Summary. 'Interstitial fluid' containing high levels of testosterone (60–250 ng/ml) was recovered from the testes of rats, the amounts increasing with increase in age and testis weight. Injection of 170 i.u. hCG/kg resulted 20 h later in significant increases in interstitial fluid and its testosterone content (300–800 ng/ml). In immature rats this effect of hCG was dose-dependent and time-related and the accumulated fluid contained high levels of potassium and phosphate; levels of sodium, calcium and protein were similar to those in serum. At 20 h after injection of hCG, other testicular changes were (1) increased 'adhesiveness', (2) reduced in-vitro binding of 125I-labelled hCG, and (3) an hCG-induced increase in the testis: blood ratio of hCG in vivo.
R. M. Sharpe
Summary. The specific testicular uptake in vivo of 125I-labelled hCG was compared in control adult rats and adult rats made bilaterally cryptorchid 5 weeks previously. Although a similar temporal pattern of uptake was observed in both groups, uptake of hCG by cryptorchid testes was reduced at all times after injection by up to 70%. The possible causes of this impairment were investigated. It could not be accounted for by differences in the rate of absorption or clearance of 125I-labelled hCG in the two groups. Therefore, because hCG-induced increase in the permeability of testicular capillaries is a crucial factor in determining hCG uptake by the testis, this change was compared in control and cryptorchid testes. Although hCG induced a characteristic increase in testicular capillary wall permeability in both groups, this change was temporally delayed in cryptorchid testes, and occurred after hCG values in the blood had fallen. Even when hCG had crossed the capillary wall into testicular interstitial fluid, its uptake into the testicular tissue was significantly lower in cryptorchid than in control testes. These changes probably account for the impairment of gonadotrophin uptake by the cryptorchid testis and have important implications with respect to the aetiology of Leydig cell changes in cryptorchidism.
R. M. Sharpe
It is now firmly established that LH-RH and its agonists can have direct inhibitory effects on gonadal function in male and female animals. The initial reaction to such findings was one of incredulity because they challenged one of the accepted 'truths' of endocrinology, namely that LH-RH was secreted only by hypothalamic neurones and acted only on the anterior pituitary. However, LH-RH, like other hypothalamic peptides (e.g. thyrotrophin-releasing hormone, somatostatin), is quite widely distributed throughout the body. Current evidence suggests that LH-RH-like peptides occur in spinal ganglia in the frog (Jan, Jan & Kuffler, 1979; Jan, Jan & Brownfield, 1980) and, in mammals, in the pineal gland (Wheaton, 1980), pancreas (Seppala, Wahlstrom & Leppaluoto, 1979; Wahlstrom & Seppala, 1979; Seppala & Wahlstrom, 1980a), certain human mammary tumours (Seppala & Wahlstrom, 1980b), the placenta (e.g. Khodr & Siler-Khodr, 1980; Lee, Seppala & Chard, 1981), ovary (Ying, Ling, Bohlen & Guillemin, 1981) and
R. M. Sharpe and I. Cooper
Summary. 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.
R. M. Sharpe and Irene Cooper
Summary. 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 < 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 μg 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.
R. M. Sharpe and H. M. Fraser
Summary. 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 of125I-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.
R. M. Sharpe and J. M. S. Bartlett
Summary. Methods have been established and validated for quantitative assessment of the distribution of testosterone in the testis, by measurement of testosterone concentrations in whole testis, in isolated seminiferous tubules and in testicular interstitial fluid. These measurements were made in individual rats injected 2–40 h previously with saline (0·9% NaCl) or a potent antiserum to ovine LH. Testosterone concentrations in interstitial fluid and seminiferous tubules were closely correlated (r = +0·98; n = 60) and their relationship was log linear over a 200-fold range. However, although the concentrations of testosterone in interstitial fluid and seminiferous tubules decreased progressively with time after LH antiserum injection, this decrease was far more pronounced for interstitial fluid. In association with this change there was a significant increase in the amounts of a locally-produced factor in interstitial fluid which stimulates basal and hCG-stimulated testosterone production by isolated purified Leydig cells. This increase was reversed by injection of hCG but not by peripheral injection of a dose (20 mg) of testosterone propionate which restored normal intratesticular concentrations of testosterone. It is concluded that the tubular 'conservation' of testosterone, which occurs as interstitial fluid levels of this steroid decrease, may be a consequence of restricted diffusion of testosterone out of the tubules, but is also associated with increased amounts of a peptide stimulator of testosterone production.
J. M. S. Bartlett and R. M. Sharpe
Summary. Rat testes were exposed to heat (43°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 < 0·01) throughout the period of study as also where the serum and interstitial fluid concentrations ABP (P < 0·05–0·01) and levels of interstitial fluid factor (P < 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.
J. B. Kerr and R. M. Sharpe
Summary. The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 μg LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 μg LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.
S. A. R. CHOUDHURY, R. M. SHARPE, and P. S. BROWN
Injection of pimozide reduced compensatory ovarian hypertrophy and raised pituitary LH content in hemispayed cyclic and androgenized female rats. Pimozide inhibited spontaneous and induced ovulation and markedly reduced the rise in serum LH during the afternoon of pro-oestrus. Pimozide failed to reduce serum LH in male rats and caused no apparent delay in sexual maturation in males or females. The results suggest that the drug suppresses the ovulatory release of LH, presumably by antagonizing hypothalamic dopamine. The drug appears to have less striking effects on the tonic secretion of FSH and LH.