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.     

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)     

Simultaneous proliferation and differentiation of mast cells and Leydig cells in the rat testis. Are common regulatory factors involved?

The proliferation and differentiation of mast cells and Leydig cells were studied in adult sham operated or hypophysectomized rats after the administration of ethylene dimethane sulphonate (EDS) and in prepubertal rats after neonatal treatment with a gonadotropin-releasing hormone (GnRH) antagonist (Organon 30276; Oss, The Netherlands). After treatment with EDS, two proliferative waves were found. On day 3, several interstitial cell types proliferated, whereas mitotic cells corresponded to differentiating Leydig cells and mast cells around day 20. Differentiating Leydig cells showed a higher mitotic index than that of differentiating mast cells. Hypophysectomized animals showed high mitotic activity 3 days after treatment, but 21 days after treatment differentiating Leydig cells were absent and proliferative activity was reduced. The number of mast cells increased from day 15 to day 30 in EDS-treated rats and from day 15 to day 50 in hypophysectomized, EDS-treated rats. GnRH antagonist-treated rats showed poorly differentiated Leydig cells and abundant mitotic figures on day 23. Proliferation and differentiation of Leydig cells occurred concomitantly with the proliferation and differentiation of mast cells between 23 and 30 days of age. These results suggest that Leydig cells and mast cells in the rat testis share some common regulatory factors.     

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.     

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.     

Testosterone and androstanediol production by regenerating Leydig cells in the ethylene dimethane sulphonate-treated mature rat

Mature (60-65 day old) male Sprague-Dawley rats received a single intraperitoneal injection of ethylene dimethane sulphonate (EDS; 100 mg/kg) and were subsequently killed at various times from day 2 to day 40 post-treatment. Testes were removed from these animals and age-matched controls and utilized either for light and electron microscopical analyses or for in-vitro assessment of Leydig cell function. Interstitial cells were prepared by collagenase digestion and used to measure 125I-labelled human chorionic gonadotrophin (hCG) binding capacity and androgen production in the presence or absence of hCG or dibutyryl cyclic AMP (dbcAMP). At day 2 after EDS treatment, 125I-labelled hCG binding capacity was reduced to 10% of control values, while the production of testosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (adiol) were non-detectable. Histological observations confirmed the lack of identifiable Leydig cells at day 2-16 after EDS treatment. Between days 24 and 40 post-treatment, Leydig cell regeneration occurred, as indicated by a rise in 125I-labelled hCG binding capacity, increased androgen production and the presence of histologically identifiable Leydig cells. A pattern of adiol production similar to that seen in the immature rat during Leydig cell development was observed with peak synthesis occurring at day 30 post-treatment. Adiol production fell to barely detectable levels by day 36 and remained low at day 40. It is concluded that the steroidogenic pattern of regenerating Leydig cells in the EDS-treated animal is similar to that of developing Leydig cells in the immature animal.     

Intratesticular factors and testosterone secretion. Effect of treatments that alter the level of testosterone within the testis

The authors recently have reported the presence of a nongonadotropic polypeptide factor in rat testicular interstitial fluid that can exert marked stimulatory effects on Leydig cell testosterone production. To assess the potential physiologic significance of this factor, its effective levels in rat interstitial fluid have been investigated in response to treatments that either markedly reduce interstitial fluid testosterone concentrations (anti-LH treatment; transient or chronic experimental cryptorchidism; destruction of Leydig cells with ethane dimethanesulphonate) or that significantly elevate testosterone levels in interstitial fluid by injection of hCG. The possible relationship between this factor and changes in testicular weight, serum LH and FSH, and interstitial fluid volume also were monitored. When testosterone levels in interstitial fluid were decreased by 75 to 99% either acutely (5-72 hours) or chronically (20-75 days), there was an accompanying increase (P less than 0.001) in the levels of the interstitial fluid factor(s), as determined by the ability of charcoal-stripped interstitial fluid from individual rats to enhance hCG-stimulated testosterone production by Percoll-purified Leydig cells in vitro. Anti-LH treatment increased the levels of the interstitial fluid factor(s) over the ensuing 5 to 48 hours. In abdominal testes from rats made unilaterally or bilaterally cryptorchid for 20 or 55 days, a decrease in interstitial fluid testosterone levels was associated with increased levels of the interstitial fluid factor(s). The same inverse relationship was found 72 hours after treatment with ethane dimethanesulphonate in which Leydig cells had disappeared from the testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory cytokine expression and interstitial fluid formation in the normal and inflamed rat testis are under leydig cell control

