Effects of Hypophysectomy on LH Receptor Binding and Leydig Cell Sensitivity and Responsiveness to hCG/LH in the Immature (30 Day) Rat

The sensitivity (dose of hormone eliciting half maximal response) and the magnitude of the maximal response of Leydig cell suspensions prepared from immature intact and immature eight day hypophysectomized rats to hCG and dibutyryl-cyclic-3'5-adenosine monophosphate (dBC) were assessed in vitro in the presence or absence of the phosphodiesterase inhibitor, methylisobutyl-xanthine (MIX). In separate studies LH/hCG binding to dispersed Leydig cells from intact and hypophysectomized rats of the same age was evaluated by Scatchard analysis and autoradiography.
Hypophysectomy was associated with a 3–4 fold increase in the sensitivity of response, but an 80 % reduction in the maximum steroidogenic response to both hCG and dBC (when expressed per million 3β-hydroxy-steroid-dehydrogenase (3β-HSD) positive cells. Addition of MIX resulted in a greater increase in the sensitivity of response from intact Leydig cells than from cells from hypophysectomized rats, suggesting that hypophysectomy is associated with a decrease in the activity of the phosphodiesterase. Scatchard analysis of [¹²⁵I]hLH binding to Leydig cells indicated no major decrease in the average number of LH/hCG receptors per cell after 8 days of hypophysectomy. Grain counts after autoradiography of the radiolabelled LH/hCG receptor confirmed this finding.     

17 beta-estradiol inhibition of Leydig cell regeneration in the ethane dimethylsulfonate-treated mature rat.

This study was designed to determine the effects of 17 beta-estradiol (E2) on Leydig cell development in the rat. Mature (60 to 65 days old) male rats received a single intraperitoneal injection of ethane dimethylsulfonate (EDS, 100 mg/kg body weight); untreated rats served as controls. In one series of experiments, groups of EDS-treated rats also received daily injections of either E2 (25 micrograms/100 g body weight), human chorionic gonadotropin (hCG, 20 IU/day), a combination of the two, or vehicle only (EDS controls). Animals were killed on days 2, 4, 10, 16, 24, 30, and 36 after EDS treatment. In another series of experiments, groups of EDS-treated rats received daily injections of hCG and E2 during days 0 through 5, 5 through 30, or 16 through 30 after EDS treatment, and were killed on day 30. In both series of experiments, the steroidogenic capacity and hCG binding capacity of the Leydig cells were examined in short-term in vitro incubations using collagenase-dispersed interstitial cells. Testes were also prepared and examined histologically by light and electron microscopy. E2 treatment of animals during the initial 5 days after EDS administration had no effect on the regeneration of interstitial cells and Leydig cells. Treatment with E2 during days 5 to 30 post-EDS blocked the regeneration of Leydig cells and thereby significantly reduced the increase in interstitial cell numbers. Finally, when E2 treatment was delayed until 16 days post-EDS, there was no significant reduction in the regeneration of interstitial or Leydig cells. These data suggest that an important developmental process that is necessary for Leydig cell regeneration occurs between days 5 and 16 post-EDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethane dimethylsulphonate selectively destroys Leydigcells in the adult bonnet monkeys （Macaca radiata ）

Aim: To study the effect of intratesficular administration of ethane-1,2-dimethylsulphonate (EDS) which has been exten-sively used to selectively destroy Leydig cells in rats and study ~ role of gonadotropin in regulation of differentiation ofLeydig cells (LC) in the adult male bonnet monkey. Methods and Results: In vitro studies with cultured interstitialcells isolated from monkey testis revealed an inhibitory effect of EDS on LC as assessed by decrease in testosterone pro-duction. Intratesticular administration of EDS (5, 10, 20, 50 rag/testis) resulted in a dose-dependent rapid decrease inserum testosterone levels, with a 6.5 % decrease with 5 nag of EDS by the 3rd day, which returned to control levels by the45th day. EDS treatment resulted in a significant decrease in testiculiar testosterone. In addition a significant decrease in[^125 1]hCG binding and phenylesterase activity in the interstitial cells was noticed. Histological analysis of the testes onthe 5th day after administration of EDS revealed an interstitium devoid of LC indicating the destructive action of EDS.Conclusion: The monkey LC are sensitive to destructive action of EDS.     

