Ethane Dimethane Sulfonate Research Articles

Immunoregulatory Activity in Adult Rat Testicular Interstitial Fluid: Relationship with Intratesticular CD8+ Lymphocytes following Treatment with Ethane Dimethane Sulfonate and Testosterone Implants1

Regulation of T-cell traffic and function in the adult rat testis was assessed following treatment with the specific Leydig cell cytoxin, ethane dimethane sulfonate (EDS), and s.c. testosterone implants to prevent Leydig cell recovery. The distribution of T-cell subsets in the testis was determined immunohistochemically using stereological techniques. Testicular T cell-inhibiting activity in the interstitial fluid was measured using a phytohemagglutinin-activated rat thymocyte proliferation bioassay. The mostly cytotoxic CD8+ T-cell subset predominated over the CD4+ (regulatory) T-cell subset in the normal rat testis. Destruction of the Leydig cells caused a rapid preferential increase in testicular CD4+ T cells, which was followed by an increase in both the CD8+ subset and T cell-inhibiting activity in the Leydig cell-deficient testis. After Leydig cell recovery, there was a significant shift toward the CD8+ T-cell subset in the EDS-treated testis but not in the EDS-treated/testosterone-implanted testis. Total T-cell numbers and inhibitory activity in the testis returned to control levels regardless of whether the Leydig cells were allowed to recover. The level of inhibitory activity was closely related to the number of CD8+ T cells in the testis across all experimental groups, but it showed no relationship with pituitary hormones, macrophage numbers, or intratesticular testosterone levels. The data suggest that 1) cytotoxic lymphocytes have a potentially significant role in testicular function and 2) T cell-inhibiting activity in the testis interstitium is not substantially affected by changes in pituitary hormones or Leydig cell function, but appears to be related to local changes in immune activity.

Localization ofd-Aspartic Acid in Elongate Spermatids in Rat Testis

In the current study, localization ofd-aspartic acid (d-Asp) in rat testis was studied by immunohistochemical and biochemical techniques. Immunohistochemical staining of this tissue using specific polyclonal antibody tod-Asp revealedd-Asp immunoreactivity (IR) in the cytoplasm of germ cells, especially around the region rich in elongate spermatids, the most mature of the germ cells. Weak IR was also noted in cytoplasm of spermatocytes and round spermatids; however, it was negligible in interstitial cells and Sertoli cells. The intensity of immunostaining in each seminiferous tubule differed according to its distinct germ cell composition. In testis of young rats, seminiferous tubules lack elongated spermatids, andd-Asp was found to be localized in spermatocytes, the most mature population of germ cells at that age. We used various toxicants to destroy specific testicular cell populations and to confirm the localization ofd-Asp in rat testis. Administration of ethane dimethane sulfonate induced a selective destruction of all Leydig cells in this tissue. This resulted in a significant decrease in thed-Asp level, which was probably due to a drop in testosterone brought about by this treatment, and this was followed by a modulation of spermatogenesis. Three days after treatment with methoxyacetic acid (MAA), many seminiferous tubules were found to lack or to have severe depletions of pachytene spermatocytes, but not of elongate spermatids. This caused reductions in protein content and in the total amount ofl-Asp, but not that ofd-Asp. Twenty days after treatment with MAA, the depleted population of germ cells progressed through the spermatogenic cycle from pachytene spermatocytes to elongate spermatids. At this time, the level ofd-Asp decreased significantly, as did that ofl-Asp and protein, consistent withd-Asp localization in elongate spermatids. This decrease in thed-Asp level was also seen with immunostaining.

Gene expression of luteinizing hormone receptor and steroidogenic enzymes during Leydig cell development.

