Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat

The objective of this study was to determine whether postnatal increases in rat Leydig cell number result from differentiation of precursor cells, division of existing Leydig cells, or both. Our approach was 1) to examine changes in the absolute number of Leydig cells and potential precursor cells (macrophages, pericytes, and mesenchymal, endothelial, and myoid cells) per testis on day 19 of gestation (day -2) and days 7, 14, 21, 28, and 56 postpartum; 2) to examine the frequency with which mesenchymal and Leydig cells divide during prenatal and postnatal development; and 3) to identify and examine the fate of the progeny of Leydig and mesenchymal cell divisions during prenatal and postnatal development. Stereological methods were used to show that mesenchymal cells comprised 44% of the total interstitial cell population and Leydig cells 16% on day -2, whereas by day 56 postpartum the relationship had reversed; mesenchymal cells comprised 3% and Leydig cells 49%. These results suggested a precursor-product relationship between mesenchymal and Leydig cells because no such reciprocal relationship was observed between Leydig cells and macrophages, pericytes, endothelial, or myoid cells. Autoradiographic analysis of [3H]thymidine incorporation into mesenchymal and Leydig cells was consistent with this interpretation. In a series of pulse-chase experiments, the percentage of labeled mesenchymal and Leydig cells was measured after a single injection of [3H]thymidine on days 2, 14, 28, and 56 postpartum, each followed by sampling at timed intervals (between 1 h and 14 days) thereafter. Starting on day 14, the percentage of labeled Leydig cells was approximately 1% immediately after injection of [3H]thymidine and increased significantly to approximately 6% by 6 days after injection. No such increase was observed when rats were similarly injected starting on days 2, 28, and 56 postpartum. The rise in Leydig cell labeling between days 14 and 28 postpartum did not result in a decline in the number of silver grains over labeled Leydig cell nuclei, indicating that the increase in the percentage of labeled cells was not caused by Leydig cell division. These observations led us to conclude that the increase in Leydig cell labeling from days 14 to 28 was the result of recruitment from a compartment of labeled mesenchymal cells. In contrast, our analysis indicated that from day 28 postpartum and thereafter until the mature number of Leydig cells is attained, Leydig cells are generated by division of morphologically recognizable Leydig cells.     

Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: the role of LH/human chorionic gonadotrophin

The influence of LH levels on the proliferation and differentiation of possible Leydig cell precursors was investigated in adult rats, after the destruction of the existing Leydig cells with the cytotoxic drug ethane dimethyl sulphonate (EDS). In rats bearing a testosterone implant which prevented the rise in plasma LH levels and kept them within the normal range after the destruction of the Leydig cells, the proliferative activity of possible Leydig cell precursors still increased seven- to eightfold 2 days after EDS administration. Apparently, in this situation, locally produced factors, and not LH, may play a role in the stimulation of proliferation. The proliferative activity of the possible precursor cells could be further stimulated by treating rats with daily injections of human chorionic gonadotrophin (hCG) following EDS administration. It was concluded that the proliferative activity of possible Leydig cell precursors is probably regulated by both paracrine and endocrine factors. Almost no Leydig cells were formed in the rats bearing a testosterone implant during the first 4 weeks after EDS administration. When these rats were treated with hCG, starting 28 days after administration of EDS, a substantial number of Leydig cells was found after 2 days, and these cells also showed 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and alpha-naphtyl esterase (alpha-NE) activity. When hCG treatment was started at 14 or 21 days after EDS administration, some cells with the nuclear characteristics of Leydig cells were present after 2 days, but no 3 beta-HSD or alpha-NE activity could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone-induced resistance of rat Leydig cells to the cytotoxic effects of ethane-1,2-dimethane sulphonate.