Leydig cells have been implicated in several inflammation-related responses of the testis. Specifically, these cells produce the proinflammatory cytokines interleukin-1 (IL-1) and IL-6, stimulate macrophage recruitment, and promote interstitial fluid formation. In addition, the immunoregulatory cytokines macrophage migration inhibitory factor (MIF), transforming growth factor-beta1 (TGFbeta1), and interferon-gamma (IFNgamma) are constitutively expressed by testicular cells, including the Leydig cells. In the present study, the contribution of the Leydig cell to testicular inflammatory responses was examined in adult male rats treated with the Leydig cell-specific toxin, ethane dimethane sulfonate (EDS). Intratesticular testosterone levels were modulated by subcutaneous testosterone implants. After 10 days, animals received an injection of lipopolysaccharide (LPS) to induce an inflammatory response, or saline alone, and were killed 3 hours later. Both depletion of Leydig cells by EDS and LPS treatment caused a decrease in collected testicular interstitial fluid to about 35% of control levels, but the effects were not additive. Maintenance of intratesticular testosterone reversed the interstitial fluid decline following EDS treatment and partially prevented the LPS-induced effect. MIF, TGFbeta1, and IFNgamma were expressed in both the normal and inflamed testis at similar levels. In contrast, EDS treatment caused a significant decline in expression of all 3 cytokines, which was prevented by the testosterone implants. These data indicate that 1) expression of TGFbeta1, MIF, and IFNgamma in the testis is not dependent on the presence of intact Leydig cells but is under direct testosterone control and 2) the decline in testicular interstitial fluid during inflammation involves the Leydig cells, acting via both androgens and nonandrogenic secretions. These data provide further support for a significant role for the Leydig cell in modulating the testicular response to inflammation.     

Evaluation of the relative importance of endocrine and paracrine factors in control of the levels of inhibin in testicular interstitial fluid

This study aimed to identify the role of endocrine (FSH, LH, testosterone) or paracrine (Leydig or germ cell) factors in control of the secretion of inhibin into testicular interstitial fluid (IF). This was done by measuring inhibin and testosterone levels in IF, and serum gonadotrophin and testosterone levels in adult rats following the destruction of Leydig cells with ethane dimethane sulphonate (EDS), alone or in combination with testosterone ester (TE) supplementation at various doses initiated at various times after EDS treatment. The effect of germ cell loss (induced by local testicular heating) on its own or in combination with the above treatments was also assessed. Treatment with EDS led to major increases in the levels of inhibin in IF and of FSH and LH in serum whilst testosterone levels in IF and serum fell to undetectable levels. Supplementation with TE (1-25 mg) for 21 days from the time of EDS treatment failed to prevent the initial (+3 days) increase in IF levels of inhibin but thereafter suppressed inhibin to control levels or lower and grossly suppressed FSH and LH levels, irrespective of whether the dose of TE administered did (25 or 5 mg) or did not (1 mg) prevent major seminiferous tubule damage. Partial regeneration of Leydig cells and normalization of testosterone levels occurred in rats 21 days after treatment with EDS alone but this failed to normalize inhibin and gonadotrophin levels. When supplementation with TE (25 mg) was initiated at 3, 6 or 9 days after EDS treatment, IF levels of inhibin were normalized within 3 days and maintained thereafter in parallel with suppression of serum FSH and LH to below control levels. Seminiferous tubule damage induced by local testicular heating (43 degrees C for 30 min) led to increased IF levels of inhibin 3 and 14 days later, in parallel with increased serum levels of FSH (but not LH). Suppression of FSH to subnormal levels in heat-exposed rats by TE treatment (25 mg) restored IF inhibin to control levels or below, a change which still occurred when Leydig cells were destroyed by EDS treatment. It is concluded that secretion of inhibin via the base of the Sertoli cell into testicular IF is controlled primarily by FSH, although local factors may play a minor role. These findings have important implications regarding the possible paracrine role(s) of inhibin in IF during puberty and in the normal adult testis.     