Testicular Leydig cells (LC) are rapidly and selectively destroyed by an injection of ethane dimethane sulfonate (EDS). LC regeneration occurs in the testis of the EDS-treated rats from the differentiation of the precursor Leydig cells (PLC). This study was designed to investigate the patterns of change in the mRNAs for the luteinizing hormone receptor (LHR) and the steroidogenic enzymes, cholesterol side chain cleavage (P-450scc) and 17 alpha-hydroxylase (P-450(17 alpha)) during LC regeneration from PLCs. Mature (60 days of age) Sprague-Dawley male rats received a single intraperitoneal injection of EDS and were killed at different times between days 2 and 60 post-treatment. PLC- and LC-enriched fractions were isolated from the testes of the EDS-treated rats and age-matched control rats using a collagenase digestion-Percoll gradient method. Total RNA was extracted from these cell populations and subjected to Northern blot analysis. The LC fraction isolated from testes of control rats expressed four major transcripts of the LHR, sized 1.8, 2.5, 4.2 and 7.0 kb. The undifferentiated PLC fraction from controls expressed only a truncated form, the 1.8 kb transcript. This truncated LHR transcript was also the only LHR mRNA species detected in PLCs at day 2 post-EDS treatment. In contrast, all four transcripts of the LHR were detected in the PLC fraction at day 10 post-EDS treatment. The levels of the full length 7.0 kb transcript increased thereafter and reached a peak between days 24 and 36 post-EDS treatment in the PLC fraction. Concomitant with the increase in the 7.0 kb transcript, the truncated 1.8 kb transcript decreased in amount and reached a nadir between days 16 and 36 post-treatment. The changes observed in this cell fraction reflect the process of differentiation of PLCs into LCs. At day 45 post-EDS treatment, the level of the 7.0 kb transcript decreased while the 1.8 kb form increased in the PLC fraction, reflecting the completion of LC regeneration from this cell fraction. By day 60 post-EDS treatment, the levels of the 1.8 kb transcript rose to the value observed in undifferentiated control PLCs and the other transcripts were no longer detected in the PLC fraction, indicating that cells in the PLC fraction were again in an undifferentiated stage. Messenger RNAs for both the steroidogenic enzymes, P-450scc and P-450(17 alpha) were expressed in the control LC fraction. Neither of these two mRNAs were detected in the PLC fraction of the control rats. P-450scc and P-450(17 alpha) mRNAs were first expressed in the PLC fraction at day 10 post-EDS treatment. Thereafter, the levels of P-450scc and P-450(17 alpha) mRNAs increased in the PLC fraction and reached a peak between days 24 and 36 and days 24 and 45 post-EDS treatment respectively. P-450scc and P-450(17 alpha) mRNAs were no longer expressed in the PLC fraction at day 60 post-EDS treatment. These patterns also reflect the process of differentiation of PLCs into functional LCs. These results demonstrate for the first time that PLCs in the control testis are undifferentiated and do not express functional LHR and steroidogenic enzymes or their mRNAs. The PLCs are characterized, however, by the expression of a truncated 1.8 kb transcript of the LHR mRNA. Functional LHR and steroidogenic enzymes are expressed in PLCs only during their differentiation into LCs after EDS treatment. Subsequent to LC regeneration, the PLCs return to an undifferentiated stage.

Ethane dimethane sulfonate and NNN′N′-Tetrakis-(2-Pyridylmethyl)Ethylenediamine inhibit steroidogenic acute regulatory (StAR) protein expression in MA-10 leydig cells and rat sertoli cells

Apoptosis inhibits steroid biosynthesis, but it is not clear how the Steroidogenic Acute Regulatory (StAR) protein, is affected. To characterize StAR expression during apoptosis, mouse MA-10 Leydig tumor cells were treated with ethane dimethane sulfonate (EDS), an inducer of apoptosis, and the metal ion chelator NNN′N′-tetrakis-(2-pyridylmethmyl)ethylenediamine (TPEN), an inducer of cell death. Both chemicals induced cell death and similarly inhibited dbcAMP-stimulated steroidogenesis and accumulation of the 30 kDa form of StAR. Utilizing the dye JC-1, it was found that TPEN and EDS also impaired the mitochondrial electrochemical potential (Δψ). In Sertoli cells, which also express StAR, EDS induced cell death and attenuated StAR expression. We conclude 1) steroidogenesis and accumulation of mature StAR protein are inhibited as a consequence of the induction of apoptosis; 2) reduced levels of StAR may be partially attributed to inhibition of import because of the loss of Δψ; 3) loss of steroidogenesis is probably due to loss of StAR synthesis and disruption of Δψ.

