Ethane Dimethanesulphonate Treatment Research Articles

Identification of Leydig cell-specific mRNA transcripts in the adult rat testis

The adult population of Leydig cells acts to secrete testosterone which is essential for reproductive health and fertility in the adult male. However, other physiological functions of these cells are uncertain, and to address this issue a cell ablation model has been used to identify Leydig cell-specific mRNA transcripts. Ethane dimethane sulphonate (EDS) was synthesised by a novel process and was used to ablate Leydig cells in adult male rats previously treated with butane dimethane sulphonate (busulphan) to delete the germ cell population. Levels of mRNA transcripts were measured in the testis using microarrays 1, 3, 5, 8 and 12 days after EDS injection. During this period, there was a significant change in the levels of 2200 different transcripts with a marked decline in the levels of canonical Leydig cell transcripts, such as Cyp11a1, Cyp17a1 and Insl3. A total of 95 transcripts showed a similar decline in expression after EDS treatment, suggesting that they have a Leydig cell-specific origin. Analysis of selected transcripts confirmed that they were expressed specifically in Leydig cells and showed that most had a late onset of expression during adult Leydig cell development. Apart from transcripts encoding components of the steroidogenic apparatus, the most common predicted function of translated proteins was endogenous and xenotoxicant metabolism. In addition, a number of transcripts encode acute-phase proteins involved in reduction of oxidative stress. Results show that, in addition to androgen secretion, Leydig cells may have a critical role to play in protecting the testis from damage caused by toxicants or stress.

Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate.

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.

Effects of thyroid hormone on Leydig cell regeneration in the adult rat following ethane dimethane sulphonate treatment.

We tested the effects of thyroid hormone on Leydig cell (LC) regeneration in the adult rat testis after ethane dimethyl sulphonate (EDS) treatment. Ninety-day-old, thyroid-intact (n = 96) and thyroidectomized (n = 5) male Sprague-Dawley rats were injected intraperitoneally (single injection) with EDS (75 mg/kg) to destroy LC. Thyroid-intact, EDS-treated rats were equally divided into three groups (n = 32 per group) and treated as follows: control (saline-injected), hypothyroid (provided 0.1% propyl thiouracil in drinking water), and hyperthyroid (received daily subcutaneous injections of tri-iodothyronine, 100 microg/kg). Testing was done at Days 2, 7, 14, and 21 for thyroid-intact rats and at Day 21 for thyroidectomized rats after the EDS treatment. Leydig cells were absent in control and hyperthyroid rats at Days 2, 7, and 14; in hypothyroid rats at all ages; and in thyroidectomized rats at Day 21. The LC number per testis in hyperthyroid rats was twice as those of controls at Day 21. 3beta-Hydroxysteroid dehydrogenase (LC marker) immunocytochemistry results agreed with these findings. Mesenchymal cell number per testis was similar in the three treatment groups of thyroid-intact rats on Days 2 and 7, but it was different on Days 14 and 21. The highest number was in the hypothyroid rats, and the lowest was in the hyperthyroid rats. Serum testosterone levels could be measured in control rats only on Day 21, were undetectable in hypothyroid rats at all stages, and were detected in hyperthyroid rats on Days 14 and 21. These levels in hyperthyroid rats were twofold greater than those of controls on Day 21. Serum androstenedione levels could be measured only in the hyperthyroid rats on Day 21. Testosterone and androstenedione levels in the incubation media showed similar patterns to those in serum, but with larger values. These findings indicate that hypothyroidism inhibits LC regeneration and hyperthyroidism results in accelerated differentiation of more mesenchymal cells into LC following the EDS treatment. The observations of the EDS-treated, thyroidectomized rats confirmed that the findings in hypothyroid rats were, indeed, due to the deficiency of thyroid hormone.

Mitosis of resident macrophages in the adult rat testis.

