consequence of the removal of a stimulatory factor(s) in testicular interstitial fluid (IF) to which Leydig cells are normally exposed. When Percoll-purified rat Leydig cells were cultured for 3 days in vitro, there was a progressive reduction in their ability to respond to hCG in terms of either testosterone or progesterone production. In contrast, culture of the cells in the presence of 10% charcoal-stripped IF maintained responsiveness to hCG either partially or completely. This effect was attributable to a factor(s) in IF with a molecular weight of greater than 30 kDa; a comparable fraction from serum had little or no effect. Crude Leydig cells from rats injected 24 h previously with 50 IU hCG showed a 70% reduction in their testosterone response to hCG in vitro and, compared to controls, increased testosterone production poorly in response to increasing concentrations of IF. However, progesterone secretion was increased considerably in response to IF. As in controls, fractionation of crude Leydig cells from hCG-desensitized rats on Percoll gradients resulted in three bands of Leydig cells, except that the yields of band 2 and 3 cells (containing about 40 and 85% Leydig cells, respectively) were reduced by 75% and 90%, respectively, with the majority of Leydig cells remaining in band 1 (which comprises poorly responsive cells). Band 2 and 3 cells from hCG-desensitized rats were not greatly different to cells from control rats in terms of their testosterone response to hCG +/- IF, although they produced considerably more progesterone. It is concluded that the reduced capacity of Leydig cells to secrete testosterone during culture may be attributable to some extent to the removal of a factor(s) in IF to which the cells are normally exposed. In contrast, the reduced in vivo exposure of Leydig cells to such factors, as occurs after hCG injection, cannot explain the poor testosterone response of these cells when they are isolated and cultured.

Animals↗
PubMed1987

Enhancement of Leydig cell testosterone secretion by isolated seminiferous tubules during co-perifusion in vitro: comparison with static co-culture systems.

The aim of this study was to identify an in-vitro test system for the reproducible demonstration of a modulatory effect of isolated seminiferous tubules (s-tubules) on testosterone production by purified rat Leydig cells. Co-incubation of s-tubules with various numbers of Leydig cells had no significant effect on basal and hCG-stimulated testosterone production over 4-24 h incubation. In contrast, addition of s-tubule conditioned medium (STCM) to Leydig cells enhanced both basal and hCG-stimulated testosterone production over 5 h, but this effect was variable in magnitude and was not completely reproducible. Co-perifusion of isolated s-tubules with Percoll-purified Leydig cells for 6 h produced significant and consistent increases in Leydig cell testosterone secretion compared with Leydig cells perifused on their own. In six experiments, s-tubules enhanced Leydig cell testosterone secretion by 26 +/- 5% (P less than 0.001) in the absence of LH stimulation and by 48 +/- 11% following pulsatile stimulation with 1 ng/ml ovine LH (oLH). The presence of s-tubules enhanced (P less than 0.01-0.001) testosterone secretion by Leydig cells in response to pulses of oLH at doses ranging from 0.1 to 10 ng/ml, but the magnitude of enhancement was greatest with 0.1 and 1 ng/ml doses. These stimulatory effects were not explained by Leydig cell contamination or by testosterone leakage from the isolated s-tubules. Co-perifusion of Leydig cells with isolated epididymal tubules as a control tissue had no significant effect on LH-stimulated Leydig cell testosterone production. Stimulatory effects of s-tubules on Leydig cell testosterone secretion were observed at a 'physiological' ratio of s-tubules to Leydig cells (200 cm tubules/3 million cells) and was mediated by a humoural agent(s), since perifusion of s-tubules and Leydig cells in series gave similar results to co-perifusion of these tissues. This system proved to be robust and, in contrast to static culture systems, gave highly reproducible results, which should allow detailed investigation of the dynamic interactions between s-tubules and Leydig cells and the hormonal control of these events.

Animals↗
PubMed1987

Changes in the secretion of ABP into testicular interstitial fluid with age and in situations of impaired spermatogenesis.

Androgen-binding protein (ABP) was measured in serum and testicular interstitial fluid (IF) from rats during sexual maturation or in adult rats in which impairment of spermatogenesis had been induced by (i) testosterone withdrawal following Leydig cell destruction, (ii) local heating (43 degrees C) of the testes for 30 min or (iii) induction of unilateral cryptorchidism (UCD). The changes observed were related to the IF levels of testosterone and, in most instances, to the serum levels of FSH. The levels of ABP in serum and IF decreased together with age, being highest at 30 days, falling steeply by 40 days and then slowly but progressively up to 100 days of age. A similar pattern was observed for serum FSH, except that the initial fall occurred beyond 40 days of age. Treatment with EDS or exposure to local heating caused comparable reductions in testicular weight (25-30% by 7 days after treatment, 50% by 21-28 days) and raised the serum levels of FSH. In both groups the levels of ABP in IF were increased by two- to three-fold while the levels of testosterone were either reduced markedly (EDS-treatment) or remained unchanged (local heating). In rats made UCD for 60 days, the weight of the abdominal testis was reduced by 75%, compared with the contralateral scrotal testis, while the IF levels of ABP and testosterone were significantly increased (55%) and decreased (90%), respectively. Short-term (3 days) deprivation of testosterone in adult rats, following immunoneutralization of LH, was without significant effect on IF levels of ABP. It is concluded that ABP secretion into IF is increased in situations of subnormal (or sub-adult) numbers of germ cells and this is usually associated with high levels of FSH. Measurement of ABP levels in IF should prove of value for the monitoring of Sertoli cell function in vivo and may be of diagnostic use for the detection of changes in germ cell numbers.