Ventral Prostate Structure and Serum Testosterone Levels After Chronic Treatment with Isoproterenol in Adult Rats with Different Androgen Status

The effects of chronic administration of the beta-adrenergic agonist isoproterenol (ISO) (120 microg/kg per day; subcutaneously) on the ventral prostate structure and serum testosterone concentrations were examined in adult rats with different androgen status: intact, intact testosterone-injected (1 mg/rat), surgically and chemically castrated rats. Chemical castration was evoked by an intraperitoneal injection of ethane dimethanesulfonate (EDS) (75 mg/kg). A ventral prostate response was only observed in intact and chemically castrated animals. Stereological analysis revealed atrophic changes in the glandular compartment of the prostate of ISO-treated intact rats, but they were probably the consequence of significantly decreased serum testosterone levels. In addition, in these animals alterations were found in the morphometrical parameters of the ventral prostate blood vessels, their relative and total volumes being increased. In chemically castrated rats, administration of ISO from the day of EDS application partially prevented the postcastrational regression of the ventral prostate without affecting blood testosterone level. However, it seems that the discharge of glandular secretion was attenuated at the same time. These results show that chronic treatment with ISO may have both stimulatory and inhibitory effects on the rat ventral prostate depending on the androgen status of the animals and, accordingly, on the site of ISO-action.

Expression of selenoprotein-P messenger ribonucleic acid in the rat testis.

It has been suggested that a plasma protein, selenoprotein P, functions as an antioxidant and that its mRNA is expressed ubiquitously, including in the testis. To determine its physiological function, we have investigated the expression of selenoprotein-P mRNA in the rat testis. Northern blot analysis showed that selenoprotein P was exclusively expressed in the Leydig cell fraction. In situ hybridization experiments further supported this observation. Testes of rats administered ethylene dimethane sulfonate (EDS) were also examined by Northern blot analysis. Selective degeneration of Leydig cells by EDS treatment resulted in disappearance of selenoprotein-P mRNA from the testis. Furthermore, upon recovery, in association with regenerative differentiation of Leydig cells, reappearance of the selenoprotein-P mRNA was observed. These results indicated that selenoprotein-P mRNA was predominantly expressed in the interstitial Leydig cells.

Molecular Mechanisms of Reappearance of Luteinizing Hormone Receptor Expression and Function in Rat Testis after Selective Leydig Cell Destruction by Ethylene Dimethane Sulfonate

Considering the major role of LH in the control of Leydig cell (LC) development and function, we aimed to characterize further the pattern of LH receptor (LHR) expression in two experimental paradigms: the rat treated with ethylene dimethane sulfonate (EDS), in which the selective destruction of preexisting mature LCs induces the proliferation and differentiation of newly formed LCs, a process that takes place in the presence of high levels of gonadotropins; and the EDS-rat treated with a high dose of testosterone (EDS + T), in which the LH secretion is suppressed, and consequently LC development after EDS arrested. In EDS rats, serum T was suppressed and testicular LHR binding became undetectable on days 5 and 15 after treatment. The pattern of LHR messenger RNA (mRNA) expression was profoundly modified: only one of the splice variants [1.8-kilobase (kb)] persisted, whereas the others disappeared. On days 20 and 45 after EDS, along with LC repopulation, serum T and LHR binding recovered, and the pattern of LHR mRNA expression gradually returned to that resembling controls. In EDS + T rats, a similar drop in testicular LHR binding and change in the pattern of LHR mRNA expression was detected on days 5 and 15 after treatment. However, on days 20 and 45, no recovery either in LHR binding or in expression of the longer LHR mRNA splice variants was observed, showing that LH is needed to induce LHR expression in repopulating LCs, at least to a quantitatively significant level. To gain further insight into the mechanism(s) by which LH acts on LC precursors, the translational status of the 1.8-kb LHR transcript, persistently expressed after EDS, was analyzed and compared with that of the 6.8-kb message. In polysome distribution analysis of total testicular RNA, the 6.8-kb LHR message was highly associated with polysomes, whereas the 1.8-kb variant was mainly localized to prepolysomal fractions, both in control and EDS testes, thus predicting lower translational efficiency. In addition, considering that only LCs express LHRs in the testis, the time course of the reappearance of functional receptors was mapped by evaluating testicular responsiveness to human recombinant LH in vitro. No response to LH stimulation was detected 5 days after EDS. However, cAMP response to LH was observed on days 15 and 20, regardless of the presence of high (EDS) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of functional LHRs, qualitatively, can take place in the absence of measurable LH levels. In EDS-treated rats, the appearance of the cAMP response coincided with those of pregnenolone, progesterone, and T. In contrast, no LH-induced steroid release was observed in EDS + T rats, indicating that steroidogenic response in developing LC requires LH priming. In conclusion, the appearance of functional LHRs, at a low level of expression, in LC precursors is an LH-independent developmental event, essential for the subsequent LH-dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results cast doubt on a major functional role of the truncated (1.8-kb) form of LHR mRNA, which persists after EDS at a high level of expression, in the early Leydig cell precursors.