Resident macrophages are maintained at a comparatively high, yet stable, tissue concentration in the adult rat testis. After destruction of Leydig cells by ethane dimethane sulphonate treatment, the number of resident macrophages increases briefly and then decreases to below normal values, but returns to normal after the reappearance of Leydig cells. The mechanisms by which the adult testicular macrophage population is maintained, either by monocyte recruitment or by mitosis of the resident macrophages, have not been examined. An immunohistochemical dual labelling approach using a specific monoclonal antibody for resident macrophages, ED2, and markers of mitotic activity (bromodeoxyuridine incorporation and expression of the proliferating cell nuclear antigen) was used to investigate resident macrophage proliferation in Bouin's-fixed paraffin wax-embedded adult rat testes. Detection of the normally fixation sensitive antigen recognized by ED2 was achieved by using a decreased fixation time and antigen retrieval. Peaks of resident macrophage mitotic activity were observed during the phases of macrophage proliferation immediately after ethane dimethane sulphonate treatment and during the recovery phase associated with Leydig cell restoration. These data demonstrate that resident macrophages have the capacity to proliferate within the adult rat testis and, thus, this population of resident macrophages is maintained, at least in part, by mitotic division in situ.

Inhibin-related proteins in rat prostate.

Inhibin and activin are members of the transforming growth factor beta (TGF beta) family which can regulate cell proliferation in a number of tissues. The presence of inhibins and the related proteins, activins, in the prostate has been implicated by the detection of activin type II receptors. The aim of this study was to determine whether or not immunoactive (ir) inhibin and ir-activin are present in the rat prostate and to study the acute regulation by androgens. The results showed that mRNAs for the alpha and beta inhibin subunits were detected in rat prostate by reverse transcription-PCR together with ir-inhibin and ir-activin in prostate cytosols. The levels of ir-activin in the prostate (223 +/- 44 ng/gland) were greater than the levels of ir-inhibin (6.89 ng/gland), and activin immunoreactivity was localised to the epithelial cells. The presence of these proteins and the subunit mRNAs suggests that these proteins are produced in the prostate and may have a role in prostate function. The study of the effect of androgen withdrawal on the levels of ir-activin and ir-inhibin in these tissues showed no change in the content of ir-inhibin or ir-activin (ng/g tissue) after 3 days of castration or following the administration of the cytotoxic drug ethane dimethane sulphonate (EDS), although there was a significant (P < 0.01) decline in prostate weight. Fourteen days after EDS treatment, as the prostate weight fell significantly lower, the amount of ir-inhibin and ir-activin per prostate gland was significantly (P < 0.01) reduced although the concentration was unaffected. These data demonstrate, for the first time, that inhibin alpha and beta subunit mRNA and ir-inhibin and ir-activin are present in the prostate; the role of these proteins in prostate function remains to be established.

Temporal changes in sulphated glycoprotein-2 (clusterin) and ornithine decarboxylase mRNA levels in the rat testis after ethane-dimethane sulphonate-induced degeneration of Leydig cells.

Short- (3-24 h) and long-term (4-50 days) changes in sulphated glycoprotein-2 (SGP-2) and ornithine decarboxylase (ODC) mRNA levels in the adult rat testis were studied following a single dose of ethane-dimethane sulphonate (EDS), to destroy the Leydig cells. Distribution patterns of SGP-2 and ODC labelling were consistent with prevailing expression of the two transcripts in Sertoli cells and germ cells, respectively. This pattern did not show appreciable changes following EDS administration. No labelling of SGP-2 mRNA was noted in the interstitium of control and EDS-treated rats. This finding indicates that Leydig cell death induced by EDS is not associated with increased SGP-2 mRNA levels, a phenomenon related to apoptotic cell death in many tissues. Semi-quantitative densitometric analysis of the preparations demonstrated differential changes in SGP-2 and ODC mRNA levels in the tubular compartment following EDS treatment. At 6, but not at 3 and 12, h following EDS administration, SGP-2 mRNA levels showed a significant increase, possibly secondary to a direct effect of the alkylating agent on Sertoli cells. A significant decrease in ODC mRNA levels was observed from day 7 to day 28, matching degenerative changes in the seminiferous epithelium. In contrast, a decrease in SGP-2 transcript levels was observed from days 21-35 after treatment. In conclusion, our findings demonstrate that SGP-2 mRNA, a putative marker of apoptosis, is not altered in the testicular interstitium during EDS-induced degeneration of Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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 microliters, 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 microliters, respectively). Regeneration of Leydig cells in hCG-treated rats significantly (p < 0.05) favoured peritubular locations (18.6 +/- 2.8 microliters/testis) compared to central or perivascular sites of origin (11.6 +/- 1.2 microliters/testis). Partial restoration of spermatogenesis occurred in hCG-treated rats (tubule diameters usually > 250 microns) 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.