Inhibin secretion is influenced by Ley dig cells: evidence from studies using the cytotoxin ethane dimethane sulphonate (EDS)

Adult male rats given a single intraperitoneal injection of the Leydig cell cytotoxin ethane dimethane sulphonate (EDS) show a significant decrease in testosterone from 7 to 14 days, and elevation of serum FSH and LH levels commencing 7 days after treatment, returning to normal at 28 days for LH and 49 days for FSH. A significant rise in serum inhibin levels was seen at day 14 after EDS treatment with levels returning to normal at day 49. In a second series of experiments, silastic implants of testosterone, either 2.5 cm or 22.5 cm in length, were introduced subcutaneously into adult male rats which were treated with EDS 10 days later. Both doses of testosterone suppressed basal LH levels but did not significantly change FSH levels. The rise in FSH and LH levels seen in normal rats after EDS treatment did not occur in either group of testosterone-implanted rats. However, serum inhibin levels rose significantly in both groups after EDS treatment, suggesting that the rise in serum inhibin levels was not due to stimulation arising from the increase in FSH levels after EDS treatment. The data suggest that the rise in serum inhibin levels after EDS treatment is linked to destruction of the Leydig cells through mechanisms that require further investigation.     

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.     

The levels and possible involvement of leukotriene B4 and prostaglandin F2α in the control of interstitial fluid volume in the rat testis

The possible involvement of two arachidonic acid metabolites, prostaglandin F2 alpha (PGF2 alpha) and leukotriene B4 (LTB4), in the stimulatory effect of human chorionic gonadotrophin (hCG) on the volume of interstitial fluid (IF) in the rat testis has been investigated. Administration of hCG caused a time- and dose-dependent increase in the IF levels of PGF2 alpha while LTB4 showed no clear dose-dependence, but did decrease significantly at 2-8 h after injection of 100 IU hCG. Administration of ethane dimethane sulphonate (EDS), which specifically destroys Leydig cells, decreased the volume of IF but the IF levels of LTB4 and PGF2 alpha were unchanged. This indicates that although the absence of Leydig cells results in significant changes in IF volume, LTB4 and PGF2 alpha are probably not involved in these changes. Furthermore, these findings suggest that Leydig cells are not the only contributors to LTB4 and prostaglandins E2 and F2 alpha in testicular IF. In rats injected peripherally with hCG, intratesticular administration of the cycloxygenase inhibitor, indomethacin, decreased PGF2 alpha levels in IF after 2 and 4 h but not at later times. However, no inhibitory effect of indomethacin on the hCG-stimulated increase in IF volume was detected; indeed, at 2 h after injection of indomethacin + hCG there was a significant increase in IF volume in the indomethacin-injected testis. It is concluded that, although prostaglandins and LTB4 are secreted into IF in the rat testis, these arachidonic acid metabolites are apparently not the primary mediators of the increase in IF volume that follows hCG treatment.     

Effects of repeated injections of human chorionic gonadotrophin on vascular permeability, extracellular fluid volume and the flow of lymph in the testes of rats

Testicular lymph flow, interstitial fluid volume and vascular permeability have been measured in adult male rats injected subcutaneously with hCG daily for up to 4 consecutive days. Albumin clearance was also measured in rats given two or three hCG injections at 2- or 3-day intervals, or every 2 days for 22 days. While a single dose of hCG increased vascular permeability and lymph flow within 24 h, subsequent daily injections did not produce any additional response and, in fact, values returned towards control levels. A second hCG dose 2 days after an initial dose did produce another similar increase in the clearance of albumin injected directly into the testis and, with continued injections every second day, a response was still evident after 22 days. The response to the second dose of hCG occurred at a time when down-regulation of hCG receptors on Leydig cells is reported to be maximal. These results suggest that in the testis, the vascular response to hCG does not require the normal number of luteinizing hormone (LH) receptors, although other evidence suggests that Leydig cells must somehow be involved.     