Molecular mechanisms of reappearance of luteinizing hormone receptor expression and function in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

Considering the major role of LH in the control of Leydig cell (LC) development and function, we aimed to characterize further the pattern of LH receptor (LHR) expression in two experimental paradigms: the rat treated with ethylene dimethane sulfonate (EDS), in which the selective destruction of preexisting mature LCs induces the proliferation and differentiation of newly formed LCs, a process that takes place in the presence of high levels of gonadotropins; and the EDS-rat treated with a high dose of testosterone (EDS + T), in which the LH secretion is suppressed, and consequently LC development after EDS arrested. In EDS rats, serum T was suppressed and testicular LHR binding became undetectable on days 5 and 15 after treatment. The pattern of LHR messenger RNA (mRNA) expression was profoundly modified: only one of the splice variants [1.8-kilobase (kb)] persisted, whereas the others disappeared. On days 20 and 45 after EDS, along with LC repopulation, serum T and LHR binding recovered, and the pattern of LHR mRNA expression gradually returned to that resembling controls. In EDS + T rats, a similar drop in testicular LHR binding and change in the pattern of LHR mRNA expression was detected on days 5 and 15 after treatment. However, on days 20 and 45, no recovery either in LHR binding or in expression of the longer LHR mRNA splice variants was observed, showing that LH is needed to induce LHR expression in repopulating LCs, at least to a quantitatively significant level. To gain further insight into the mechanism(s) by which LH acts on LC precursors, the translational status of the 1.8-kb LHR transcript, persistently expressed after EDS, was analyzed and compared with that of the 6.8-kb message. In polysome distribution analysis of total testicular RNA, the 6.8-kb LHR message was highly associated with polysomes, whereas the 1.8-kb variant was mainly localized to prepolysomal fractions, both in control and EDS testes, thus predicting lower translational efficiency. In addition, considering that only LCs express LHRs in the testis, the time course of the reappearance of functional receptors was mapped by evaluating testicular responsiveness to human recombinant LH in vitro. No response to LH stimulation was detected 5 days after EDS. However, cAMP response to LH was observed on days 15 and 20, regardless of the presence of high (EDS) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of functional LHRs, qualitatively, can take place in the absence of measurable LH levels. In EDS-treated rats, the appearance of the cAMP response coincided with those of pregnenolone, progesterone, and T. In contrast, no LH-induced steroid release was observed in EDS + T rats, indicating that steroidogenic response in developing LC requires LH priming. In conclusion, the appearance of functional LHRs, at a low level of expression, in LC precursors is an LH-independent developmental event, essential for the subsequent LH-dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results cast doubt on a major functional role of the truncated (1.8-kb) form of LHR mRNA, which persists after EDS at a high level of expression, in the early Leydig cell precursors.

A switch in the cellular localization of macrophage migration inhibitory factor (MIF) in rat testis after ethane dimethane sulfonate treatment

A switch in the cellular localization of macrophage migration inhibitory factor (MIF) in rat testis after ethane dimethane sulfonate treatment

Is Leydig cell steroidogenic function affected by the germ cell content of the seminiferous tubules?