Response of the seminiferous epithelium of the rat testis to withdrawal of androgen: evidence for direct effect upon intercellular spaces associated with Sertoli cell junctional complexes

The morphological response of the Sertoli cells to partial or complete withdrawal of testosterone was studied in adult rats following hypophysectomy or administration of ethane dimethanesulphonate (EDS), a toxicant known to destroy selectively the Leydig cells of the testis. To assess the role of germ cells in effecting changes to Sertoli cells following withdrawal of testosterone, germ cell-deficient rats with Sertoli-cell-only testes (SCO) were treated with EDS to remove the source of testosterone. At 6 days after hypophysectomy or 4, 6 and 8 days after EDS treatment, stage VII and VIII seminiferous tubules showed degenerating germ cells and numerous basally-located vacuoles approximately 1-15 microns in diameter. Ultrastructural analysis indicated that most of the vacuoles were multiple focal dilations of the intercellular space associated with Sertoli cell junctional complexes. In SCO rats, treatment with EDS resulted in a significant (P < 0.05) increase in the formation of many vacuoles particularly in the base but also in the trunk of the Sertoli cells and again electron microscopic analysis showed multiple, localized expansions of the intercellular space associated with Sertoli cell junctional complexes. The appearance of intercellular spaces in SCO testes following androgen withdrawal cannot be attributed to shrinkage of degenerating germ cells since the seminiferous tubules did not contain germ cells. It is concluded that withdrawal of androgen induces early morphological alterations of the Sertoli cell junctional complexes in which the sites of membrane fusions representing tight junctions remain intact whereas the intercellular spaces exhibit major focal dilations.(ABSTRACT TRUNCATED AT 250 WORDS)

Stage‐Dependent changes in spermatogenesis and sertoli cells in relation to the onset of spermatogenic failure following withdrawal of testosterone

Rapid and complete withdrawal of intratesticular testosterone was achieved via the destruction of all Leydig cells with the specific Leydig cell cytotoxin ethane dimethanesulphonate (EDS). Restoration of testosterone levels was accomplished by administration of a single dose (25 mg) of testosterone esters (T) known to reverse the antispermatogenic effects of androgen withdrawal. Quantitation of the degenerating germ cells in cross sections of seminiferous tubules (ST) at stages IV-V, VII, IX, and X-XI of the spermatogenic cycle was used as a sensitive biological index of the effects of testosterone withdrawal and restoration upon the function of the Sertoli cells. Compared to control testicular tissues, the mean numbers of pyknotic germ cells per ST cross section at stages VII, IX and X-XI increased significantly (P < 0.01-0.001) between 4 to 8 days post-EDS treatment, but only in stage VII tubules was this trend reversed significantly (P < 0.005) within 2 days by T supplementation. In EDS-treated rats, stages VII, VIII, IX, and X-XI also exhibited significant (P < 0.05-0.001) increases (compared to controls) in the volumetric proportions by which intraepithelial vacuoles appeared within the seminiferous tubules. Again, in EDS+T supplemented rats, the appearance of vacuoles was significantly (P < 0.001) suppressed in stage VII and VIII. In contrast to tubules at stages VII-XI, those at stages IV-V were completely unaffected by testosterone withdrawal or replacement. The results show that at selected time intervals after EDS treatment, testosterone supplementation is capable of preventing/reversing these morphological changes within 2 days in stage VII tubules. It is suggested that the induction and subsequent prevention of seminiferous epithelial damage will serve as an important in vivo and in vitro approach for studies on the androgen-mediated changes in Sertoli cell biology during phases of impairment and recovery of their function. Manipulation of adult Sertoli cell function as provided by our model should permit identification of androgen-regulated gene products together with an understanding of their role(s) in normal and abnormal spermatogenesis.

Reduced testosterone during puberty results in a midspermiogenic lesion.