Response of testicular macrophages to EDS-induced Leydig cell death

Summary.  The response of testicular macrophages to massive Leydig cell death was studied by the administration of the specific Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) to sham-operated (SO), short-term (STHX), and long-term (LTHX) hypophysectomized rats. EDS-killed Leydig cells showed the morphological features of the programmed cell death or apoptosis. A 2-fold increase in the number of macrophages was found on days 1–2 after treatment in both SO and STHX rats, and dead Leydig cells were completely eliminated by day 3 after treatment. Otherwise, in LTHX rats, there was a delay in the increase in the number of macrophages, and EDS-killed Leydig cells remained in the testicular interstitium for several days. These results indicate that the phagocytic capacity of the macrophage population was diminished in hypophysectomized rats, and particularly after long-term hypophysectomy.     

Response to acute hCG stimulation and steroidogenic potential of Leydig cell fibroblastic precursors in humans

The process of early testosterone (T) secretion and Leydig cell differentiation in humans was studied to explore the steroidogenic capacity of Leydig cell fibroblastic precursors. Seven cryptorchid boys received hCG prior to orchidopexy. Patients CP, PB, and MR received one injection of 1000 IU; patients JR and GG, three daily injections of 1000 IU, and patients MP and MM, five daily injections of 1000 IU. A testicular biopsy was obtained at the time of operation, 24 hours after the last injection. Serum T (ng/dl) before and after hCG stimulation and testicular T (ng/g) were determined by RIA. A control prepubertal testis (tumoral orchidectomy) was incubated in vitro and showed a time-dependent accumulation of T both in the medium and the testicular tissue. Testosterone released into the medium at 1, 2, and 4 hours was 0.76, 1.43, and 4.03 ng/ml, respectively. Tissue T at 0, 1, 2, and 4 hours was 9, 11, 16, and 24 ng/g, respectively. This indicates synthesis and secretion of T into the medium. Control testes showed abundant fibroblastic precursors with scanty cytoplasm, few organelles, heterochromatic nuclei, and minute nucleoli. No Leydig cells were present. After 1 day of hCG stimulation, numerous fibroblasts were activated, displaying enlarged cytoplasms with increased numbers of organelles, nuclei rich in euchromatin, and bigger nucleoli. No Leydig cells were present. Basal serum testosterone was 58.2 +/- 45.3 ng/dl and 87.3 +/- 42.0 after hCG administration, while testicular T was 974.0 +/- 686.0 ng/g (control prepubertal testicular T is 10-50 ng/g). After 3 days of hCG, activated fibroblasts increased and immature Leydig cells appeared. Basal serum T was 35.5 +/- 7.8 ng/dl and 394.0 +/- 24.0 after hCG stimulation, while testicular T rose to 2797.5 +/- 1222.6 ng/g. After 5 days, mature Leydig cells appeared for the first time. Serum T was 58 +/- 59.3 ng/dl (basal) and 641.5 +/- 390 ng/dl (after hCG); testicular T was 789 ng/g (patient MM did not have a value for testicular T). HCG induced numerous coated pits and endocytic vesicles in activated fibroblasts and young Leydig cells, suggesting receptor aggregation and internalization of hormone-receptor complexes. Peroxidase-antiperoxidase (PAP) localization of T was positive in peritubular fibroblasts and Leydig cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

大鼠睾丸Leydig细胞体外增殖研究

目的:探讨通过优化细胞培养体系,实现Leydig细胞的体外增殖培养。方法:联合应用胶原酶消化、不锈钢滤网过滤及差速贴壁法获得3周龄雄性Wistar大鼠睾丸Leydig细胞,贴壁细胞以DMEM/F12培养液及优化培养体系培养,MTT法、细胞计数法检测培养细胞的增殖能力;分别对原代培养2h、4d细胞进行3β-HSD免疫化学染色及流式细胞术分析,检测细胞成分,同时检测培养细胞睾酮在hCG刺激下分泌能力变化。结果:优化培养体系能够明显促进3周龄大鼠睾丸Leydig细胞大量增殖,群体倍增时间为(2.26±0.31)d,传统培养体系培养细胞群体倍增时间为(16.32±2.14)d,两者差异有显著性(P<0.05);原代细胞经流式细胞术鉴定,3β-HSD阳性细胞所占比例分别为(54.3±7.1)%,培养4d后,增殖细胞3β-HSD阳性细胞率为(93.6±4.6)%。增殖细胞均有睾酮生成功能,在hCG刺激下睾酮分泌均明显上升(P<0.05)。结论:优化培养体系能够促进差速贴壁法获得的睾丸Leydig细胞大量增殖。     

The importance of the Leydig cells in the vascular response to hCG in the rat testis

The increase in permeability of the testicular blood vessels following an injection of hCG into rats is abolished completely if the animals are treated 3 days earlier with ethane dimethane sulphonate (EDS), a compound that effectively eliminates Leydig cells from the testes. As there is other evidence that androgens or prostaglandins are not involved in this vascular response, further studies will be necessary to determine whether these data mean that another vasoactive substance is secreted by the Leydig cells or whether the EDS also eliminates other cells besides the Leydig cells, for example the mast cells found in the vicinity of the testicular artery.     

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.     

Intratesticular factors and testosterone secretion: The effect of treatment with ethane dimethanesulphonate (EDS) and the induction of seminiferous tubule damage

The role of seminiferous tubule dysfunction in regulating the levels of a factor (or factors) in testicular interstitial fluid (IF) which stimulates Leydig cell testosterone secretion in vitro, was assessed by injecting rats with the Leydig cell toxin, EDS. Within 72 h of treatment EDS destroyed the Leydig cells and concomitantly reduced IF testosterone to undetectable levels. This was associated with nearly a 2-fold increase (P less than 0.001) in levels of the IF-factor(s) as judged by the enhancement of hCG-stimulated testosterone production (= IF bioactivity). By 3 weeks, and thereafter up to 10 weeks post-EDS, Leydig cells regenerated within the testis, and testosterone levels returned to control values, but IF-bioactivity remained significantly increased. The latter was associated with seminiferous tubule dysfunction as indicated initially by testicular morphology, raised serum levels of FSH and reduced testicular weight. For animals with normal testosterone levels, there was a significant negative correlation (r = -0.57, N = 46; P less than 0.001) between testicular weight and IF bioactivity. A similar increase in IF bioactivity in the presence of normal testosterone levels was observed in rats in which patchy severe seminiferous tubule damage had been induced by short-term cryptorchidism. It is concluded that, in addition to testosterone, seminiferous tubule function may dictate the intratesticular levels of the testosterone-stimulating factor(s) in IF.     

Variation in the effect of a nongonadotropic Leydig cell stimulating factor in testicular interstitial fluid after exposure of the testis to a single episode of heat treatment

The activity of a nongonadotropic factor present in rat testicular interstitial fluid and capable of stimulating Leydig cell testosterone production was measured at intervals for 8 weeks after exposure of the testis to a single episode of heat treatment (43 C for 15 minutes). The activity of this factor was determined using a Leydig cell bioassay and the testicular albumin space (a measure of the interstitial fluid volume) was determined after the injection of 125I-labeled bovine serum albumin. Additionally, the response of interstitial cells to hCG stimulation was measured at each time point after heat treatment. The response of interstitial cells to hCG and the activity of the interstitial fluid were both increased transiently 2 or 3 weeks after heat treatment coincident with the disruption of spermatogenesis. These data provide further evidence for the local interaction of the tubules and interstitium and imply a role for the testicular interstitial fluid factor in these local regulatory mechanisms.