The effect of testicular germ cell content on Leydig cell steroidogenic function in vivo in adult rats was examined. Three experimental paradigms were used to effect germ cell changes. First, a vitamin A-depletion/repletion regimen was used to achieve synchrony at different stages of the cycle of the seminiferous epithelium and thus produce testes with widely differing germ cell contents. Second, long term vitamin A depletion was used to effect germ cell, but not Leydig cell, loss. Third, Leydig cells and germ cells first were eliminated from the testes of adult rats by the administration of ethane 1,2-dimethane sulfonate (EDS) along with testosterone- and estradiol-filled Silastic capsules; Leydig cells were then restored to the germ cell-depleted testes by removal of the luteiniging hormone (LH)-suppressing capsules. Serum, interstitial fluid, and seminiferous tubule fluid testosterone concentrations did not differ between rats in which at least 70% of the seminiferous tubules contained germ cells at stages VII-VIII or at stages XII-III of the cycle. The capacity of the testes of these rats to produce testosterone, assessed by their in vitro perfusion with maximally stimulating LH, also showed no differences despite the differences in germ cell content. Elimination of germ cells throughout the testes by long term vitamin A depletion also did not affect the steroidogenic function of the testes. Finally, the steroidogenic function of Leydig cells restored to germ cell-depleted testes was indistinguishable from that of germ cell-containing controls. These results, taken together, provide evidence supporting the contention that the germ cell content of the testis has little or no effect on testicular steroidogenic function.

Restoration of Leydig cells after repeated administration of ethane dimethanesulfonate in adult rats.

Adult male rats were repeatedly treated with ethane dimethanesulfonate (EDS), an agent known to destroy Leydig cells selectively. Following a second injection, changes in serum testosterone levels and histological and morphometric changes of Leydig cells showed the time course to be similar to those after the first treatment. The number and volume of Leydig cells markedly decreased at day 2, began to increase from day 7, and recovered to the values of the control rats at day 30, concomitant with the changes of serum testosterone levels. Cells in the interstitial tissue labeled with bromodeoxyuridine markedly increased in number at day 2, gradually decreased thereafter, and returned to the values of the controls at day 14. During this period, cells undergoing mitosis were seen, their type unable to be determined, but were presumed to be regenerating Leydig cells. Even 30 days following four treatments with intervals of 30 days each, serum testosterone levels were the same as those in the controls. Also the numerical and volume densities of Leydig cells and the volume of an average Leydig cell were the same as those of the controls. Mitosis was observed in mature Leydig cells at this period, if any. It appears that new Leydig cells began to proliferate by division earlier than 14 days after EDS, allowing that there were several stages of proliferation, and that the source of reappearing Leydig cells may not be a limited number of precursor cells, implying the presence of stem cells for Leydig cells.

EFFECTS OF ETHANE DIMETHANESULFONATE ON THE STRUCTURE OF ADULT MALE RAT ADRENAL CORTEX

The influence of ethane dimethanesulfonate (EDS), a specific toxicant for Leydig cells, on the morphometric characteristic of adult male rat adrenal cortex was examined on day 15 after administration. As expected, the dose of 75 mg kg −1of EDS produced drastic reduction in serum testosterone levels followed by a decrease in testis and seminal vesicle weight. However, a considerable drop (over 50%) in adrenal weight was also found. Stereological analysis of the adrenal cortex, performed under light microscope, revealed atrophy of all adrenocortical zones. The changes were most prominent in the inner zones. Thus, in the zona fasciculata the number of parenchymal cells was markedly decreased. In the zona reticularis a layer consisting mostly of atrophic parenchymal cells was localised at the border between zona reticularis and zona fasciculata. The remaining small number of cortical cells in this zone displayed a notable hypertrophy. It is concluded that EDS at a dose that destroys Leydig cells has a strong deleterious effect on steroidogenic cells of adult male rat adrenal cortex.

Expression of apolipoprotein E mRNA in the epithelium and interstitium of the testis and the epididymis.

