ABSTRACT

Weekly doses of the Leydig cell cytotoxic ethylene dimethanesulphonate (EDS) were administered to adult male rats in an attempt to study the endocrine activity of the testis in the absence of Leydig cells. One week after the first dose serum testosterone and LH concentrations and seminal vesicle weights were close to levels in castrated rats and testicular human chorionic gonadotrophin (hCG) binding was severely depressed. These changes were maintained for a further week but subsequently began to return to, but did not achieve, control levels. After six weekly doses seminal vesicle weight and serum testosterone concentrations were significantly higher than in the castrated rats. Serum LH concentrations were declining towards control values at 4 weeks but had risen again at 6 weeks. Serum FSH concentrations were raised to about 50% of the value in castrated rats throughout the period studied. Testis weight and hCG binding, which initially fell, were partially restored at 6 weeks and spermatogenesis was recovering. The data show that responses of the testis to multiple doses of EDS are similar to those after a single dose. This apparent resistance indicates that the regenerating Leydig cells are functionally different from the mature Leydig cell. The similarities between the maturing Leydig cell seen after EDS destruction and those in the immature rat suggest that EDS will provide a valuable model for the investigation of Leydig cell physiology.

J. Endocr. (1985) 105, 311–316

SUMMARY

The oestrogen specific, high-affinity cytosol receptor (HAR) from amygdala, anterior, middle and posterior hypothalamus, pituitary and uterus was studied in the ovariectomized rat. A single in-vivo injection of oestradiol-17β produced significant changes in both the tissue HAR concentrations and the apparent dissociation constants (K_d ) determined in vitro. Four hours after oestradiol-17β treatment (20 μg/kg), the HAR concentration was depleted in all tissues except the posterior hypothalamus. A lower dose of oestradiol-17β (4 μg/kg) produced similar changes in HAR concentration with the exception of those in the amygdala and posterior hypothalamus. Twenty-four hours after oestradiol-17β, HAR concentrations had returned to pre-injection levels in all tissues except the uterus. The uterine HAR concentrations were raised after both doses of oestradiol-17β. The apparent tissue cytosol K_d values were decreased by both doses of oestradiol-17β. The results suggest that brain, pituitary and uterine oestrogen cytosol HARs react to plasma oestrogen in a manner predictable by the steroid receptor hypothesis. The oestradiol-17β-induced differential effects upon the tissue cytosol concentration may contribute to the overall spectrum of action of oestrogen in the central and peripheral reproductive processes.

ABSTRACT

Insulin-like growth factor-I (IGF-I) peptide, receptors and binding proteins are present in the rodent testis, which strongly implies that IGF-I has one or more testicular functions. In the present study we provide further information to support the concept that IGF-I is an important local mediator in the testis. High concentrations of IGF-I were measurable in interstitial fluid by radioimmunoassay, and IGF-I-binding proteins (IGFBPs) were readily detectable in interstitial fluid by ligand blotting, the predominant type being IGFBP-2. In vitro, IGF-I bound to testicular interstitial cells which did not have 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and which were resistant to ethane dimethanesulphonate treatment. In vitro, IGF-I receptor-mediated actions increased both steroidogenesis and DNA synthesis. Insulin stimulated DNA synthesis at concentrations appropriate to cross-react with the IGF-I receptor, and this effect was greater in a testicular interstitial Leydig cell-depleted cell population compared with a Leydig cell-enriched cell culture. Furthermore, combinations of epidermal growth factor or transforming growth factor -α together with insulin appeared to act synergistically, causing extremely large increases in [³ H]thymidine incorporation in the interstitial cells. These results support a paracrine and/or autocrine role for IGF-I in interstitial cell growth and development.

Journal of Endocrinology (1993) 138, 107–114

The improved survival rate in patients treated for certain malignant diseases such as Hodgkin's disease and testicular teratoma is due predominantly to the use of cytotoxic chemotherapy. Unfortunately the cytotoxic drugs affect normal tissues as well as tumour cells and, in particular, those with a high proliferative rate such as skin, gut, bone marrow and testes. The improvement in survival has led to greater emphasis being paid to the quality of life amongst the survivors. The males treated for Hodgkin's disease and testicular tumours are usually young. The sterility imposed by the most widely used combination chemotherapy (mustine, vincristine or vinblastine, procarbazine and prednisolone) persists for at least 10 years in well over 90% of males treated for Hodgkin's disease and only reverses in 50% of males within 3 years of treatment with bleomycin, vinblastine and cis-platinum for testicular teratoma.

One approach to solve this problem has been to seek

ABSTRACT

The epidermal growth factor (EGF) receptor is expressed in a wide variety of cell types and is known to be present in the testis of many species including man. In the present study, specific ¹²⁵ I-labelled EGF binding was observed in isolated interstitial cell preparations from both the intact and Leydig cell-depleted rat testis. It was demonstrated that the population of cells to which ¹²⁵I-labelled EGF binds has a different buoyant density from either of the two adult Leydig cell populations, and remains unchanged in the absence of Leydig cells following in-vivo treatment with ethane dimethane sulphonate (EDS). Cells of this density (1·064 g/ml) identified by electron microscopy were fusiform mesenchymal cells, identical to those suggested by others to be able to differentiate into Leydig cells in vitro, i.e. Leydig cell precursors. In a culture system using two interstitial cell preparations of different buoyant densities from immature rats, both EGF and transforming growth factor-α (TGF-α) caused increased [³H]thymidine incorporation in the less dense cell preparation. TGF-α was more potent than EGF. EGF increased testosterone production in both fractions in amounts which could be related to the amount of 3β-hydroxysteroid dehydrogenase (3β-HSD)-positive cells. This study demonstrated that rat Leydig cells (defined as those cells which bind ¹²⁵I-labelled human chorionic gonadotrophin, have distinct buoyant densities, are 3β-HSD positive and are sensitive to EDS), do not bind ₁₂₅I-labelled EGF. Rather, EGF binds to a mesenchymal cell without LH receptors which is resistant to EDS. Growth factors which act via the EGF receptor increased [₃H]thymidine incorporation in a Leydig cell-depleted interstitial fraction which may reflect an action upon the progenitor of the mature Leydig cell.

Journal of Endocrinology (1993) 136, 439–446

ABSTRACT

Ethane-1,2-dimethanesulphonate (EDS) destroys Leydig cells in the testis of the adult rat and subsequently a new population of Leydig cells develops. It has been reported that EDS is not cytocidal to the new immature Leydig cell population. In the present study, the effect of increasing the time-interval between injections of EDS on cytotoxicity to Leydig cells was examined. At time-intervals of 4–10 weeks between injections the response was similar to that seen after a single injection of EDS to the adult rat. Four days after the second injection, EDS was found to reduce substantially serum testosterone concentrations and in-vitro binding of ¹²⁵I-labelled human chorionic gonadotrophin (hCG) to testicular LH receptors which can be correlated with Leydig cell destruction. However, when the interval was only 2 or 3 weeks there was no reduction in serum testosterone, and ¹²⁵I-labelled hCG binding was not so markedly reduced. During days 1–6 after a second injection of EDS, administered 3 weeks after the first, there were marked reductions in serum testosterone concentrations and in ¹²⁵I-labelled hCG binding to testis homogenates within 24 h. Recovery from the effects of EDS was rapid, and increased Leydig cell activity was seen from 2 to 6 days after injection. In contrast to the established changes in the adult rat, there was only a 50% reduction in the number of Leydig cells positive for 3β-hydroxysteroid dehydrogenase 2 days after the second injection of EDS, and after 6 days the number of cells had increased. These experiments show that the immature Leydig cell of the rat is sensitive to the cytotoxic effects of EDS but that the temporal changes in Leydig cell activity after EDS treatment are different in developing and mature Leydig cell populations. The data are consistent with the view that EDS is preferentially cytotoxic towards steroidogenically active Leydig cells, allowing the resident population of precursor cells to continue to respond to the prevailing homeostatic mechanisms.

Journal of Endocrinology (1989) 123, 197–203

ABSTRACT

Replicative DNA synthesis (¹²⁵I-labelled iododeoxy-uridine incorporation) was measured in interstitial cells prepared from rat testes and separated by Percoll density gradient centrifugation. Leydig cells were identified by ¹²⁵I-labelled human chorionic gonadotrophin (hCG) binding and 3β-hydroxysteroid dehydrogenase histochemistry. Continuous density gradients indicated that interstitial cell DNA synthesis was not associated with Leydig cells, and was greater in cells from the immature than from the mature rat testis. Fractionation of cells by discontinuous density gradients into Leydig cell-rich and -depleted pools did not result in a similar enrichment of DNA synthesis. Treatment of the adult rat with hCG increased DNA synthesis into both fractions but oestrogen had no effect. DNA synthesis was greater in cells from the immature rat but, in contrast to the adult, in-vivo hCG treatment had no effect, whilst oestrogen decreased synthesis. To characterize the cells synthesizing DNA further, interstitial cells were prepared from testes in which the Leydig cells were depleted by in-vivo treatment with ethane dimethanesulphonate (EDS) or depleted in their germ cells by treatment in utero with busulphan. EDS treatment had no effect on DNA synthesis by the interstitial cells in spite of the ¹²⁵I-labelled hCG binding being markedly reduced. Similarly, busulphan treatment was also without effects upon DNA synthesis. Fluorescence-activated cell cycle analysis of cells from both fractions from germ cell-depleted testes indicated that only a small proportion (3%) of the interstitial cells were actively dividing and this was almost doubled in cells from the germ cell-depleted immature rat testes. The experiments showed that the majority of cells in the interstitium of the rat testes do not synthesize DNA and are not undergoing cell division. The small proportion of cells that are dividing are probably not Leydig cells. The experiments have identified a dividing interstitial cell population and, in consideration of the changes in the immature and mature rat as well as the effects of hormone treatment, they may be regarded as putative Leydig cell precursors.

Journal of Endocrinology (1992) 134, 247–255

ABSTRACT

The Leydig cells repopulating the adult rat testis after destruction by a single injection of the cytotoxic ethylene-1,2-dimethanesulphonate (EDS) were investigated. After 14 days, serum concentrations of LH and FSH were significantly raised and concentrations of testosterone in the serum and testis reduced. At 21 days, hormone concentrations had returned to within the normal range. Binding of ¹²⁵I-labelled human chorionic gonadotrophin (hCG) to testis homogenate, however, was still less than 10% of normal. After 21 or 28 days the ¹²⁵I-labelled hCG binding profiles of isolated Leydig cells from EDS-treated rats, separated on a Percoll gradient, showed a single peak similar to that of immature (25 days old) rats. After 49 days, ¹²⁵I-labelled hCG binding resolved into two peaks more like that of normal adult rats. Using a quantitative cytochemical method, 3β-hydroxysteroid dehydrogenase activity in individual Leydig cells of unfixed testis sections was determined. Activity was increased by 70% (P < 0·05) in repopulating Leydig cells 21 days after EDS treatment compared with cells from vehicle-treated rats. In addition, Leydig cells were still capable of further 'in-vivo' stimulation by pharmacological doses of hCG. These data indicate that Leydig cells repopulating the testis are homogenous. Fewer cells from the newly formed population are capable of maintaining normal serum concentrations of testosterone and must thus be individually more active in secreting testosterone. In these respects, the Leydig cells repopulating the adult rat testis after EDS treatment more closely resemble those of the fetal rat testis.

J. Endocr. (1988) 117, 11–18

ABSTRACT

The influence of various endocrine and environmental factors on pituitary-ovarian function was studied in peripubertal rats treated with pregnant mare serum gonadotrophin (PMSG). Pregnant mare serum gonadotrophin induced ovulation in rats aged 27 days provided they weighed over 60 g. The response was preceded by a marked hypersecretion of LH which was detectable by radioimmunological and biological assay methods. In contrast, smaller rats of the same age did not ovulate in response to PMSG apparently because of the secretion of a pleiomorphic form of LH which, although immunoreactive, appeared to be biologically inactive. Ovarian function, assessed by response to exogenous gonadotrophins and by measurement of ¹²⁵I-labelled human chorionic gonadotrophin binding, was normal despite the presence of the biologically inactive pleiomorph. Exposure of the small PMSG-treated rats to a high environmental temperature (39 °C) or treatment with corticosterone or GH altered the nature of the LH in the blood so that it was active in both assay systems and facilitated ovulation as also did ACTH. The results suggest that the abrupt change in the nature of the LH released by the pituitary gland essential for the initiation of ovulation may be affected by GH, corticosterone or a raised environmental temperature.

J. Endocr. (1985) 104, 179–183

The oestrogen agonist and antagonist activity of a single dose of en-clomiphene (0·5–50 mg/kg) was studied in peripheral and brain tissue in the 3 week ovariectomized rat. 17β-Oestradiol benzoate (100 pg/kg) or vehicle was injected 24 h after en-clomiphene administration and data collected at 72 h. En-clomiphene produced a dose-related (agonist) fall in body weight and food intake. Agonist action was not observed upon sexual receptivity and prolactin secretion; oestrogen antagonism of these parameters was only seen at the higher doses. The effects of en-clomiphene upon serum LH and FSH were complex, both agonist and antagonist activity being demonstrated in the absence and presence of oestrogen. En-clomiphene was uterotrophic at all doses tested; however, oestrogen antagonism was only seen at the higher doses. Inhibition of the accumulation of uterine luminal fluid at the higher doses of en-clomiphene was a sensitive index of oestrogen antagonism. The results are discussed in relation to previous studies of the effects of enclomiphene on oestrogen receptors. With the exception of the antagonism of oestrogen-induced sexual receptivity no correlation could be made between biological activity and the status of oestrogen receptors in tissue.