The aim of this study was to determine the role of testosterone, as reflected in the testicular interstitial fluid, in the completion of the first wave of spermatogenesis and to further elucidate its role in spermiogenesis. At weekly intervals beginning with 26-day-old rats, body and testis weights were obtained, testicular interstitial fluid testosterone (TIF-T) was assayed, daily sperm production (DSP) was determined, and testicular tissue was structurally analyzed by light and electron microscopy. At 40 days postpartum, half the rats were treated with ethane dimethanesulphonate (EDS) to temporarily reduce Leydig cells. The other half served as controls and were treated with the vehicle. The timing of EDS treatment was just prior to the elongation of spermatids. At Day 47 (1 week after EDS treatment), TIF-T, testis weight, DSP, and number of Leydig cells were significantly reduced. At Day 54 (2 weeks after treatment), TIF-T had returned to the normal adult level, Leydig cell repopulation was apparent, and testis weight was normal. The DSP returned to normal by Day 61 (3 weeks after treatment). At 1 and 2 weeks after treatment, Step 8-9 spermatids were partially or completely detached from Sertoli cells. Results indicate that a temporary reduction of testosterone during the peripubertal period leads to a temporary reduction of the DSP approximately 1 week later. It is suggested that reduced testosterone is associated with a mid-spermiogenic lesion interfering with stable attachment of Step 8-9 spermatids to Sertoli cells during Stage VIII-IX of the spermatogenic cycle.

In-vitro DNA synthesis in Leydig and other interstitial cells of the rat testis.

Replicative DNA synthesis (125I-labelled iododeoxy-uridine incorporation) was measured in interstitial cells prepared from rat testes and separated by Percoll density gradient centrifugation. Leydig cells were identified by 125I-labelled human chorionic gonadotrophin (hCG) binding and 3 beta-hydroxysteroid dehydrogenase histochemistry. Continuous density gradients indicated that interstitial cell DNA synthesis was not associated with Leydig cells, and was greater in cells from the immature than from the mature rat testis. Fractionation of cells by discontinuous density gradients into Leydig cell-rich and -depleted pools did not result in a similar enrichment of DNA synthesis. Treatment of the adult rat with hCG increased DNA synthesis into both fractions but oestrogen had no effect. DNA synthesis was greater in cells from the immature rat but, in contrast to the adult, in-vivo hCG treatment had no effect, whilst oestrogen decreased synthesis. To characterize the cells synthesizing DNA further, interstitial cells were prepared from testes in which the Leydig cells were depleted by in-vivo treatment with ethane dimethanesulphonate (EDS) or depleted in their germ cells by treatment in utero with busulphan. EDS treatment had no effect on DNA synthesis by the interstitial cells in spite of the 125I-labelled hCG binding being markedly reduced. Similarly, busulphan treatment was also without effects upon DNA synthesis. Fluorescence-activated cell cycle analysis of cells from both fractions from germ cell-depleted testes indicated that only a small proportion (3%) of the interstitial cells were actively dividing and this was almost doubled in cells from the germ cell-depleted immature rat testes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Ethane Dimethane Sulphonate and Orchidectomy on Luteinizing Hormone Secretion

Abstract The aim of this study was to determine whether testicular products of non-Leydig cell origin modulate rat luteinizing hormone (LH) secretion in vivo. We therefore compared the effects of ethane dimethane sulphonate (EDS), a toxin regarded as highly selective for Leydig cells, with that of bilateral orchidectomy on LH secretion in mature male Wistar rats. The intention was thereby to compare the effects of selective removal of Leydig cells with that of removing both Leydig cells and seminiferous tubules, respectively. Following a single dose of EDS (75 mg/kg, ip), plasma LH concentrations rose equally with those of castrated rats for the first 3 days. After that time, however, plasma LH concentrations in the EDS-treated rats fell progressively below those of the orchidectomized rats despite the continuing castrate level of circulating testosterone until Day 17. The effects of EDS treatment or orchidectomy on pulsatile LH secretion were then compared after 11 days to castrate levels of testosterone. EDS-treated rats demonstrated reduced LH pulse amplitude, mean plasma LH levels and net LH secretion compared with castrate rats, although LH pulse frequency was unaltered. However, a further group of rats treated with EDS and orchidectomized failed to demonstrate that these changes were fully reversed by the castration and therefore EDS may have direct effects upon pituitary LH secretion. In order to determine the mechanism of the reduced LH pulse amplitude after EDS treatment, a further study was conducted to determine whether EDS treatment resulted in reduced pituitary sensitivity to gonadotropin-releasing hormone (GnRH). Responsiveness of pituitary LH to exogenous GnRH (0.01 to 10 mug/kg body wt) was studied 11 days after removal of testicular testosterone feedback by either EDS or castration. Plasma LH response was linearly related to the log of the GnRH dose. At 10 min after GnRH administration, the plasma LH response in EDS-treated rats was less sensitive than in castrate rats. We conclude that the lesser augmentation of LH secretion between Days 3 and 17 after EDS treatment compared with castrate rats cannot be explained solely by changes in Leydig cell secretion but may involve direct effects of EDS on pituitary LH secretion or non-Leydig cell testicular products. Dampening of LH pulse amplitude without change in LH pulse frequency together with the reduced sensitivity to GnRH in EDS-treated rats suggests that this toxin may have direct effects on pituitary LH secretion independent of its effects on Leydig cell function.