Apolipoprotein E (apo E) is an important constituent of plasma lipoproteins and is believed to be involved in the regulation of lipid transport and distribution between tissues. The production of this apolipoprotein in extra-hepatic tissues such as the testis and epididymis could facilitate specific local functions. Apo E mRNA was detected in testis, epididymis, seminal vesicles, and prostate. In the epididymis, apo E was detected using in situ hybridization in epithelial cells and in some cells in the interstitium throughout the organ (i.e., caput, corpus, and cauda). Northern blot analysis showed that apo E mRNA is present in Sertoli cells and germ cells, but not peritubular myoid cells. Interstitial cells of the testis displayed the most intense signal for apo E message using in situ hybridization. Messenger RNA for apo E was also detected in the interstitium of rat testes at 3 and 6 days after animals were treated with ethylene dimethanesulfonate (EDS) to eliminate Leydig cells. Thus, in addition to Leydig cells, other cell types within the interstitium are capable of producing apo E message. Levels of testicular apo E mRNA increased between 30 and 60 days pc during which the germ cell population is increasing. As determined by northern blot analysis of RNA from stage synchronized testes, the levels of apo E mRNA fluctuate in relation to the cycle of the seminiferous epithelium. The cells responsible for this stage-specific variation in message could not be identified by in situ hybridization. Apolipoprotein Al (apo Al) mRNA was also found to be expressed in the epididymis but not in the testis of adult rats. The role of apolipoproteins in spermatogenesis and sperm maturation has not been elucidated. The results of this study demonstrate the specific tissues and cells types which play a role in the production and possible regulation of apo E mRNA in the male reproductive tract. These data will help in the elucidation of the function of apo E in spermatogenesis and sperm maturation.

The Detection of Thyrotropin-Releasing Hormone (TRH) and TRH Receptor Gene Expression in Siberian Hamster Testes

Rao, J. N., L. Debeljuk, A. Bartke, Y.-P. Gao, J. F. Wilber and P. Feng. The detection of thyrotropin releasing hormone (TRH) and TRH receptor gene expression in Siberian hamster testes. Peptides 18(8) 1217–1222, 1997.—Thyrotropin-releasing hormone (TRH) from the hypothalamus is the major regulator of TSH synthesis and secretion. Most recently, TRH and TRH receptors (TRH-R), as well as their mRNAs, have been identified in rat testis. To expand our knowledge on the testicular TRH and TRH receptor gene expression in different species, in the present study the mRNA levels of testicular TRH and TRH-R were investigated in Siberian hamsters. To further localize the cellular sites of the gene expression, the animal model was treated with a single injection of ethylene dimethane sulfonate (EDS) (IP, 80 mg/kg body weight), a compound known as to specifically eliminate testicular Leydig cells. The elimination of Leydig cells induced by EDS treatment was confirmed by histological studies of the testis sections and by serum hormonal analyses, which showed a dramatic reduction of serum testosterone (T) levels and significantly elevated serum LH concentrations. Messenger RNA levels of TRH and TRH-R in the testes were determined by Northern blot analyses quantitated with densitometry scanning. The results showed that specific TRH-R mRNA, 3.8 kb in size, was identified in Siberian hamster testes and the mRNA levels were significantly elevated in the EDS-treated testes compared to the controls ( p < 0.01). Testicular TRH mRNA was also detected; however, no significant differences in TRH mRNA levels were found between EDS-treated and control groups. The size of TRH mRNA was characterized as about 1.2 kb in hamster testes, which was smaller than that observed in the rat hypothalamus (1.6 kb) and in the rat testis (2.0 kb). Further studies by RNase H digestion revealed the presence of smaller TRH transcripts in the hamster testes than those in the rat testis. No hybridization signal for TRH mRNA was detected by RNase protection assay, when a rat TRH riboprobe was applied to hamster testis RNA, suggesting the limited homology of TRH gene sequences between these two species. Our results demonstrate that both TRH and TRH-R genes are expressed in Siberian hamster testes, and a significant increase of TRH-R mRNA levels occurs in the Leydig cell eliminated hamster testes. Unlike the rat testicular TRH mRNA mainly detected in Leydig cells, in hamster TRH mRNA could also be detected in other testicular compartment.