Several studies have shown that the cytotoxic agent ethane-1,2-dimethane sulphonate (EDS) specifically destroys Leydig cells in the adult rat testis. It has also been reported that when rats are pretreated with human chorionic gonadotrophin (hCG), administration of EDS does not result in the complete destruction of the Leydig cell population. It has been suggested that hCG pretreatment 'protects' Leydig cells against the cytotoxic action of EDS. In the present study the underlying principles for this resistance to the cytotoxic effects of EDS have been investigated. Within 48 h of the start of daily hCG treatment the number of nuclear profiles of Leydig cells (henceforth called relative number of Leydig cells) had increased from 1014 +/- 40 to 1368 +/- 30 cells per 1000 Sertoli cell nuclei. Previous experiments have indicated that these newly formed Leydig cells probably develop from differentiating Leydig cell precursors. When EDS is administered concomitantly with the third injection of hCG (2 days after the start of hCG treatment), the relative number of Leydig cells surviving EDS treatment was 388 +/- 52 per 1000 Sertoli cells. Hence, there is a similarity between the increase in the relative number of Leydig cells after 2 days of hCG treatment and the relative number of EDS-resistant Leydig cells. The Leydig cells that survived EDS administration showed characteristics which also occur in developing Leydig cells in the immature testis. It is concluded that, in rats pretreated with hCG for 2 days before EDS administration, new Leydig cells with some immature characteristics are formed. One of these characteristics is that these cells are insensitive to EDS.     

Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro.

The number of interstitial macrophages in the testis fluctuates according to age, increasing gradually during prepubertal development to reach 15-20% in the interstitial compartment in the adult rat. These macrophages are in close morphological association with Leydig cells. Macrophage products, interleukin-1 (IL-1) and tumor necrosis factor alpha stimulate and/or inhibit steroid production in cultures of Leydig cells. We have studied the effects of macrophage products on DNA synthesis in rat Leydig cells to investigate a possible paracrine role of testicular macrophage products on the proliferation of Leydig cells. Leydig cells isolated from 10-, 20-, and 70-day-old rats were cultured for 48 h in serum-free medium, washed, and treated with different cytokines for 18 h. The medium was then removed, fresh medium containing 0.5 microCi [3H]thymidine was added, and cells were incubated for 4 h prior to determining the incorporation of [3H]thymidine into DNA. Human recombinant IL-1 beta caused a dose-dependent stimulation in the incorporation of [3H]thymidine into DNA in the Leydig cells from 10- and 20-day-old rats but had no effect on DNA synthesis in interstitial cells from adult rats. Maximum stimulation of DNA synthesis in immature Leydig cells was observed with 1-2 ng/ml IL-1 beta. Autoradiography after incubation with [3H]thymidine showed a dramatic increase in the number of labeled Leydig cells after treatment with IL-1 beta (19.27 +/- 3.77% vs. 1.44 +/- 0.52% in control cultures) indicating that IL-1 beta recruited more cells to enter the cell cycle and initiate DNA synthesis. Human recombinant IL-1 alpha and tumor necrosis factor alpha also caused significant stimulation of DNA synthesis in Leydig cells but these cytokines were much less potent (1-10%) than IL-1 beta. IL-1 beta enhanced the effects of maximally effective concentrations of growth-promoting agents previously known to stimulate DNA synthesis in immature rat Leydig cells, i.e. human CG, steroidogenesis-inducing protein, and transforming growth factor alpha plus insulin. On the basis of these results it is concluded that IL-1 may play an important role in the proliferation of Leydig cells during prepubertal development in immature rats.     