Hormonal regulation of epidermal growth factor and insulinlike growth factor-I in adult male hypophysectomized rats treated with ethane dimethane sulphonate

There is increasing evidence implicating growth factors in the regulation of spermatogenesis and in-vitro studies have shown that epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) interact with the gonadotrophins in regulating testicular function. In the present study, the effect of FSH, testosterone and GH treatment on serum IGF-I and intratesticular EGF and IGF-I concentrations in adult male hypophysectomized rats treated with ethane dimethane sulphonate (EDS) to destroy Leydig cells has been investigated. Hypophysectomy alone or followed by EDS treatment was associated with a significant increase in intratesticular EGF concentrations compared with normal controls (P less than 0.05). Treatment of hypophysectomized animals with a combination of GH, FSH and testosterone resulted in a return of intratesticular EGF concentrations to normal control levels. In a second group of animals treated with a 5 cm silicone elastomer implant of testosterone and FSH, intratesticular EGF concentrations were also not significantly different from those of normal controls. Following hypophysectomy alone or hypophysectomy and EDS treatment, a significant increase in circulating IGF-I concentrations occurred, which was only reversed following the administration of GH. In addition, increases in testicular IGF-I concentrations were evident in all treated animals compared with controls, an effect which was only partially reversed in animals treated with a combination of FSH and testosterone (1.5 or 5 cm implant). Similar results were obtained with a combination of GH and FSH or GH and testosterone, although GH alone had no effect on testicular IGF-I concentrations. A combination of GH, FSH and testosterone restored testicular IGF-I to concentrations not significantly different from controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [ 3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [ 3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.

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.

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.

Interstitial fluid volume in the rat testis: androgen-dependent regulation by the seminiferous tubules?

Regulation of testicular interstitial fluid (IF) volume has been investigated in adult male rats in which the Leydig cells were selectively destroyed with a single i.p. injection of ethane dimethane sulphonate (EDS). Following this treatment, some animals also received testosterone supplementation by s.c. injection every 3 days, beginning either from the time of EDS injection, or 3-12 days afterwards. The volume of IF obtained by drip collection was determined, and testosterone and gonadotrophin concentrations measured in blood and in IF. Testosterone levels in IF and serum became undetectable by 3 days after EDS treatment. IF volume was reduced by 50% (P less than 0.01) to reach a minimum level between 6 and 9 days after treatment. However, this decline was prevented in the absence of Leydig cells by supplementation with testosterone from the time of EDS injection, a treatment which also kept gonadotrophins at minimum or undetectable levels. Furthermore, the reduced IF volume seen up to 9 days after treatment with EDS alone could be restored to control levels within 3 days by a single injection of testosterone. The results obtained demonstrate that androgens, but not Leydig cells or gonadotrophins, are required for the maintenance of interstitial fluid volume in the adult rat testis. It is suggested that the seminiferous tubules may mediate this response, through an androgen-dependent mechanism.

Factors affecting the secretion of immunoactive inhibin into testicular interstitial fluid in rats.