Depletion and repopulation of Leydig cells in the testes of aging brown Norway rats

The capacity of Brown Norway rat Leydig cells to produce testosterone has been shown to decrease with aging. Our objectives herein were to determine 1) whether ethane dimethanesulfonate (EDS) administration would eliminate the hypofunctional Leydig cells of the aged Brown Norway rat testis; 2) if so, whether a new generation of Leydig cells subsequently would appear; and 3) if so, whether the steroidogenic capacity of the new Leydig cells would be at the relatively low level of the cells they replaced or at the high level of young adult Leydig cells. Young (3-month-old) and aged (18-month-old) rats received an injection of EDS (8.5 mg/100 g BW). One, 5, and 10 weeks thereafter, the serum testosterone concentration and the capacity of the testes and of isolated Leydig cells to produce testosterone were determined. One week after EDS treatment, Leydig cells were not seen in the testes of young or aged rats, and the serum testosterone concentration and testicular testosterone production were reduced to undetectable levels. Five weeks after EDS treatment, serum testosterone levels at both ages were restored to those in age-matched controls, and the capacity of the testes to produce testosterone was restored partially (young rats) or completely (aged rats). By 10 weeks after EDS treatment, the serum testosterone concentration in young rats and the ability of their testes to produce testosterone were at the levels of age-matched controls. In aged rats, however, serum testosterone and testicular testosterone production were at levels that significantly exceeded those of aged-matched controls and, indeed, were not significantly different from those of young control or EDS-treated rats. Consistent with this, the ability of Leydig cells isolated from the testes of young rats and that of cells from aged rats to produce testosterone 10 weeks after the rats were treated with EDS were equivalent. The enhanced ability of the Leydig cells restored to the aged testes to produce testosterone was not a consequence of exposure to increased levels of LH. Thus, although situated in an aged testis and in the environment of an aged hypothalamic-pituitary axis, the steroidogenic function of the Leydig cells restored to aged rat testes was equivalent to that of young rat Leydig cells.

Depletion and repopulation of Leydig cells in the testes of aging brown Norway rats.

The capacity of Brown Norway rat Leydig cells to produce testosterone has been shown to decrease with aging. Our objectives herein were to determine 1) whether ethane dimethanesulfonate (EDS) administration would eliminate the hypofunctional Leydig cells of the aged Brown Norway rat testis; 2) if so, whether a new generation of Leydig cells subsequently would appear; and 3) if so, whether the steroidogenic capacity of the new Leydig cells would be at the relatively low level of the cells they replaced or at the high level of young adult Leydig cells. Young (3-month-old) and aged (18-month-old) rats received an injection of EDS (8.5 mg/100 g BW). One, 5, and 10 weeks thereafter, the serum testosterone concentration and the capacity of the testes and of isolated Leydig cells to produce testosterone were determined. One week after EDS treatment, Leydig cells were not seen in the testes of young or aged rats, and the serum testosterone concentration and testicular testosterone production were reduced to undetectable levels. Five weeks after EDS treatment, serum testosterone levels at both ages were restored to those in age-matched controls, and the capacity of the testes to produce testosterone was restored partially (young rats) or completely (aged rats). By 10 weeks after EDS treatment, the serum testosterone concentration in young rats and the ability of their testes to produce testosterone were at the levels of age-matched controls. In aged rats, however, serum testosterone and testicular testosterone production were at levels that significantly exceeded those of aged-matched controls and, indeed, were not significantly different from those of young control or EDS-treated rats. Consistent with this, the ability of Leydig cells isolated from the testes of young rats and that of cells from aged rats to produce testosterone 10 weeks after the rats were treated with EDS were equivalent. The enhanced ability of the Leydig cells restored to the aged testes to produce testosterone was not a consequence of exposure to increased levels of LH. Thus, although situated in an aged testis and in the environment of an aged hypothalamic-pituitary axis, the steroidogenic function of the Leydig cells restored to aged rat testes was equivalent to that of young rat Leydig cells.

Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis.

Endogenous TRH-like products were analyzed in rat testis using a TRH enzyme immunoassay coupled to molecular sieve filtration and HPLC identification. Of the three immunoreactive peptides detected, the two major forms exhibited the same chromatographic properties as synthetic TRH and pGlu-Phe-Pro-NH2. These peptides accounted respectively for 33% and 54% of the total TRH immunoreactivity in the testis. In rat serum, HPLC analysis showed the presence of only one immunoreactive peak with a retention time similar to that of authentic TRH. Ethylene dimethanesulfonate treatment of adult rat was used to assess the effect of Leydig cell destruction on the TRH immunoreactivity content. The concentration of the three TRH immunoreactive peptides fell gradually after treatment and reached a minimum at day 21, where a marked decrease (98%) was observed. At day 41, the regeneration of Leydig cells was achieved, as shown by histochemistry and measurements of serum testosterone and testicular weight. However, no restoration of the TRH immunoreactive content was achieved, the three TRH-related peptides being only detectable in trace amount. On the other hand, no significant change in hypothalamic levels of TRH was observed at any treatment time, indicating that hypothalamic TRH biosynthesis was not influenced by testosterone. By using the indirect immunofluorescence technique, TRH immunoreactive cells were found in the interstitial space of the testis. These results suggest that Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis. A paracrine, or autocrine role for these products is suggested.

Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis

Endogenous TRH-like products were analyzed in rat testis using a TRH enzyme immunoassay coupled to molecular sieve filtration and HPLC identification. Of the three immunoreactive peptides detected, the two major forms exhibited the same chromatographic properties as synthetic TRH and pGlu-Phe-Pro-NH2. These peptides accounted respectively for 33% and 54% of the total TRH immunoreactivity in the testis. In rat serum, HPLC analysis showed the presence of only one immunoreactive peak with a retention time similar to that of authentic TRH. Ethylene dimethanesulfonate treatment of adult rat was used to assess the effect of Leydig cell destruction on the TRH immunoreactivity content. The concentration of the three TRH immunoreactive peptides fell gradually after treatment and reached a minimum at day 21, where a marked decrease (98%) was observed. At day 41, the regeneration of Leydig cells was achieved, as shown by histochemistry and measurements of serum testosterone and testicular weight. However, no restoration of the TRH immunoreactive content was achieved, the three TRH-related peptides being only detectable in trace amount. On the other hand, no significant change in hypothalamic levels of TRH was observed at any treatment time, indicating that hypothalamic TRH biosynthesis was not influenced by testosterone. By using the indirect immunofluorescence technique, TRH immunoreactive cells were found in the interstitial space of the testis. These results suggest that Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis. A paracrine, or autocrine role for these products is suggested.

Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate.

The quantitative effects of ethane dimethane sulfonate (EDS), a Leydig cell toxin, on apoptosis in adult rat seminiferous epithelium were studied by the improved transillumination method. Nonradioactive in situ end labeling of fragmented DNA in squash preparations revealed significant increases in apoptotic cells in stages II-XI, whereas controls showed 0.5-2.3 apoptotic cells/mm tubule. Seven days post-EDS treatment, the highest numbers of apoptotic cells were scored in stages VIIab and VIIcd (74.7 +/- 23.8 and 61.3 +/- 16.0 cells/mm, respectively). The effects were suppressed by testosterone (T) supplementation, except in stages II-III and VIIcd. An opposite effect was found in stage XII, where the number of apoptotic cells decreased 1, 3, and 7 days after EDS treatment and returned to control levels in T-supplemented rats. Electrophoretic analysis of internucleosomal DNA fragmentation revealed a biphasic apoptotic process after 1 and 5-7 days due to Leydig and germ cell apoptosis, respectively. The specific germ cell apoptosis was also confirmed by electron microscopic analysis. The results suggest that T withdrawal induces apoptotic cell death in most stages of the cycle and that the effects are largely preventable. In stage XII, however, T seems to promote apoptosis in premeiotic cells.

Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate

Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate