A transplantable rat Leydig cell tumour. Properties of the prostaglandin E receptor

In the present paper we have examined the properties of the prostaglandin E (PGE) receptor in a transplantable rat Leydig cell tumour (H-540). It appears that PGE1 and PGE2 share a common receptor in membrane particles from this Leydig cell tumour. From saturation analysis and modified Hofstee plots, the specific binding sites for PGE1 can be divided into a high (25%) and low affinity state (75%) with apparent equilibrium constants of dissociation (Kd) of 2.4.10(-7) mol/l and 4.4.10(-6) mol/l, respectively. Association rate kinetics at different temperatures employing 5.10(-9) mol/l [3H]PGE1 showed that specific binding was time- and temperature-dependent. At 37 degrees C an apparent steady state was achieved after approximately 4 h incubation. The binding of [3H]PGE1 was very tight and no dissociation was observed at 20 degrees C during the first 20 h. The free PGE1 receptor appears to be very unstable. Binding was reduced rapidly by storage at 0 degrees C, by freezing and thawing of membrane particles, and by incubation of concentrated membrane particles. Specificity curves showed that PGA1 and PGA2 displaced [3H]PGE1 from receptor to a somewhat lesser degree than PGE2 and PGE1, whereas PGs of the B, D, I and F series had little or no effect. The fact that inhibition of [3H]PGE1 binding by cold PGE1 occurred in the same concentration range as PGE1 activation of adenylate cyclase, indicates that the specific binding of PGE observed here represents functional receptors coupled to the adenylate cyclase.     

Effect of luteinizing hormone on Leydig cell structure and testosterone secretion

Hypophysectomy or sc implantation of testosterone-estradiol 17 beta (T-E) filled polydimethylsiloxane capsules for 5 days caused a dramatic reduction in testosterone secretion when testes subsequently were perfused in vitro. The diminution in testosterone-secreting capacity of testes from T-E treated rats was coupled closely with reductions in the membrane surface areas of Leydig cell cytoplasmic organelles, particularly those of the smooth endoplasmic reticulum. Simultaneous treatment of T-E implanted rats with LH (12 micrograms/day), but not with FSH, PRL, TSH, or GH, maintained both the Leydig-cell cytoplasmic membranes and the capacity of testes to secrete testosterone in vitro. Testosterone secretion by testes from hypophysectomized rats treated simultaneously with T-E plus LH was identical to that in control rats. Therefore, T-E did not inhibit directly the Leydig cell steroidogenic apparatus. Taken together these results suggest that one of the trophic effects of LH in the Leydig cell is to maintained the integrity of smooth endoplasmic reticulum and enzymes responsible for the conversion of pregnenolone to testosterone.     

Gigantism associated with a pituitary tumour secreting growth hormone and prolactin and cured by transsphenoidal hypophysectomy.

An 18-year old male is reported who presented with a history of a growtn spurt over the year preceding his admission. His height was above the 97th percentile, and he had incompletely developed secondary sexual characters. Pituitary evaluation demonstrated a moderately elevated level of growth hormone (hGH) not suppressible by a glucose load and not stimulable by TRH or by L-DOPA. Serum prolactin (PRL) concentration was also increased while gonadotrophin, thyroid and adrenal function were all subnormal. There was clear radiological evidence of a large pituitary tumour with suprasellar extension and transsphenoidal total hypophysectomy was performed. A mixed chromophobe and acidophilic adenoma was found and both growth hormone and prolactin were demonstrable in different cells of the tumour by the immunoperoxidase technique. Post-operatively the patient has hypopituitarism and levels of growth hormone and prolactin have remained low or undetectable after 6 months. Thus early diagnosis and surgical treatment of gigantism of this mixed hGH-PRL secreting pituitary tumour was associated with a cure, which contrasts with the unfavourable outcome of many of the patients previously reported.     

Transforming growth factor-beta effects on morphology of immature rat Leydig cells

Transforming growth factor-beta (TGF beta) has been shown to regulate steroid production and DNA synthesis in rat Leydig cells. We have investigated the effects of TGF beta on the secretion of extracellular matrix (ECM) proteins and on the cytoskeleton of immature rat Leydig cells in vitro. TGF beta caused significant morphological changes in Leydig cells, which were accompanied by significant increases in secretion of fibronectin, laminin and collagen IV and rearrangement of actin filaments in TGF beta-treated cells. The cells cultured on plates pre-coated with fibronectin or fibronectin plus laminin and collagen IV, displayed morphological and cytoskeletal changes similar to those induced by TGF beta. Immunofluorescence localization studies revealed significantly higher fibronectin staining in Leydig cells in adult animals and in LH-treated immature animals than those in untreated immature animals. We conclude that TGF beta participates in the morphological differentiation of immature Leydig cells into adult Leydig cells in the rat testis by inducing the expression of ECM proteins.     