Ethylene dimethanesulfonate destroys Leydig cells in the rat testis

Ultrastructural changes in the interstitial cells of the adult rat testis were studied up to 45 days after administration of a single dose (100 mg/kg) of the antifertility compound ethylene dimethanesulfonate (EDS). Most Leydig cells showed degenerative changes 12 h after treatment. Twenty-four and 48 h after injection, all Leydig cells observed showed gross degenerative changes. At 4 and 14 days, intact Leydig cells could not be identified in the interstitial spaces. Twenty-one days after treatment with EDS, small Leydig cells were visible, and at 45 days, Leydig cells appeared normal. The seminiferous epithelium appeared morphologically normal until 4 days after injection of EDS, when slight abnormalities were observed. At 14 and 21 days, the seminiferous epithelium was grossly abnormal, but at 48 days, spermatogenesis appeared normal. Twelve, 24, and 48 h after treatment, large quantities of material, presumably from dead Leydig cells, were observed within the macrophage cytoplasm. The predominant cell in the interstitial space 4 and 14 days after EDS was the macrophage. Inclusions from the dead Leydig cells within the cytoplasm of the macrophages had almost disappeared. LH receptors (hCG binding) in testicular homogenates were consistent with the cytological changes in Leydig cells. Receptor concentration was low at 24 h and was almost zero at 4 days. This change was accompanied by a decrease in serum testosterone to castrate levels by 2 days. The responses of the endocrine system to destruction of the Leydig cell by EDS, as monitored by serum FSH, LH, and testosterone, were slower than those after castration, indicating that the response to EDS reflects the time required to kill the Leydig cell rather than direct impairment of the steroidogenic pathway. These experiments demonstrate that Leydig cells can be specifically destroyed by a cytotoxic drug. The availability of a specific cytotoxic agent for Leydig cells offers further opportunities to study the interrelationships between the Leydig cell and the seminiferous tubule.     

Gonadotropin regulation of Leydig cell DNA synthesis

Adult male rats were injected s.c. with either saline, 100 IU hCG, 100 micrograms FSH, 50 micrograms LH, 100 micrograms PRL, 50 micrograms estradiol-17 beta, 500 micrograms or 10 mg testosterone; 50 micrograms estradiol-17 beta; animals were sacrificed at 12-120 h post-injection. Collagenase-dispersed interstitial cells (150-200 X 10(6) cells/2 ml) were incubated in vitro with 10 microCi [3H-methyl]thymidine for 1 h at 32 degrees C. Centrifugation of the cells on discontinuous 11-27% metrizamide gradients revealed thymidine incorporation in the regions of population I and II Leydig cells. A significant increase in thymidine incorporation into DNA after treatment with either hCG or LH was first detectable at 48 h, was equivalent to control values at 72 h and was again significantly increased at 96 h in population I and at 120 h in population II cells. [3H]Thymidine incorporation at 48 h, expressed as dpm/10(6) cells, was 2205 +/- 432 and 4119 +/- 929 vs. 16473 +/- 3795 and 11648 +/- 3427 for control and hCG-treated population I and II cells, respectively. Addition of 20 mM hydroxyurea suppressed [3H]thymidine incorporation, 97% and 96% in hCG-treated population I and II cells, respectively. Autoradiographic analyses revealed that nuclei from control and 48 h hCG-treated population I and II cells exhibited 1.2% and 2.3% vs. 7% and 6.8% silver grains, respectively. PRL had no influence on LH/hCG-enhanced DNA synthesis; however, estradiol-17 beta administration for 48 h dramatically suppressed thymidine incorporation. Population I Leydig cells exhibited a higher level of LH/hCG-stimulated DNA synthesis compared to population II cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of Leydig cells in the regulation of inhibin secretion by the testis

The stimulation of Leydig cells by the administration of a single injection of 100 IU human CG (hCG) to adult male rats caused a significant biphasic stimulation of serum testosterone levels at 2 h and 3 days after injection. Serum immunoreactive (IR)-inhibin levels were elevated by 6 h and peaked at 24 h after hCG, then progressively declined thereafter. The removal of Leydig cells in vivo by the injection of the cytotoxic drug ethane dimethane sulfonate (EDS) causes a significant decrease in serum testosterone levels within 4 days, which is sustained 1 and 2 weeks after EDS. Serum IR-inhibin levels, however, rise significantly 2 and 4 weeks after injection of EDS. An injection of 100 IU hCG, 4 days after EDS (when no Leydig cells were present in vivo), failed to provoke an elevation of either testosterone or IR-inhibin levels in serum. But 2 or 4 weeks after administration of EDS, as a new population of Leydig cells develops in the interstitium, an injection of 100 IU hCG provokes a significant increase in serum testosterone and IR-inhibin levels. The possibility that the failure of IR-inhibin levels to rise after EDS and hCG treatment could be due to changes in the seminiferous epithelium, caused by testosterone deprivation induced by Leydig cell destruction after EDS, was examined by administering high doses of testosterone known to maintain spermatogenesis. Under such conditions, hCG failed to induce a rise of IR-inhibin after EDS treatment had destroyed the Leydig cells. These data strongly support the concept that the Leydig cells are involved in the regulation of IR-inhibin secretion in vivo through factors other than testosterone.     