Developmental pattern and sensitivity to down-regulation of testicular LH receptors in the pig

Testicular receptors for luteinizing hormone (LH) were quantified in 40 pigs between the day of birth and 22 weeks of age. The highest specific binding capacity (fmol hLH bound/mg protein) occurred at about 2--3 weeks of age which coincides with the age when the relative volume of Leydig cells is at a maximum. Significantly lower binding capacity was observed at 7 weeks of age which is the time of maximum Leydig cell regression. hCG was injected intramuscularly to immature (3000 IU, 22 days old) and adult boars (10 000 IU, 30--40 weeks old). Twentyfour h after injection the numbers of available LH receptors were reduced to 6 and 18% of control values, respectively. At this time occupied receptors were at a maximum (90% and 67% of controls, respectively). The total number of LH receptors (available + occupied) was increased 1 h after injection after which a gradual decrease was observed. Three days after hCG administration the total receptor quantities were at a minimum in both immature and adult pigs (59% and 31% of controls, respectively) and returned to control levels 5--7 days after treatment. Thus, pig LH receptors appear to be subjected both 'positive' and 'negative' regulation as previously described in the rat.     

The regulation of the proliferation and differentiation of rat Leydig cell precursor cells after EDS administration or daily HCG treatment

The proliferation and differentiation of possible Leydig cell precursors in adult rats were studied after destruction of the existing Leydig cells with EDS or after daily treatment with hCG. After 2 days with either treatment, a 12- to 16-fold increase in the number of [3H]thymidine-incorporating interstitial cells was found. In the case of hCG treatment, this was probably due to the high plasma hCG levels. However, after EDS treatment, LH levels start to rise between days 1 and 3, suggesting a paracrine stimulation of the proliferation of interstitial cells. After hCG treatment, a substantial increase in the numbers of Leydig cells was already found at day 2. It was concluded that hCG induced a rapid differentiation, without cell division, of existing precursor cells into recognizable Leydig cells. In rats treated with both EDS and hCG, new Leydig cells were not formed during the first 10 days. This indicates that EDS destroys not only mature Leydig cells but also those Leydig cell precursors that are able to differentiate rapidly into recognizable Leydig cells.     

Prolactin and Leydig cell responsiveness to LH/hCG in the rat.

The effects of prolactin and 2-bromo-alpha-ergocryptine (CB-154) on Leydig cell function in intact and hypophysectomized male rats were studied. The conclusions can be summarized as follows: prolactin (1) has a direct stimulatory effect on the number of LH receptors on rat Leydig cells, (2) has no effect on the characteristics of the dose-response curve of isolated Leydig cells (hCG stimulated androgen production) in vitro even after treatment with pharmacological doses in vivo, and (3) acts synergistically with LH to stimulate the quantity of androgen produced by the Leydig cells in response to hCG in vitro and to increase the sensitivity of the hCG-dose-response curve. Treatment of intact rats with CB-154 reduced the quantity of androgen produced by the Leydig cells in vitro after exposure to hCG and decreased LH binding to the same cells by 50%. These results suggest that under normal conditions, endogenous prolactin plays a key role in maintaining the functional integrity of rat Leydig cells.     

Effect of cryptorchidism on testicular and Leydig cell androgen production in the mouse

Unilateral cryptorchidism was induced surgically in adult mice and the effects on testicular and Leydig cell steroidogenesis were studied after 7 weeks. There was a 60% reduction in weight of the cryptorchid testis and this was associated with a significant reduction in intratesticular androgen content, both under basal conditions and following an injection of hCG. Testicular androgen production in vitro was also significantly lower in the cryptorchid testis compared to the scrotal testis, again under both basal conditions (29 +/- 6% of control) and in the presence of hCG (46 +/- 9% of control). Scrotal testes from the unilaterally cryptorchid animals did not show any significant difference in steroidogenic capacity compared to testes from untreated control animals. The decrease in steroidogenic capacity of the cryptorchid testis was due, at least in part, to a reduction in activity for each Leydig cell. In four experiments, androgen production by Leydig cells isolated from cryptorchid testes was 48 +/- 9% of cells from scrotal testes in the presence of a saturating dose of hCG. Under basal conditions the effect was more variable between experiments with steroid secretion by Leydig cells from cryptorchid testes being 58 +/- 32% of that for cells from scrotal testes. Leydig cell steroidogenesis in the scrotal testes of unilaterally cryptorchid animals did not differ significantly from untreated controls. These results show that induced cryptorchidism in the mouse causes a significant reduction in Leydig cell activity. This is apparently different from the effects of this procedure on the rat and raises the possibility that intratesticular regulation differs between the two species.     