The effect of castration and testosterone replacement on specific proteins and androgen levels of the rat epididymis.

The normal weight increase of the epididymis during sexual maturation and its maintenance through adulthood were found to be dependent on the provision of androgens. Binding of [3H]dihydrotestosterone (DHT) to the epididymal 8S cytoplasmic receptor gradually decreased after castration to become undetectable after 25 days. Binding to the androgen binding protein (ABP) was absent 4 days after castration and was not reinduced by 3 weeks of testosterone (T) administration. Unilateral castration for periods of up to 27 days showed the disappearance of ABP with preservation of the 8S receptor on the castrated side, indicating a testicular source for ABP and the epididymal origin of the 8S receptor. The tissue concentrations of T and DHT in the epididymis became undetectable 30 days after castration and were restored to normal values by administration of testosterone in large doses (1.5 mg/100 g BW). Similar results were obtained in rats castrated at 10 days of age and injected with testosterone until 60 days old. The ratio DHT/T was depressed in the castrate and increased with testosterone treatment. The protein content of the epididymis (mg of protein/g wet weight) was also found to be influenced by androgens. Our results show evidence of some mechanisms involved in the trophic effect of androgens upon the epididymis and suggest the possible androgenic control of epididymal 5alpha-reductase activity. They also indicate that a testicular factor is required for the maintenance of the 8S cytoplasmic androgen receptor. It is not known whether this factor is testosterone or some other testicular secretion.     

Influence of rat seminiferous tubules on Leydig cell testosterone production in vitro

The interaction of seminiferous tubules and Leydig cells was investigated in the rat in vitro. Crude collagenase-dispersed, or Percoll-purified, Leydig cells were incubated together with seminiferous tubules at different stages of the cycle, isolated by transillumination-assisted microdissection. The testosterone production was measured. Seminiferous tubules inhibited testosterone production of crude Leydig cell preparations, but induced a clear stimulation (30–100%) in Percoll-purified cells. The stimulation was maximal at stages VII and VIII of the cycle, significantly higher than at stages II–VI (P < 0.05). The stimulation by seminiferous tubules was observed both in basal and hCG-stimulated testosterone production. The effect was independent of FSH or GnRH action. These results demonstrate the presence of paracrine regulatory interaction between seminiferous tubules and Leydig cells, and are in agreement with the concept of a perferential androgen requirement of stages VII and VIII of the cycle.     

Effects of castration and testosterone on the pituitary and adrenal responses of the newborn rat to ether inhalation

In 8-day-old rat newborns, the pituitary response to 2 min of ether inhalation was noted to vary according to sex. Plasma ACTH levels were similarly increased in males and females at the end of ether exposure; however, during the following 30 min, ACTH levels were always higher in females than in males. In order to verify that the putative masculinization of some neuroendocrine pathways involved in the pituitary response to ether stress was the result of the transitory surge of testosterone at birth, fetuses at term were delivered by cesarean section and thereafter immediately castrated or sham-castrated under cold anesthesia (males), injected with testosterone heptylate (1 mg s.c.) or olive oil used as solvent (females) before being put in the care of a nurse. The rise in plasma testosterone levels during the 1st h after birth was prevented or stopped in males put at 2 degrees C. At the 8th postnatal day, the newborns were subjected to 2 min of ether inhalation; they were sacrificed either just, before or after the end (0 and 30 min) of the stress procedure. Plasma immunoreactive ACTH level and adrenal corticosterone content were measured. The pituitary response, shown by the ACTH increase, in castrated or sham-castrated males and testosterone-injected females was similar to that of intact males but very different from that observed in olive-oil-injected or intact females. The rise in adrenal corticosterone content 30 min after ether inhalation was greater in intact and olive-oil-injected females than in testosterone-injected ones or in males; adrenal response was well correlated with the maintenance of ACTH release in the former and the decrease following transitory surge in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoreactive alpha-MSH and ACTH levels in rat plasma and pituitary.

A radioimmunoassay is described for the measurement of alpha-melanocyte-stimulating hormone (alpha-MSH). The antibody was produced in rabbits by immunization with alpha-MSH coupled to bovine serum albumin with carbodiimide. The antibody did not react significantly with ACTH, beta-MSH, or 6 fragments of ACTH. The sensitivity and reliability of the assay were improved by employing a simple plasma extraction procedure. When applied to a 2 ml plasma sample, the detection limit of the radioimmunoassay was 6 pg/ml. ACTH was measured with a sensitive and specific radioimmunoassay previously described for humans and adapted for the rat. The anti-ACTH serum cross-reacted with the biologically active portion of alpha-p ACTH and not with alpha-MSH, beta-MSH or the alpha-p 17-39 and alpha-p 25-39 fragments of ACTH. The detection limit was 20 pg/ml. Plasma and pituitary alpha-MSH and ACTH had the same immunoreactivity as synthetic alpha-MSH and ACTH. alpha-MSH and ACTH contents of the rat neurointermediate lobe were 1398 +/- 360 (SE) ng and 28.2 +/- 2.9 ng, respectively, while in the anterior lobe they were 102 +/- 31 ng and 551 +/- 36 ng, respectively. The plasma alpha-MSH concentration at 8 AM in male rats was 64 +/- 8 pg/ml when the plasma ACTH concentration was 92 +/- 15 pg/ml. Over a 24-hour period two peaks of plasma alpha-MSH were observed, one at 4 AM (142 +/- 35 pg/ml) and the other at 4 PM (139 +/- 26 pg/ml). Plasma ACTH was higher at noon (151 +/- 43 pg/ml) and 4 PM (130 +/- 48 pg/ml). Short-term exposure to ether induced a transient increase in alpha-MSH level 5 min later and a rapid return to normal levels. Plasma ACTH increased significantly 2.5 min after the onset of ether stress and remained high for 30 min. Two hours' exposure to ether did not change plasma alpha-MSH, although a 3-fold increase in plasma ACTH was observed. Haloperidol injection was followed by a large increase in plasma alpha-MSH, whereas ACTH levels increased similarly after saline and Haloperidol injection. Corticoid administration reduced ACTH, but not alpha-MSH. Three weeks after adrenalectomy, alpha-MSH levels had not changed but ACTH levels had increased ten-fold. These data indicate that alpha-MSH is secreted in the rat, and that the regulation of its secretion is different from that of ACTH.     

Effect of aldrin on spermatogenesis, plasma gonadotrophins and testosterone, and testicular testosterone in the rat

Quantitative evaluation of the different varieties of germ cells at stage VII of the seminiferous epithelium cycle, namely type-A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7Sd), along with radioimmunoassay of plasma gonadotrophins (FSH and LH), testosterone and testicular testosterone were performed in Wistar rats following treatment with aldrin (polycyclic chlorinated hydrocarbon insecticide) for approximately one (13 days) or two cycles (26 days) of the seminiferous epithelium. Extensive degeneration of all varieties of germ cells at stage VII, reduction in the sperm count and significant reductions in plasma concentrations of LH and testosterone were observed following aldrin treatment. The reduction in plasma concentrations of FSH was statistically significant only after treatment for two cycles. The inhibitory effect of aldrin on plasma gonadotrophins, testosterone levels, testicular testosterone content and numbers of 7Sd and ASg was maximum after treatment for two cycles. Administration of human chorionic gonadotrophin along with aldrin treatment for two cycles partially prevented the degeneration of germ cells and enhanced testosterone production. The results indicate that aldrin may have a direct inhibitory influence on gonadotrophin release, but the possibility of a direct action of the insecticide at the level of the testes is also discussed.     

Effect of warfarin on cell kinetics, epithelial morphology and tumour incidence in induced colorectal cancer in the rat.

The effect of low dose warfarin and high dose warfarin on epithelial cell kinetics (as determined by stathmokinetic techniques), and preneoplastic morphological changes was studied during azoxymethane induced carcinogenesis in the rat. Warfarin, at either low or high dose, had no effect on crypt cell production rate (CCPR) at any time interval whereas tumour incidence in both low dose warfarin and high dose warfarin groups was significantly reduced. Morphological changes were observed using scanning electron microscopy, which by conventional histology were shown to be adenoma precursors. In the control group the number of microadenomas increased with time after starting azoxymethane. In warfarin treated animals, the number of microadenomas also increased with time, but the actual incidence was reduced when compared with controls. These results suggest that the effects of warfarin on tumour development is unrelated to its anticoagulant effect, because increased dose did not result in greater tumour reduction. Furthermore, there was no overall change in CCPR when warfarin was administered. Warfarin may exert a specific effect, by preventing neoplastic change in cells which have undergone morphologically undetectable changes associated with early carcinogenesis.     

Effect of intraventricular injection of 5,6-dihydroxytryptamine on spermatogenesis and plasma testosterone levels in the rat

Quantitative evaluation of spermatogenesis at stage VII of the cycle of the seminiferous epithelium and radioimmunoassay of plasma testosterone were performed in adult Wistar rats after intraventricular injection of 5,6-dihydroxytryptamine (5,6-DHT). The rats were killed 2, 10 and 21 days after injection. Brain 5-hydroxytryptamine (5-HT) and plasma testosterone levels were found to be significantly lower in all rats treated with 5,6-DHT. A significant reduction in step 7 spermatid count was also observed after 10 and 21 days. Supplementation with human chorionic gonadotrophin for 21 days in rats injected with 5,6-DHT partially prevented the step 7 spermatid degeneration and increased testosterone levels without producing any effect on brain concentrations of 5-HT. These results suggest that changes in testicular steroidogenesis and spermatogenesis are secondary to pituitary gonadotrophin release which, in turn, is under the influence of brain 5-HT neurones.