Aging↗
PubMed1987

Isolation of human Leydig cells which are highly responsive to human chorionic gonadotropin.

Leydig cells were purified on discontinuous Percoll gradients after collagenase digestion of human or rat testes. Leydig cells from both species were found in three bands. As determined by positive staining for 3 beta-hydroxysteroid dehydrogenase, band 1 (lowest density cells) from both species contained only 12-28% Leydig cells. However, while band 3 was the most Leydig cell-enriched fraction in rat cell preparations (70-90% Leydig cells), human band 2 (48-70% Leydig cells) was consistently more Leydig cell enriched than was band 3 (30-56% Leydig cells). Despite their slightly different fractionation pattern, Leydig cells prepared from five men with prostatic carcinoma were similar to those from the rat, both in terms of the amount of testosterone produced basally per 10(6) Leydig cells (80-234 ng/20 h) and in terms of the magnitude of their response to hCG (764-1342 ng/10(6) Leydig cells X 20 h; 5- to 17-fold stimulation above basal). Cells prepared from five other men with prostatic carcinoma produced much lower amounts of testosterone, but still had up to a 6-fold response to hCG. Plasma LH, FSH, and testosterone concentrations in the latter group could not be distinguished from those in the group whose Leydig cells produced large amounts of testosterone in vitro. Morphologically, the testes from the latter group appeared to contain more darkly staining than lightly staining Leydig cells than did the former group. Rat Leydig cells responded in a dose-dependent fashion to hCG over the range 0.03-0.5 mU/mL, whereas human Leydig cells were 10- to 100-fold less sensitive, responding to hCG in the range 0.4-100 mU/mL. The number and affinity of Leydig cell LH (hCG) receptors were assessed from Scatchard analysis of [125I]hCG binding. Compared with rat cells, human Leydig cells contained approximately 20% of the number of LH receptors, while the affinity of the receptors (Kd, approximately 10(-10) M) was similar to that in rats. In conclusion, a method for the isolation of highly responsive human Leydig cells has been developed. The results so far suggest that the function of human Leydig cells may be more similar to that of the rat than thought previously.

Aged↗
PubMed1987

Effect of local heating of the rat testis on the levels in interstitial fluid of a putative paracrine regulator of the Leydig cells and its relationship to changes in Sertoli cell secretory function.

Rat testes were exposed to heat (43 degrees C) for 15 or 30 min to induce moderate or severe disruption of spermatogenesis, respectively. Over 3-42 days after treatment, testicular morphology and weight, the serum concentrations of FSH and the concentrations in interstitial fluid of testosterone, androgen-binding protein (ABP) and a factor(s) capable of stimulating Leydig cell testosterone secretion were monitored. Moderate seminiferous tubule damage induced by 15 min heat exposure caused a small decrease (20%) in testicular weight, but did not affect the other measures, other than transiently. In contrast, after exposure of testes to heat for 30 min there was a major and progressive decline in testicular weight throughout the experimental period, reaching 39% of control values by 42 days. In these animals, the serum concentrations of FSH were significantly increased (P less than 0.01) throughout the period of study as also where the serum and interstitial fluid concentrations ABP (P less than 0.05-0.01) and levels of interstitial fluid factor (P less than 0.01). It is concluded that the activity of the interstitial fluid factor(s) can be increased by inducing severe but selective disruption of spermatogenesis, whereas moderate disruption has no effect. Moreover, as ABP secretion into interstitial fluid was increased after severe but not moderate disruption, this suggests that in such animals proportionately more ABP may be secreted via the base of the Sertoli cell. The parallel changes in activity of the interstitial fluid factor(s) and concentrations of ABP in interstitial fluid also provides further circumstantial evidence that these products may have a common (Sertoli cell) origin.

Androgen-Binding Protein↗
PubMed1987

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

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Macrophages in the interstitial tissue of the rat testis.

Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3 beta-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3 beta-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12-14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Paracrine control of the testis.

The mammalian testis is under the overall control of pituitary gonadotropins but the utilization of these signals to achieve normal testicular function involves complex local interactions between the Sertoli and germ cells, the Sertoli and peritubular cells, and the Sertoli and Leydig cells as well as local control of the testicular vasculature. These interactions serve two purposes: (1) to coordinate the functions of the three testicular compartments (seminiferous tubules, interstitium and the vasculature); and (2) to control the complex but orderly sequence of events that constitutes the spermatogenic cycle. This process, which involves multiplication, differentiation and translocation of the germ cells is organized into a sequence of stages, each of which is composed of a constant association of germ cells at four or five different stages of development. At each stage of the spermatogenic cycle, different events occur and the function of the Sertoli cells alters, probably in accordance with the changing requirements of the associated germ cells. As yet, our understanding of these many local events is extremely limited, particularly with respect to the identity of the hormones/factors involved in controlling the various processes. Our knowledge of paracrine control mechanisms in the testis is derived mainly from studies of the rat, but as the process of spermatogenesis is essentially the same in most mammals and involves the same sequence of events, then findings in the rat can probably be applied in general, if not in detail, to the human testis; the limited direct information available on the human testis supports this view. As most cases of infertility in men occur despite normal or raised serum gonadotropin levels and are characterized by the production of reduced or normal numbers of sperm, then it seems likely that malfunction of one or more of the intricate paracrine processes within the testis may be involved in the aetiology of idiopathic oligospermia. It is therefore argued that advances in our knowledge of the paracrine control of the testis should have major repercussions on our ability to understand, and eventually treat, idiopathic infertility in men, and also to induce infertility for contraceptive purposes.

Animals↗