Evidence for an androgen receptor in porcine Leydig cells

Cytosol and nuclear androgen receptor concentrations were measured in freshly prepared and cultured Leydig cells of immature pig testis with exchange assays using [3H]methyltrienolone as labelled ligand. Androgen receptors in Leydig cells had high affinity for [3H]methyltrienolone and steroid binding specificity typical of an androgen receptor. The mean receptor concentrations were 76 fmol/mg protein and 210 fmol/mg DNA for cytosol and nuclei, respectively. In sucrose gradients, cytosol androgen receptors sedimented in the 4 S region. The cells maintained androgen receptors under culture conditions. Exposure of cultured cells to [3H]methyltrienolone (10 nmol/l) resulted in accumulation of androgen receptors in the nuclei with maximal uptake by 1 h. We conclude that methyltrienolone binding sites with characteristics of androgen receptors were identified in both cytosol and nuclei of porcine Leydig cells.     

Leydig cell recovery following a second challenge with ethane-1,2-dimethanesulphonate is enhanced by short intervals between cytotoxin administration to rats

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 3 beta-hydroxysteroid dehydrogenase 2 days after the second injection of EDS, and after 6 days the number of cells had increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of long-term LH deprivation on testosterone secretion and Leydig cell volume, number and proliferation in adult rats

The purpose of this study was to determine whether Leydig cell volume and function could recover fully from long-term LH deprivation upon restoration of endogenous LH secretion, and whether the restoration of LH would elicit a mitogenic response, i.e. stimulate Leydig cell proliferation or affect Leydig cell number per testis. LH secretion was inhibited by treating adult rats with testosterone and oestradiol-filled (TO) silicone elastomer implants (16 weeks), and was restored by removing the implants. Changes in serum concentrations of LH and FSH, LH-stimulated testosterone secretion by testes perfused in vitro, Leydig cell volume and number per testis, average Leydig cell volume and Leydig cell [3H]thymidine incorporation were measured at weekly intervals following implant removal. The TO implants inhibited (P less than 0.01) LH secretion, but serum concentrations of FSH were not significantly different (P greater than 0.10) from control values. After implant removal, serum LH returned to control values within 1 week, whereas serum FSH increased twofold (P less than 0.01) and returned to control values at 4 weeks. LH-stimulated in-vitro testosterone secretion was inhibited by more than 99% in TO-implanted rats, but increased (P less than 0.01) to 80% of control values by 8 weeks after implant removal. The total volume of Leydig cells per testis and the volume of an average Leydig cell were 14 and 19% of control values respectively, after 16 weeks of TO implantation (P less than 0.01), but returned to 83 and 86% of controls (P greater than 0.10) respectively, by 6 weeks after implant removal. Leydig cell proliferation ([3H]thymidine labelling index) was low (less than 0.1%) in both control and TO-implanted rats, increased (P less than 0.01) fivefold from 1 to 4 weeks after implant removal and then declined to control values at 6 weeks. The increase in Leydig cell [3H]thymidine incorporation was mimicked by treating TO-implanted rats with exogenous LH, but not FSH. Leydig cells were identified in both the interstitium and the lamina propria of the seminiferous epithelium. The proportion of Leydig cell nuclei in the lamina propria was 30-fold greater (P less than 0.01) at 1 and 3 weeks after implant removal (3%) compared with that for control and TO-implanted rats (0.1%). Total Leydig cell number per testis was marginally but not significantly (P = 0.06) decreased in rats treated with TO implants for 16 weeks when compared with controls (18.4 +/- 2.2 vs 25.4 +/- 1.2 x 10(6)).(ABSTRACT TRUNCATED AT 400 WORDS)     

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.     

Phorbol ester and vasopressin activate phospholipase D in Leydig cells

In the present study evidence is provided for the existence of phospholipase D (PLD) activity in rat Leydig cells. Leydig cells were cultured and labelled with [3H]myristic acid. In the presence of ethanol, phorbol 12-myristate 13-acetate (PMA) stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) in a dose-dependent manner at the expense of [3H]phosphatidic acid ([3H]PA). In cells prelabelled with [3H]choline, PMA caused a rapid increase in intracellular free [3H]choline. The time course of [3H]PEt formation was similar to the time course of intracellular [3H]choline formation. The data taken together support the notion that PMA stimulates phosphatidylcholine (PC) hydrolysis by a mechanism, which principally involves PLD. Activation of PLD by PMA was inhibited by long-term pretreatment of cells with PMA to downregulate protein kinase C (PKC) and by pretreatment with staurosporine. These data support the notion that activation of PLD by PMA is dependent on PKC. Arginine vasopressin (AVP) caused a rapid stimulation of PLD activity in the cells. This activation was inhibited after down-regulation of PKC, indicating that the agonist acts by a mechanism similar to that of PMA.     

Steroidogenic enzyme activity in the rat testis following Leydig cell destruction by ethylene-1,2-dimethanesulphonate and during subsequent Leydig cell regeneration.

Ethylene-1,2-dimethanesulphonate (EDS) rapidly destroys Leydig cells in the rat testis, although repopulation occurs within 5-7 weeks. In this study we have examined the activity of testicular steroidogenic enzymes after Leydig cell destruction and during regeneration. This was designed to measure the contribution of cells, other than Leydig cells, to steroidogenic activity in the testis, and to determine whether changes in steroidogenic enzyme activity during Leydig cell regeneration after EDS parallel those which occur during normal Leydig cell development. The enzymes studied are those responsible for androgen synthesis and metabolism in the testis. Adult male Wistar rats (300-350 g) were injected with EDS (100 mg/kg, i.p.) and testicular steroidogenic enzyme activity was measured on days 0, 3, 7, 14, 21 and 35. On day 3, when no Leydig cells remain in the testis, cholesterol side-chain cleavage (CSCC) activity, per testis, declined to undetectable levels, while 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase retained only 0.04 and 0.15% of control activity respectively. In contrast, 17-ketosteroid reductase (17-KSR) and 5 alpha-reductase retained 33 and 10% of control activity respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arginine vasopressin stimulates phosphoinositide turnover in an enriched rat Leydig cell preparation

An enriched rat Leydig cell preparation was preincubated with [14C]arachidonic acid. Stimulation of the cells with arginine vasopressin (AVP) (1 microM) for 2 min caused a significant increase in labelled phosphatidic acid and a significant fall in radioactivity in phosphatidylinositol and phosphatidylinositol 4-monophosphate + phosphatidylinositol 4,5-bisphosphate. Preincubation with dibutyryl cyclic AMP had no effect on the AVP-induced phospholipid turnover. Leydig cells were preincubated with myo-[2-3]inositol for 22 h and then with 10 mM LiCl for 10 min. Exposure to AVP (1 microM) induced a rise in labelled inositol phosphates. The response was inhibited when the cells were preincubated with the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (0.16 microM) for 10 min. These results provide evidence for an AVP-induced phospholipase C stimulation in rat Leydig cells and suggest a protein kinase C-dependent feedback inhibition of the stimulation. Other agonists that might have a regulatory function in the testis were tested for possible effects on phosphoinositide metabolism. Of prostaglandin E2 (10 microns,) angiotensin II (0.1 microM), and bradykinin (0.9 microM), only the latter induced a significant increase in the labelled inositol phosphates. This suggests that Leydig cells possess a bradykinin receptor which can activate phospholipase C.     

Thyroid epithelial cell proliferation in xenotransplanted human toxic nodular goitre is increased by Graves' IgG

Human toxic nodular goitre tissue was xenotransplanted to athymic mice. Transplant function was analysed as 18-h thyroid transplant uptake of iodide-125 at day 21 and again at 10 weeks after transplantation. Graves' IgG or IgG from healthy donors was given intraperitoneally daily day 22-35. Epithelial cell proliferation in thyroid tissue transplants from human toxic nodular goitre and from normal thyroid glands was analysed by continuous [3H]thymidine administration for 4 days between day 21 and 24 and for 12 days between day 21 and 33 in separate series given daily injections of Graves' IgG or normal IgG during the same period. After administration of Graves' IgG, the 18-h iodide-125 uptake by the toxic nodular tissue transplants was 7 times higher at 10 weeks than at 3 weeks. Control IgG gave a corresponding 1.6-fold increase. The fraction of labelled cells after [3H]thymidine incorporation was 18 and 56% in toxic nodular goitre transplants and 4 and 48% in normal thyroid tissue transplants after daily Graves' IgG administration for 4 and 12 days, respectively, but only 1.3% in both types of tissue transplants after administration of normal IgG. Graves' IgG therefore seems to be able to stimulate cell proliferation in toxic nodular goitre tissue.     

Cell kinetics of thyroid epithelial cells during hyperplastic goitre involution

Thyroid epithelial cell kinetics were investigated in rats when a normal iodine diet was re-established after a long period of iodine deficiency supplemented with propylthiouracil (0.15%) for the last 2 months. In the first (prelabelling) experiment, all rats were labelled with a single injection of [3H]thymidine 2 days before iodine refeeding in order to follow the fate of the prelabelled cells. In the second experiment, the pulse-labelling index at the time of killing was measured; for this purpose the rats received [3H]thymidine 1 h before death. In these two experiments autoradiography was performed on histological sections. Thyroids were excised on day 0 and then at various intervals up to day 73 of iodine refeeding. Plasma tri-iodothyronine (T3) and thyroxine (T4) were very low until day 4 and then increased to reach control values on day 30. Thyroid concentration of iodide rose to 20 times the value on day 0, remained at this high level until day 2, and then diminished on day 4 to reach the control value on day 16. Plasma TSH concentrations were very high in iodine-deficient rats and did not vary significantly until day 8, when they fell rapidly to reach the control value on day 30. Thyroid weight, raised on day 0, decreased relatively quickly until day 4, then more slowly until day 30. Total thyroid epithelial cell number, high on day 0 (30.7 x 10(6) cells) was constant until day 16, then decreased until day 30 at which time a plateau was reached.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated recovery of Leydig cells of the immature rat testis after administration of the cytotoxic ethylene dimethanesulphonate

A single injection of ethane-1,2-dimethanesulphonate (EDS; 100 mg/kg) selectively destroys Leydig cells in the testis of the adult rat; however, unconfirmed reports indicate that Leydig cells in the immature rat are not affected. In this study the effect of EDS was examined 2 days after treatment of rats aged 20, 25 or 35 days. There was a large reduction in the in-vitro binding of 125I-labelled human chorionic gonadotrophin (hCG) to the homogenates of testes from EDS-treated immature rats. EDS reduced the testosterone content of the testes at all ages studied, but 2 days after injection had only significantly lowered the serum testosterone concentration of 25- or 35-day-old animals. Light microscopic examination of the testis of the 22-day-old rat, 2 days after treatment with EDS, indicated that there were still many cells staining for 3 beta-hydroxysteroid dehydrogenase. The interstitium also contained numerous atypical cells which did not stain for 3 beta-hydroxysteroid dehydrogenase. Electron microscopy of testes from the 22-day-old EDS-treated rat showed that Leydig cells were still present in the interstitium together with macrophages and fibroblast-like cells. Six days after EDS treatment of 20-day-old rats, but not 35-day-old rats, there was an increase in the binding of 125I-labelled hCG to testis homogenate to 70% of control value. Testicular testosterone content 6 days after treatment of the 20-day-old rat had risen to 50% of the control testis value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the regenerated Leydig cell population of the rat after destruction by ethylene-1,2-dimethanesulphonate

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, 3 beta-hydroxysteroid dehydrogenase activity in individual Leydig cells of unfixed testis sections was determined. Activity was increased by 70% (P less than 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 homogeneous. 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.     

Hormonal regulation of type I insulin-like growth factor receptors of Leydig cells in hypophysectomized rats

The effects of hCG and various pituitary hormones on type I insulin-like growth factor (IGF) receptors of purified Leydig cells of hypophysectomized rats were studied. The number of type I IGF receptors of Leydig cells obtained from hypophysectomized rats (18.0 +/- 1.5 fmol/10(6) cells) was lower than that in normal rats (54.6 +/- 5.3 fmol/10(6) cells; P less than 0.05). After a single administration of hCG (10 U, ip), specific binding of [125I]IGF-I to purified Leydig cells increased 3-fold. Scatchard analyses of the binding data suggested that increased binding was the result of an increase in receptor number, whereas binding affinity remained unaltered. Type I IGF receptor increased within 12 h and remained persistently elevated 96 h after hCG treatment. Administration of hCG (10 U, ip) daily for 5 days increased type I IGF receptor levels to 73.2 +/- 8 fmol/10(6) cells (P less than 0.001). FSH caused a small but significant increase in type I IGF receptors. Concomitant administration of FSH and hCG further enhanced IGF-I-binding capacity. IGF-I-binding affinity of Leydig cells treated with FSH or FSH plus LH was not significantly different from that in the control hypophysectomized rats. Daily administration of GH for 5 days also upregulated type I IGF receptors, whereas PRL had no effect. FSH, GH, and PRL administration had no effect on serum testosterone levels. Serum testosterone levels increased to 3.99 +/- 0.35 ng/ml after 5 days of treatment with hCG. Concomitant administration of FSH and hCG caused a further increased in serum testosterone levels (6.13 +/- 0.46 ng/ml; P less than 0.01). The present study suggests that type I IGF receptors of Leydig cells can be up-regulated by LH, FSH, and GH. However, hCG/LH seems to be the most important factor in maintaining and regulating type I IGF receptors of Leydig cells. Steroidogenic and growth-promoting effects of hCG and pituitary hormones on Leydig cells may be mediated by increased type I IGF receptors.     

Bromobenzene accelerates hepatocyte streaming in rats.

Sixty male adult rats weighing between 250 and 300 g were divided in two equal experimental groups. The first experiment was designed to evaluate hepatocyte streaming. Animals were injected with 0.5 microCi [3H]thymidine and 1 hour later received one intraperitoneal injection of bromobenzene (3.8 mmol/kg, dissolved in corn oil). The animals were then killed in groups of five at 1 hour and 2, 4, 7, 14, and 30 days. The aim of the second experiment was to evaluate labeling index changes with time. Animals received one intraperitoneal injection of bromobenzene and were killed in groups of five at 1, 2, 3, 4, 7, and 14 days. They received [3H]thymidine 1 hour before killing. Bromobenzene induced a necrosis in the third acinus zone that disappeared within a week. On day 0 the labeling index of hepatocytes and littoral cells was 0.3% +/- 0.04% and 0.4% +/- 0.05%, respectively. On the second day, it reached 14.6% +/- 2.6% and 10.1% +/- 3.1% and returned to its initial value after 1 week. Dead cells in the third zone were replaced by inflowing cells from the intact zones. Hepatocytes and littoral cells streamed at the same velocity of 6 +/- 0.5 micron/day, faster than in untreated animals with velocity of 3.2 microns/day. Parenchyma and stroma responded to injury in a coordinated fashion. From the functional point of view, hepatocytes and littoral cells operate as a unit that is called proliferon. The maximal proliferon life span was 57 days after bromobenzene treatment and 108 days in controls.