Sites of Action of Cyproterone and Cyproterone Acetate in the Immature Male Rat

Plasma testosterone (Leydig cell function), LH and FSH (pituitary function), the epididymal content of androgen binding protein (ABP) (Sertoli cell function) and plasma corticosterone (adrenal cortical function) were determined after 10 daily injections of varying doses of cyproterone and cyproterone acetate, beginning at 21 days of age. Daily doses of 0.5 mg or greater of either antiandrogen resulted in a marked depression in the levels of plasma testosterone and intra-testicular testosterone (measured only in the cyproterone group) with a dose-dependent decrease in testis and epididymal weights; both effects occurring with only minor changes in the levels of circulating gonadotrophins. In addition, these compounds caused a marked decrease in Sertoli cell secretion as reflected in a significant fall in the epididymal content of ABP.
A more detailed examination of the apparently direct effects of cyproterone on Leydig cell function revealed: 1) no major effects of in vivo treatment for 6 days (5 mg/day) on [I¹²⁵]hLH binding to testis membrane particles or on the in vitro response of enriched Leydig cell suspensions to hCG, 2) a dose-dependent inhibition of the hCG responsiveness of normal Leydig cells in the presence of the drug in vitro . The inhibitory effects on steroidogenesis in vivo can well be explained by a direct inhibition of the 3β-steroid-dehydrogenase-isomerase exerted by both antiandrogens.     

Characterization of insulin binding and comparative action of insulin and insulin-like growth factor I on purified Leydig cells from the adult rat

Insulin binding and insulin action were characterized in adult rat Leydig cells, purified on discontinuous Percoll gradients. Binding of [125I]-porcine insulin was found to be dependent on time, temperature, cell concentration and Leydig cell specific gravity. Competition relative to porcine insulin (100) was as follows: insulin-like growth factor I (IGF-I) : less than 1; proinsulin : 5; guinea-pig insulin : 2; hCG, ovine prolactin and bovine GH : 0. High and low affinity binding sites for insulin were identified on purified Leydig cells with Ka values of 1.2 X 10(9) and 0.3 X 10(8) M-1, with 10,300 and 34,000 binding sites per cells, respectively. Using primary cultures of Leydig cells in serum-free medium, the action of insulin on steroidogenesis was studied and compared with IGF-I action. Insulin and IGF-I used at 1-35 nM enhanced basal testosterone production in a dose-dependent manner; the effect was significant 4 h after administration. Insulin or IGF-I also potentiated the effect of hCG on steroidogenesis during short-term incubation (4 h). Insulin was shown to improve hCG responsiveness without modifying sensitivity to hCG. Moreover, neither cell number nor hCG-binding was altered by insulin, IGF-I or a combination of the two. Concomitant treatment with insulin and IGF-I at half-maximal and maximally effective doses, in the presence or absence of hCG, indicated that the two factors synergized in the stimulation of testosterone production via a common saturable mechanism.     

Developmental changes in testicular interstitial cell populations, LH receptors and in the response to hCG in the rat

High doses of hCG were administered to immature rats of different ages and the animals killed 48 h later. Serum testosterone increased 2 to 4-fold over control values 48 h after hCG. In-vitro androgen production showed different patterns according to age. Animals younger than 35 days, when treated with hCG, retained the ability to respond to in-vitro gonadotrophic stimulation. This ability was lost in testes from rats aged 45 days. The number of free LH-receptors 48 h after hCG diminished with increasing age to become non-detectable at 35 and 45 days. In control animals the proportion of differentiated Leydig cells in relation to their mesenchymal precursors increased progressively with age to reach highest values at 45 days. hCG administration induced a shift of the cellular composition of the interstitium toward the more mature cell types. hCG has a predominantly trophic action on mesenchymal precursors in young rats, promoting their differentiation. These effects are minimal in the differentiated Leydig cells in older animals. It is proposed that the observed biochemical responses are the result of the balance between the increase in LH receptors and steroidogenic enzymes in the developing new generation of young Leydig cells and the down-regulation of receptors and enzymatic lesions in fully differentiated Leydig cells.     

Changes in testicular function induced by short-term exposure of the rat testis to heat: further evidence for interaction of germ cells, Sertoli cells and Leydig cells

This study was designed to determine the effects of a short episode of testicular heating (43 degrees C for 15 min) on spermatogenesis and Sertoli and Leydig cell function. Rats killed at intervals up to 156 days after heating were assessed by histological examination, and by measurement of serum FSH and LH, and by tests of Sertoli cell function consisting of fluid production, androgen binding protein (ABP) content of the ligated and unligated tests, together with the binding of [125I]FSH. Leydig cell function was assessed by in vitro testosterone production, serum testosterone levels and [125I]hCG binding to testes homogenates. Testis weight declined 7 days after heating to 70% of control and remained lower until 82 days, whereas epididymal weight did not decrease significantly until 26 days and also recovered by 82 days. Fluid production was significantly lower in heated testes at 26 days and returned to normal at 56 days. ABP production measured as the difference between the ABP content of ligated and unligated testes was significantly reduced at 14 and 26 days, but subsequently recovered. Serum FSH levels were significantly elevated from 14-26 days in the heat treated group and the binding of [125I]FSH was reduced at 26 days post-heating. Basal and stimulated in vitro T production was significantly increased in the heat-treated testes at 14 days and subsequently returned to normal whilst [125I]hCG binding was significantly lower in the heat-treated testes from 7-26 days. Serum T and LH did not alter significantly during the study. Primary spermatocytes and young spermatids were the most heat sensitive germ cell type and a reduction in spermatogenesis was noted from 7 to 26 days, although recovery appeared complete by 56 days and thereafter. These results demonstrate that the transient spermatogenic disruption induced by heating is accompanied by significant alterations in Sertoli and Leydig cell function which are identical to those produced in other models of spermatogenic dysfunction. The results suggest that the duration of these changes appears to correlate closely with alterations occurring in the germ cell compartment.     

Characterization of functional Leydig cells after purification on a continuous gradient of percoll

Two human chorionic gonadotropin (hCG) responsive cells from rat testicular interstitium were previously isolated on a discontinuous gradient of Percoll. The light cells were non-steroidogenic and bound 125I-labeled hCG with high affinity (Kd 3.0 x 10(-10) mol/L), whereas the steroidogenic heavier cells (Leydig cells) produced cyclic adenosine monophosphate (cAMP) and testosterone in response to hCG stimulation with very little hCG binding. In that study, the heavier cell fraction was contaminated with germ cells, red blood cells, and other cells. These cells have now been further purified on a continuous gradient of Percoll (20 to 60%, v/v), and have resolved into three visible bands. The cells in subfraction I, predominantly damaged Leydig cells, germ cells, and/or residual light cells, bind 125I-labeled hCG with high affinity (Kd 4.09 x 10(-10) mol/L) without producing cAMP and testosterone in response to hCG. Subfraction III consists mainly of red blood cells. The cells in subfraction II, identified as typical Leydig cells by electron microscopy, produce cAMP and testosterone in response to hCG but, again, bind only a small amount of hCG (4.5 +/- 0.3 fmol/2 x 10(6) cells/250 microliters/per hour at 37 degrees C). Thus, further purification of the heavier cell fraction from a discontinuous gradient of Percoll on a continuous gradient of Percoll yields Leydig cells, free of contaminating germ cells and red blood cells, which actively produce cAMP and testosterone with a very low level of hCG binding, the affinity of which is undetectable by current binding techniques.     

Heterogeneity of adult mouse Leydig cells with different buoyant densities.

The authors investigated the morphologic characteristics and human chorionic gonadotrophin (hCG)-stimulated testosterone production of adult mouse Leydig cells in vitro, which have different buoyant densities. Leydig cells from five testes of Swiss outbred male mice (15 weeks old) were isolated and purified by mechanical dispersion followed by density gradient centrifugation using Percoll. Two groups of Leydig cells were obtained with different buoyant densities: group 1 had densities of 1.0667 to 1.0515 g/cm3 and group 2 had densities of 1.0514 to 1.0366 g/cm3. In vitro testosterone production of these Leydig cells, in response to different doses of hCG (0, 5, 25, 125, 625, and 3125 pg/mL), was determined by radioimmunoassay. Leydig cells were fixed and processed for electron microscopic stereology to quantify the organelles by volumes and surface area. In Leydig cells of Group 1, testosterone production per cell in vitro in response to 0 and 5 pg/mL hCG was not significantly different (P greater than 0.05). Increases in the dose up to 25 pg/mL produced a significant increase (P less than 0.05) in testosterone production, although hCG doses of 125 and 625 pg/mL did not produce further increases in testosterone levels. However, 3125 pg/mL hCG further elevated the testosterone production by those Leydig cells with high buoyant density. In Leydig cells in group 2, the patterns of testosterone production in response to hCG doses of 0, 5, and 25 pg/mL were similar to those of Leydig cells in group 1. Those Leydig cells with low buoyant density, however, were unable to stimulate further testosterone production by an hCG dose of 3125 pg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠睾丸Leydig细胞的培养和鉴定

目的:研究体外培养大鼠睾丸Leyd ig细胞的有效方法。方法:原代培养大鼠睾丸Leyd ig细胞,用4 U/m l人绒毛膜促性腺激素(hCG)作用细胞,对照组未用hCG,放射免疫法测定培养液中睾酮浓度,3β羟类固醇脱氢酶(3β-HSD)免疫组化染色观察睾丸Leyd ig细胞形态和生物学特性。结果:培养细胞成分均一、增殖旺盛、分化率高。接种72 h后大鼠睾丸Leyd ig细胞纯度达95%。接种后24 h内,hCG刺激组较对照组睾酮分泌量明显提高(P<0.05)。结论:体外培养的睾丸Leyd ig细胞可分泌高浓度的睾酮;睾丸Leyd ig细胞的纯化和培养方法的建立,可为中老年男性雄激素部分缺乏综合征睾酮替代治疗的基础和临床研究提供一条可行的思路。     

Effect of seminiferous tubule size on hCG-induced regeneration of peritubular Leydig cells in hypophysectomized, EDS-treated rats

Following their selective destruction 3 weeks previously by administration of ethane dimethanesulphonate (EDS) the regenerative capacity of Leydig cells was assessed in relation to seminiferous tubule morphology in hypophysectomized adult rats administered 7 daily injections of 100 iu hCG. Total Leydig cell volume per testis in hCG-treated rats (30.2 ±3.2 μl, mean ± SEM) was significantly ( p <0.01) greater than in the testes of rats at 3 and 4 weeks after EDS-treatment (7.6 ± 0.7 and 22.7 ± 1.4 μl, respectively). Regeneration of Leydig cells in hCG-treated rats significantly ( p <0.05) favoured peritubular locations (18.6 ± 2.8 μl/testis) compared to central or perivascular sites of origin (11.6 ± 1.2 μl/testis). Partial restoration of spermatogenesis occurred in hCG-treated rats (tubule diameters usually >250μm) and a significant inverse correlation was found between peritubular Leydig cell percentage, or total volume per testis, and the volumetric proportion of seminiferous tubules ( r =-0.94, p <0.001) or the seminiferous epithelium ( r =-0.73 to -0.79, p <0.05–0.01). No significant ( p >0.4–0.9) correlation existed between centrally-regenerated Leydig cells and these parameters. The results show that in response to hCG stimulation, Leydig cells are more likely to develop around smaller seminiferous tubules, suggesting that hCG alone cannot mimic the expected pattern of Leydig cell regeneration (central and peritubular origins) which occurs during normal sexual maturation or at 3–4 weeks after EDS treatment. It is concluded that other factors, possibly FSH, are required for typical Leydig cell development which in turn may be influenced by local cellular growth factors originating from either the seminiferous tubules or the adjacent intertubular tissue.     

Leydig Cell Function in Rats Chronically Deprived of Normal Gonadotrophic Stimulation: The Effect of Treatment with hCG

Leydig cell function was assessed in adult rats 11 months after active immunization against LH-RH. Immunized rats were divided into 2 groups according to whether the serum levels of LH and FSH were always undetectable (Group A) or were detectable at some time during the 11 months (Group B). Compared with controls, testicular weight was reduced by 84% (Group A) and 78% (Group B), but the number of Leydig cells per unit area of testis was increased by a factor of 7. Despite the latter change, the binding of ¹²⁵I-hCG per 20 mg testicular homogenate was 40% lower in immunized than in control rats. The serum level of testosterone in immunized rats was ≤ 0.1 ng/ml whilst levels ranged from 0.7 to 2.9 ng/ml in controls. In the same rats the testicular production of testosterone in vitro , both basally and after incubation with hCG or dibutryl cyclic AMP, was reduced by over 90% in immunized animals, although this impairment was more severe in Group A than in Group B animals.
Leydig cell function was also assessed at 48 h after a single injection of 100 IU hCG. This treatment reduced the testicular binding of ¹²⁵I-hCG by over 89% in controls as well as both groups of immunized rats and caused a 3- to 4-fold increase in the serum levels of testosterone in all animals. Following injection of hCG, the basal production of testosterone by the testis in vitro was doubled in controls whereas this increase was over 50-fold in Group A immunized rats. In the latter, the maximum steroidogenic response of the testis in vitro was more than trebled by prior injection of hCG whilst this treatment caused a 30% reduction in the maximum response of testes from controls.     

Stimulation of the proliferation and differentiation of Leydig cell precursors after the destruction of existing Leydig cells with ethane dimethyl sulphonate (EDS) can take place in the absence of LH

In hypophysectomized rats, 2 days after the administration of the cytotoxic drug ethane dimethyl sulphonate (EDS), the proliferative activity of Leydig cell precursors increased six-fold. Thus, factors other than LH act locally to stimulate the proliferation of precursor cells after EDS. Twenty-six days after EDS administration, neither cells with the morphological characteristics of Leydig cells nor histochemical enzyme activities, such as 3 beta-HSD and alpha-naphtyl esterase, could be detected in testis tissue. In hypophysectomized rats treated daily with hCG (100 iu) for 7 days, starting at 26 days after EDS, the number of Leydig cells was increased to 48 +/- 11 cells (per 1000 Sertoli cells), which is approximately 4.5% of the intact control level. 3 beta-HSD and alpha-naphtyl esterase activity could be detected, and plasma testosterone levels had increased 15-fold compared with the hypophysectomized controls. These results show that proliferation and some differentiation of precursor cells along the Leydig cell lineage can occur independent of LH, but the final stages of the differentiation process require hCG stimulation.     

Binding and internalization in vivo of [125I]hCG in Leydig cells of the rat

The present study was performed to demonstrate the binding, mode of uptake, pathway and fate of iodinated human chorionic gonadotropin ([125I]hCG) by Leydig cells in vivo using electron microscope radioautography. Following a single injection of [125I]hCG into the interstitial space of the testis, the animals were fixed by perfusion with glutaraldehyde at 20 minutes, 1, 3, 6 and 24 hours. The electron microscope radioautographs demonstrated a prominent and qualitatively similar binding of the labeled hCG on the microvillar processes of the Leydig cells at 20 minutes, 1, 3, and 6 hours. The specificity of the [125I]hCG binding was determined by injecting a 100-fold excess of unlabeled hormone concurrently with the labeled hormone. Under these conditions, the surface, including the microvillar processes of Leydig cells, was virtually unlabeled, indicating that the binding was specific and receptor-mediated. In animals injected with labeled hCG and sacrificed 20 minutes later, silver grains were also seen overlying the limiting membrane of large, uncoated surface invaginations and large subsurface vacuoles with an electron-lucent content referred to as endosomes. A radioautographic reaction was also seen within multivesicular bodies with a pale stained matrix. At 1 hour, silver grains appeared over dense multivesicular bodies and occasionally over secondary lysosomes, in addition to the structures mentioned above, while at 3 and 6 hours, an increasing number of secondary lysosomes became labeled. At 24 hours, binding of [125I]hCG to the microvillar processes of Leydig cells persisted but was diminished, although a few endosomes, multivesicular bodies and secondary lysosomes still showed a radioautographic reaction. No membranous tubules that were seen in close proximity to, or in continuity with, endosomes and multivesicular bodies were observed to be labeled at any time interval. Likewise, an attempt to correlate silver grains with small coated or uncoated pits, the stacks of saccules of the Golgi apparatus and other Golgi-related elements including GERL, proved unsuccessful, since these structures were mostly unlabeled. These in vivo experiments thus demonstrate the specific binding of [125I]hCG to the plasma membrane of Leydig cells predominantly on their microvillar processes, and the subsequent internalization of the labeled hCG to secondary lysosomes. In addition, binding and internalization of hCG persisted for long periods of time.     

Study on ethane dimethane sulphonate (EDS)-induced spermatogenic damage and protective drugs against this damage in the rat]

The effects of a single administration of ethane dimethane sulphonate (EDS), which has a direct cytotoxic effect on Leydig cells, was assessed for its spermatogenic damage and intratubular androgen level in SD male adult rats. The protective effect of human chorionic gonadotropins (hCG) (s.c.), testosterone propionate (TP) (s.c.) and intratesticular administration of testosterone microcrystal suspension (Tmcs) against the spermatogenic damage in rats EDS given was also evaluated. EDS caused a decrease of the seminiferous tubular diameter and impaired spermatogenesis remarkably; moreover, it also caused significant decreases in intratubular androgen levels. These results suggest that EDS-treated SD male adult rats may be suitable as a model for hormone dependent infertility. The administration of hCG and intratesticular Tmcs prevented tubular damage and increased the intratubular T level. On the other hand, the administration of TP prevented tubular damage while remarkably decreasing intratubular androgen level. In this connection, it was inferred that priming of rats with TP caused an increase in intratubular androgen binding protein, which would stimulate spermatogenesis. The fact that a single injection of Tmcs caused no tubular damage suggests that intratubular T level is one of the factors playing an important role in spermatogenesis and that an intratesticular injection of Tmcs may be useful for the treatment of some cases of idiopathic male infertility.     

Increased testosterone production in vitro by Leydig cells from rats with severe autoimmune orchitis

We have previously observed (M. O. Suescun et al. , 1994, Journal of Andrology , 15 , 442–448) that rats with autoimmune orchitis (EAO) exhibit increased testosterone production in vitro by isolated testes. The aim of the present study was to determine whether the increase in testosterone production correlated with an enhanced number of Leydig cells and/or enhanced steroidogenic capacity per Leydig cell. For this purpose, EAO was induced in adult Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. At 80 days after the primary immunization, 60% of rats presented with severe testicular damage characterized by sloughing of the seminiferous epithelium, seminiferous tubule atrophy and interstitial mononuclear cell infiltration. At 160 days after the first immunization, testicular lesions were more severe. A morphometric study, by light microscopy, showed an increase in the number of Leydig cells in rats with EAO (45% increase at 80 days and 50% at 160 days). By electronmicroscopy, testicular sections of rats with EAO revealed the presence of numerous Leydig cells closely associated with macrophages. Most Leydig cells exhibited ultrastructural features of active steroid secreting cells.
The steroidogenic capacity of Percoll-purified Leydig cells from testes of rats with EAO, killed at 80 and 160 days, was evaluated. Leydig cells from rats with EAO exhibited an enhanced steroidogenic response to hCG in vitro at 80 days (38%) and an increase in basal (77%) and post-hCG testosterone production (115%) at 160 days compared to controls. However, these cells were less sensitive to hCG. In conclusion, the results indicate that the enhancement of in-vitro testosterone production observed in rats with EAO is accounted for both by the increased number of Leydig cells and by the increased testosterone production of each Leydig cell.