Immunoreactive inhibin was measured in testicular interstitial fluid (IF) from rats during sexual maturation or after impairment of spermatogenesis induced by ethane dimethanesulphonate (EDS), unilateral cryptorchidism or local heating (43 degrees C, 30 min) of the testes, to ascertain its usefulness as a marker of changing Sertoli cell function. Cultures of isolated seminiferous tubules were also studied. Inhibin was measured by a radioimmunoassay directed towards the first 26 amino acids of the N-terminus of the alpha-subunit, and the results confirmed for selected pools of IF by in-vitro bioassay using dispersed ovine pituitary cells. During puberty, IF levels of immunoactive inhibin fell by more than 90% (P less than 0.001) between 30 and 60 days of age, a decrease paralleled by the levels of androgen-binding protein (ABP), another Sertoli cell product secreted into IF. These changes also paralleled, but preceded, the fall (60%; P less than 0.001) in serum levels of FSH between 40 and 70 days, while the serum and IF levels of testosterone increased more than two-fold over this period. When adult rats were injected with EDS to destroy the Leydig cells, testosterone levels in IF and serum were undetectable at 3 and 7 days after treatment, were just detectable at 14 days and thereafter returned slowly towards normal by 42 days. The initial androgen withdrawal following EDS treatment caused a progressive reduction in testicular weight up to 21 days and this was accompanied by a significant increase in the serum levels of FSH and a two- to threefold increase in the IF levels of immunoactive inhibin (and also of ABP). Serum FSH and IF levels of immunoactive inhibin returned to within the normal range by 42 days when testosterone levels had normalized. In contrast, in two other experimental situations in which a marked decrease in testicular weight coupled with an increase in IF levels of ABP occurs, different results for the IF levels of immunoactive inhibin were obtained. Thus, in rats exposed to local heating of the testes, IF levels of immunoactive inhibin remained unchanged from control values at 21-40 days after treatment, a finding confirmed by bioassay results. In rats made unilaterally cryptorchid for 10 months, levels of immunoactive inhibin in IF were reduced by 60% (P less than 0.01) in the abdominal compared with the contralateral scrotal testis.(ABSTRACT TRUNCATED AT 400 WORDS)

The regenerative capacity of testicular interstitial tissue : A recurrent pattern of experimental destruction and regeneration of rat Leydig cells.

A single intraperitoneal injection of ethane dimethanesulphonate (EDS) destroys all Leydig cells in the adult rat testis but 1-2 weeks later new foetal-type Leydig cells begin to regenerate within the interstitial tissue. A further EDS treatment at 4 weeks failed to kill the new population of foetal-type Leydig cells. Between 10-20 weeks, the new Leydig cells exhibited the characteristics of adult-type Leydig cells. These cells responded to another EDS treatment by exhibiting a second phase of complete degeneration followed by regeneration of a foetal-type and subsequently an adult-type cell population. The results indicate that the testis retains the ability to replenish its supply of Leydig cells despite successive phases of total degradation of Leydig cells.

Origin of regenerating Leydig cells in the testis of the adult rat. An ultrastructural, morphometric and hormonal assay study.

Ethane dimethanesulphonate (EDS) was used as a specific cytotoxin to eliminate the Leydig cell population of the adult rat testis. Ultrastructural, morphometric and serum gonadotrophin and testosterone analysis was used to study the response of the intertubular tissue of the testis from 1 day to 10 weeks after EDS treatment. In control animals, the testis contained approximately 28 million Leydig cells and 8 million macrophages. Three to seven days after EDS treatment, Leydig cells were absent and serum testosterone was undetectable. Macrophage numbers increased three-fold by 3 days and returned to pretreatment values thereafter. At 2 and 3 weeks post-EDS, foetal-type Leydig cells (approximately 1-2 million per testis) appeared in proximity to perivascular and peritubular tissues, a feature also observed at 4 weeks when numerous such cells (approximately 15 million per testis) formed prominent clusters in perivascular and peritubular locations. Between 6 and 10 weeks after EDS treatment, the foetal-type Leydig cells were transformed morphologically into adult-type Leydig cells, they occupied central intertubular positions and their numbers were restored to pretreatment values. Regeneration of Leydig cells was reflected by elevated serum testosterone levels which returned towards the normal range. The results demonstrate the regenerative capacity of the testicular intertubular tissue and indicate a dual site of origin of Leydig cells which initially resemble foetal-type Leydig cells prior to establishing the adult-type Leydig cell population. The morphological pattern of Leydig cell regeneration suggests that in addition to gonadotrophic stimulation, local testicular factors from the seminiferous tubules may stimulate Leydig cell growth.