Ethane Dimethane Sulfonate Treatment Research Articles

Bone morphogenetic protein 4 inhibits rat stem/progenitor Leydig cell development and regeneration via SMAD-dependent and SMAD-independent signaling

Bone morphogenetic protein 4 (BMP4) is an important member of the transforming growth factor-β superfamily. BMP4 is expressed in the Leydig cell lineage. We hypothesized that BMP4 might regulate the development of stem/progenitor Leydig cells. The BMP4 receptors, BMPR1A, BMPR1B, and BMPR2 were found to be expressed in progenitor Leydig cells of prepubertal testis and isolated cells. BMP4 at 1 and 10 ng/mL significantly reduced androgen production and down-regulated steroidogenesis-related gene and protein expression possibly by activating the SMAD signaling pathway (increasing SMAD1/5 phosphorylation and SMAD4) at 24 h treatment. BMP4 at 0.1 ng/mL and higher concentrations markedly reduced the EdU labeling index of CD90+ stem Leydig cells after 24 h treatment and significantly reduced the number of EdU+ stem Leydig cells on the surface of seminiferous tubules after 7 days of culture. BMP4 at 0.01 ng/mL and higher concentrations significantly blocked the differentiation of stem Leydig cells into adult cells, as shown by the reduction of testosterone secretion and the downregulation of Lhcgr, Scarb1, Cyp11a1, Hsd11b1, and Insl3 and their function after 3D seminiferous tubule culture for 3 weeks, and this effect was reversed by co-treatment with the BMP4 antagonists noggin and doxomorphine. In addition, BMP4 also blocked stem Leydig cell differentiation through SMAD-independent signaling pathways (ERK1/2 and AMPK). Ethanedimethane sulfonate (EDS) single injection can result in reduction of testosterone, restoration can happen post treatment. In an in vivo model of Leydig cell regeneration following EDS treatment, intratesticular injection of BMP4 from day 14 to day 28 post-elimination significantly reduced serum testosterone levels and down-regulated the expression of Scarb1, Star, Hsd11b1, and Insl3 and its proteins, possibly through SMAD-dependent and SMAD-independent (ERK1/2 and AMPK) signaling pathways. In conclusion, BMP4 is expressed in cells of the Leydig cell lineage and blocks entry of stem/progenitor Leydig cells into adult Leydig cells through SMAD-dependent and SMAD-independent signaling pathways.

Preliminary Investigation on the Ameliorative Role Exerted by D-Aspartic Acid in Counteracting Ethane Dimethane Sulfonate (EDS) Toxicity in the Rat Testis.

Simple SummaryFor proper fertility, the production of good-quality spermatozoa is essential. Nowadays, many environmental pollutants affect the spermatogenetic process, at different levels. For this reason, new approaches are needed to prevent/counteract these toxic effects. Here, we showed that the excitatory amino acid D-aspartic acid (D-Asp) prevents the deadly action of ethane dimethane sulfonate (EDS) on the testosterone-secreting Leydig cells in rat testis. We found that EDS, probably via the reduced testosterone level, alters the normal histology of the seminiferous epithelium, leading to germ cells death and to the decreased protein level of two Leydig cell “markers”: steroidogenic acute regulatory and prolyl endopeptidase. In addition, the same analysis performed on rats that were pre-treated with D-Asp revealed a protective role of this compound, since all the above parameters were quite normal. Moreover, we found that the protective mechanism of action involved in this scenario may be due to the ability of D-Asp to reduce the oxidative stress induced by EDS. Based on these findings, we could affirm that D-Asp may be an encouraging candidate to be used to alleviate the harmful action due to environmental pollutants exposure, in order to maintain appropriate fertility.Herein is reported the first evidence of the protective role of D-aspartic acid (D-Asp) in preventing the toxic effect exerted by the alkylating agent ethane dimethane sulfonate (EDS) in the rat testis. We confirmed that EDS treatment specifically destroyed Leydig cells (LC), resulting in the drastic decrease of the serum testosterone level and producing morphological changes in the germinal tubules, i.e., altered organization of the epithelium, loss of cell contacts and the consequent presence of empty spaces between them, and a reduce number of spermatozoa. Moreover, an increase of TUNEL-positive germ cells, other than alteration in the protein level and localization of two LC “markers”, StAR and PREP, were observed. Interestingly, results obtained from rats pre-treated with D-Asp for 15 days before EDS-injection showed that all the considered parameters were quite normal. To explore the probable mechanism(s) involved in the protection exerted by D-Asp, we considered the increased oxidative stress induced by EDS and the D-Asp antioxidant effects. Thiobarbiturc acid-reactive species (TBARS) levels increased following EDS-injection, while no change was observed in the D-Asp + EDS treated rats. Our results showed that D-Asp may be used as a strategy to mitigate the toxic effects exerted by environmental pollutants, as endocrine disrupters, in order to preserve the reproductive function.

MiR-205 Expression Elevated With EDS Treatment and Induced Leydig Cell Apoptosis by Targeting RAP2B via the PI3K/AKT Signaling Pathway.

The adult Leydig cells (ALCs), originated from stem Leydig cells (SLCs), can secrete testosterone which is essential for germ cell development and sexual behavior maintenance. As a synthetic compound, ethane dimethane sulfonate (EDS), a well-known alkylating agent, has been reported to specifically ablate ALCs. In this study, EDS was verified to ablate differentiated pig LCs by experiments. Subsequently, the primary isolated pig LCs (containing SLCs and differentiated LCs) and EDS-treated LCs (almost exclusively SLCs) were collected for RNA-seq 4,904 genes and 15 miRNAs were differently expressed between the two groups. Down-regulated genes in the EDS-treated group were mainly related to steroid hormone biosynthesis. The highest up-regulation miRNAs was miR-205 after EDS treatment. Additionally, miR-205 was expressed more highly in pig SLCs clones compared with differentiated LCs. Through qRT-PCR, western blot (WB), TUNEL, EDU and flow cytometry, miR-205 was found to induce cell apoptosis, but did not affect proliferation or differentiation in both TM3 and GC-1spg mouse cell lines. Through luciferase reporter assays and WB, RAP2B was identified as a target gene of miR-205. Besides, overexpression of miR-205 inhibited the expressions of PI3K, Akt and p-AKT. All these findings were helpful for elucidating the regulation mechanism in pig LCs.

Zearalenone Delays Rat Leydig Cell Regeneration.

Zearalenone (ZEA), a fungal mycotoxin, is present in a wide range of human foods. By virtual screening, we have identified that ZEA is a potential endocrine disruptor of Leydig cells. The effect of ZEA on Leydig cell development is still unclear. The objective of the present study was to explore whether ZEA affected Leydig cell developmental process and to clarify the underlying mechanism. Adult male Sprague-Dawley rats (60 days old) were randomly divided into three groups and these rats received a single intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all Leydig cells. Seven days after EDS treatment, rats intratesticularly received normal saline (control) or 150 or 300 ng/testis/day ZEA for 21 days. Immature Leydig cells isolated from 35-day-old rats were treated with ZEA (0.05-50 μM) for 24 h in vitro. In vivo ZEA exposure lowered serum testosterone levels, reduced Leydig cell number, and decreased Leydig cell-specific gene or protein expression levels possibly via downregulating the steroidogenic factor 1 (Nr5a1) expression. ZEA in vitro inhibited androgen production and steroidogenic enzyme activities in immature Leydig cells by downregulating expression levels of cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), and steroid 5α-reductase 1 (Srd5a1) at a concentration as low as 50 nM. In conclusion, ZEA exposure disrupts Leydig cell development and steroidogenesis possibly via downregulating Nr5a1.

Influence of fetal Leydig cells on the development of adult Leydig cell population in rats.

Leydig cells are the main endogenous testosterone synthesis cells in the body. Testosterone is an essential hormone in males that affects metabolism, emotion, and pubertal development. However, little is known about the development of Leydig cells and relationship between fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). The aims of this study were to investigate the effect of (FLCs) on ALC development. Our study showed that FLCs in neonatal rat testis can be eliminated by 100 mg/kg ethane dimethane sulfonate (EDS) treatment without affecting the health of newborn rats. Immunohistological results showed that eliminating FLCs led to early re-generation of the ALC population (progenitor Leydig cells [PLCs] and ALCs) accompanied at first by increased and then by decreased serum testosterone, indicating that ALCs which appeared after neonatal EDS treatment were degenerated or had attenuated functions. Our results showed that FLCs were eliminated 4 days after EDS treatment, the ALC population regenerated by 21 days, and serum testosterone levels dramatically decreased at 56 days. Collectively, our results indicate that the ablation of FLCs in neonatal rat results in abnormal development of ALCs. Our study further indicates that abnormal development of Leydig cells in the fetal stage leads to steroid hormone disorders, such as testosterone deficiency, in the adult stage. Therefore, studies of Leydig cell development are important for understanding the pathogenesis of testosterone deficiency or pubertas praecox.

Testosterone regulates granzyme K expression in rat testes.

Testosterone depletion induces increased germ cell apoptosis in testes. However, limited studies exist on genes that regulate the germ cell apoptosis. Granzymes (GZM) are serine proteases that induce apoptosis in various tissues. Multiple granzymes, including GZMA, GZMB and GZMN, are present in testes. Th us, we investigated which granzyme may be testosterone responsive and possibly may have a role in germ cell apoptosis aft er testosterone depletion. Ethylene dimethane sulfonate (EDS), a toxicant that selectively ablates the Leydig cells, was injected into rats to withdraw the testosterone. The testosterone depletion effects after 7 days post-EDS were verified by replacing the testosterone exogenously into EDS-treated rats. Serum or testicular testosterone was measured by radioimmunoassay. Using qPCR, mRNAs of granzyme variants in testes were quantified. The germ cell apoptosis was identified by TUNEL assay and the localization of GZMK was by immunohistochemistry. EDS treatment eliminated the Leydig cells and depleted serum and testicular testosterone. At 7 days post-EDS, testis weights were reduced 18% with increased germ cell apoptosis plus elevation GZMK expression. GZMK was not associated with TUNEL-positive cells, but was localized to stripped cytoplasm of spermatids. In addition, apoptotic round spermatids were observed in the caput epididymis. GZMK expression in testes is testosterone dependent. GZMK is located adjacent to germ cells in seminiferous tubules and the presence of apoptotic round spermatids in the epididymis suggest its role in the degradation of microtubules in ectoplasmic specializations. Thus, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.

A brief exposure to cadmium impairs Leydig cell regeneration in the adult rat testis

Cadmium is an endocrine disruptor, impairing male reproduction. The objective of this study is to investigate whether cadmium affects rat Leydig cell regeneration and to dissect the underlying mechanism. Adult male Sprague-Dawley rats received a single intraperitoneal injection (i.p.) of 0, 0.5 or 1.0 mg/kg of cadmium chloride, followed by ethane dimethane sulfonate (EDS) treatment to eliminate adult Leydig cells 20 days later. Compared to control (0 dose), cadmium treatment reduced serum testosterone levels by days 21, 35, and 56 after EDS treatment. Serum luteinizing hormone (LH) levels were also affected by day 56, the only time point examined. There were fewer regenerated Leydig cells in the cadmium-treated testis on days 35 and 56 after EDS treatment. Further studies demonstrated that the mRNA or protein levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Hsd11b1), non-Leydig (Fshr and Dhh), and gonadotroph (Lhb) cells were also significantly lower in cadmium-treated animals. Since LH and desert hedgehog (DHH) are critical factors for Leydig cell differentiation, our result demonstrated that the lower doses of cadmium exposure, even briefly, may permanently damage Leydig cell regeneration.

Steroidogenic fate of the Leydig cells that repopulate the testes of young and aged Brown Norway rats after elimination of the preexisting Leydig cells

The capacity of Brown Norway rat Leydig cells to produce testosterone (T) decreases with aging. In a previous study, we reported that a new generation of Leydig cells can be restored in both young and old rat testes after a single injection of ethane dimethanesulfonate (EDS), and that the abilities of the new Leydig cells in young and old rats to produce T were equivalent. Our objective herein was to compare the steroidogenic fate of the new Leydig cells over time. Young (3month-old) and old (18month-old) rats were injected with EDS to eliminate the existing Leydig cells. Ten weeks after EDS, Leydig cells had been restored and T production by the new Leydig cells isolated from young and old rat testes was equivalent. Thirty weeks after EDS treatment of young rats, the ability of the new Leydig cells to produce T had not diminished from 10weeks post-EDS. In contrast, at 30weeks post-EDS, T production by new cells in old rat testes was reduced significantly from the 10-week level. Serum T levels at 10 and 30weeks were consistent with Leydig cell T production. Serum LH levels did not differ in any group. Thus, although the Leydig cells restored to both young and old rats after EDS initially produced T at high, equivalent levels, the cells in the old testes did not maintain this ability. These results suggest that: 1) the cells from which new populations of Leydig cells are derived may differ depending upon the age of the rat; and/or 2) factors extrinsic to the new Leydig cells in young and old testes differ, and it is these differences that are responsible for reductions in T by the newly formed Leydig cells in the testes of old rats.

Apoptotic and Inflammatory Role of TNF‐α in Adult Rat Testis upon Ethylene Dimethane Sulfonate Treatment

Tumor necrosis factor (TNF‐ α) is multifunctional cytokine secreted in mammalian testis by germ cells, Sertoli cells and to some extent by macrophage. TNF‐α regulates different cellular processes pertinent to spermatogenesis including steroidogenesis, germ cell apoptosis and inflammation. Inflammatory marker, IL‐1b is secreted by activated macrophages. The Fas/FasL apoptotic pathway is the current model for depletion of Leydig cells with ethylene dimethane sulfonate (EDS) treated adult rats. As TNF‐α belongs to the same cytokine family, we are investigating the role of TNF‐α in Leydig cell loss at 6, 15 and 24 hr post‐EDS. Lhr and Insl3, primarily Leydig cells markers declined by more than 85% while Hsd3b2, Leydig cell enzyme marker declined by 96% at 24 hr post‐EDS. Using TUNEL, we found 5‐fold increase in interstitial apoptotic cells at 24 hr post‐EDS. Next, genes associated with macrophage and inflammation was assayed. We observed a 2.2 fold increase in Cd163/Cd68 at 15 hr‐post EDS, suggesting an increase in testicular macrophages. Tnfa was increased 2.7‐fold at 15 hr after EDS and Il1b increased 3.3 fold, 24 hr post‐EDS. ELISA detection of TNF‐α protein in the testicular tissue showed no change in expression at 6, 15 and 24 hr post‐EDS. Immunohistochemistry for 3β‐HSD and CD163 comply with q‐PCR results. Since no change was observed in Fas and Fasl over the course of time and both the receptors CD120a and CD120b changes, we are currently studying TNF‐ α inhibitors.

Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3β-hydroxysteroid dehydrogenase (3β-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3β-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery.

Alterations of gene profiles in Leydig-cell-regenerating adult rat testis after ethane dimethane sulfonate-treatment.

Only occupying about 1%–5% of total testicular cells, the adult Leydig cell (ALC) is a unique endocrine cell that produces androgens. Rat Leydig cells regenerate after these cells in the testis are eliminated with ethane dimethane sulfonate (EDS). In this study, we have characterized Leydig cell regeneration and messenger ribonucleic acids (mRNA) profiles of EDS treated rat testes. Serum testosterone, testicular gene profiling and some steroidogenesis-related proteins were analyzed at 7, 21, 35 and 90 days after EDS treatment. Testicular testosterone levels declined to undetectable levels until 7 days after treatment and then started to recover. Seven days after treatment, 81 mRNAs were down-regulated greater than or equal to two-fold, with 48 becoming undetectable. These genes increased their expression 21 days and completely returned to normal levels 90 days after treatment. The undetectable genes include steroidogenic pathway proteins: steroidogenic acute regulatory protein, Scarb1, Cyp11a1, Cyp17a1, Hsd3b1, Cyp1b1 and Cyp2a1. Seven days after treatment, there were 89 mRNAs up-regulated two-fold or more including Pkib. These up-regulated mRNAs returned to normal 90 days after treatment. Cyp2a1 did not start to recover until 35 days after treatment, indicating that this gene is only expressed in ALCs not in the precursor cells. Quantitative polymerase chain reaction, western blotting and semi-quantitative immunohistochemical staining using tissue array confirmed the changes of several randomly picked genes and their proteins.

TNFα /TNFα receptors in adult rat testis after ethylene dimethane sulfonate treatment (609.15)

Tumor necrosis factor (TNF‐α) is a multifunctional cytokine secreted in mammalian testis by germ cells, Sertoli cells and to some extent by macrophage; however TNF‐α receptors are largely confined to Sertoli cells. TNF‐α regulates different cellular processes pertinent to spermatogenesis including steroidogenesis, germ cell apoptosis, growth hormone function and inflammation. The Fas/FasL apoptotic pathway is the current model for depletion of Leydig cells with ethylene dimethane sulfonate (EDS) treated adult rat testis. We are investigating a potential role of TNF‐α in EDS Leydig cell depletion as well as other tissue correlates. At 6, 15 and 24 hr post‐EDS, using RT‐ and q‐PCR we find that mRNA expression of Leydig cell markers, Lhr and Insl‐3 declined by more than 85% and 3β‐HSD declined by 97% at 24 hr after EDS compared to controls. Using TUNEL, a DNA fragmentation detection assay, we observed a time dependent loss of Leydig cells by apoptosis. Next, genes associated with inflammation, TNF‐α was increased modestly and IL‐1β increased 3 fold, 24 hr post‐EDS. A three‐fold increase in TNF‐α receptor superfamily member 1A mRNA (Tnfrsf1a/ CD120a) was found. However, no change was observed in tumor necrosis factor receptor superfamily member 1B mRNA (Tnfrsf1b/CD120b) at 24 hr post‐ treatment. The mRNA of the two TNF‐α receptors has a differential expression, suggesting their presence on different cell types. ELISA detection of the testicular tissue showed an increased TNF‐α protein expression at 24 hr post‐EDS. Thus, with a high percentage of Leydig cell loss at a time when TNF‐α and its receptors are changing may imply TNF‐α activity is involved with Leydig cell apoptosis following EDS, however, stronger correlates may be found with other cell types. Future work is to localize TNF‐α receptors using immunohistochemistry.

Testicular Macrophage Activity Correlates with Leydig Cell Apoptosis following Ethylene Dimethane Sulfonate (EDS) Treatment of Mature Male Sprague‐Dawley Rats

Tumor necrosis factor alpha (Tnfα), a cytokine secreted by macrophages is known to initiate cell apoptosis. Other laboratories have reported that macrophages are directly associated with Leydig cells in testicular interstitium. Using reverse transcription of total RNA (RT) and real‐time quantifiable polymerase chain reaction (qPCR) with quantification of expression using the (−) ΔΔCt method, we found increased Tnfα mRNA expression within 24 hr of EDS treatment. We also found that CD68 mRNA (a macrophage/monocyte/dendritic cell marker) increased within 24 hr of EDS treatment. Thus, not only are macrophages present in testes but macrophages may also be increasing in cell number/testis following EDS treatment. Leydig cells are lost via apoptosis following EDS treatment and analysis of the Leydig cell markers, Lhr and Insl‐3 mRNA levels declined more than 80%. Since these genes are expressed primarily in Leydig cells, this suggested an 80% loss of Leydig cells by 24 hr. Using the TUNEL assay that detects DNA fragmentation, we confirmed that Leydig cell apoptosis is time dependent and is increased with increased time following EDS exposure. We suggest a potential paracrine action of macrophages through secretion of TNFα to induce Leydig cell apoptosis.

Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

Gene expression analysis of toxicological pathways in TM3 leydig cell lines treated with Ethane dimethanesulfonate

Ethane dimethanesulfonate (EDS), a well-known alkylating agent, selectively destroys Leydig cells. To clarify the molecular pathways underlying EDS action on Leydig cells, we analyzed gene expression profiles of an EDS-treated TM3 Leydig cell line. In this study, we analyzed the representative canonical pathways and toxicity pathways/gene lists using the Ingenuity Pathways Analysis program. In TM3 cells, 677 and 6756 genes were identified as being up- or downregulated after 3 and 24 h EDS treatments, respectively, (>1.3-fold changes, p < 0.05). Toxicological pathway analysis revealed that expression of genes related to Nrf2-mediated oxidative stress response showed remarkable changes in early or later stage of EDS-treated TM3 cells. Several genes related to steroidogenesis and apoptosis were also differentially expressed at 24 h in EDS-treated TM3 cells. Overall, toxicological pathway analysis using gene expression profiling showed that oxidative stress might be an important factor in cell death in TM3 cells affected by EDS treatment.

Prolactin Receptor loss with Leydig Cell Apoptosis after EDS treatment of Adult Rats

Prolactin receptors (Prlr) are reported to be localized to plasma membranes of testicular interstitial cells including Leydig cells. Prlr is also present in Sertoli cell and cells of germinal epithelium (spermatogonia, spermatocytes and elongating spermatids) in the testis. Ethylene dimethane sulfonate (EDS) eliminates Leydig cell in adult rat testes leading to testosterone depletion and germ cell loss. In this study, two Prlr transcript variants (Prl1r &amp; Prl2r) were investigated at times 6, 15, 24 hr, and 5 and 7 days (d) following EDS using RT and qPCR. Rate of Leydig cell loss was evaluated by analysis of insulin‐like peptide 3 mRNA (Insl3), a Leydig cell‐specific marker. Prl2r and Insl3 were less than 1% of normal values in 5 &amp; 7 d EDS‐treated rats. Similar profiles for Prl2r and Insl3 were observed at earlier time points (6, 15 &amp; 24hr). Testosterone replacement after EDS, however, did not restore the levels of Prl2r and Insl3 mRNAs suggesting they are localized to only Leydig cells. In contrast, Prl1r only declined to 40% of normal levels in EDS‐treated rats suggesting that this receptor variant is located in cells of germinal epithelium, as well as Leydig cells.Texas Woman's University‐Research Enhancement Program &amp; Dept. of Biology

Identification and Function of Putative Stem Leydig Cells in the Adult Rat Testis.

Stem cells have the capacity to self-renew and to generate differentiated cells. Such cells are found in adult as well as in embryonic tissue. Previous studies have shown that a single injection of adult rats with ethane dimethanesulfonate (EDS) results in the elimination of the adult Leydig cells. Subsequently, a new generation of Leydig cells is restored to the testes, indicating that the elimination of the adult Leydig cells results in the induction of precursor cells to differentiate into functional Leydig cells. We developed an organ culture system with which to test this hypothesis. Interstitial and seminiferous tubule fractions of rat testes were separated from each other and isolated four days after the rats received an EDS injection. The two fractions were cultured in vitro with LH for four weeks. Immediately after their isolation, the tubules did not produce testosterone in response to LH. However, after two weeks in culture, testosterone was detected in the medium of the tubule fraction. Testosterone production by the tubules increased at least through four weeks. Western blots showed that the steroidogenic proteins, P450scc and StAR, which were undetectable immediately after the tubules were isolated, became detectable. Immunhistochemical staining showed that 3β-HSD positive cells were located around the tubules. In contrast to the tubules, the interstitial fraction failed to produce testosterone. The effects of hormones and/or growth factors, including IGF-1, PDGFAA, LIF, T3, TGF-a and PDGFBB, and of Notch inhibitor, on Leydig cell differentiation were tested on cultured tubules. These factors were selected based on previous studies suggesting that they may play roles in Leydig cell differentiation during normal prepubertal development. Among the factors examined, IGF-1, PDGFAA, LIF and Notch inhibitor were found to stimulate testosterone production, while T3, TGF-a and PDGFBB had no effect on Leydig cell differentiation. hese results suggest that Leydig cell differentiation in the adult testis after EDS treatment is similar to Leydig cell differentiation during normal prepubertal development. We suggest that the precursor cells that give rise to the adult cells might be stem Leydig cells. The results support the peritubular localization of these putative stem cells. The organ culture system used for these studies was particularly useful for identifying the factors (hormones and growth factors) involved in the regulation of the differentiation of the putative stem cells. Supported by grants R37 AG021092, RO1 HD050570 and RO1 AG030598. (platform)

Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place.

Studies on the Expression of 80-kDa Human Sperm Antigen in Rat Testis and Epididymis

The 80-kDa human sperm antigen (HSA) has demonstrated to be a promising candidate for development of an antifertility vaccine because it is a sperm-specific, conserved, and immunogenic protein. The present study demonstrates the androgen-regulated expression of 80-kDa HSA in testis and epididymis of rat by immunohistochemistry (IHC), using its specific antibodies. Developmental expression of 80-kDa HSA was investigated on days 10, 20, 40, 60, and 90 of age in the testis and epididymis by IHC, and relative staining intensity was estimated by image analysis using BIOVIS software. On days 10 and 20, no significant staining was observed in the testis and epididymis, whereas it gradually increased from day 40 onwards. The highest staining was seen on day 90 in both testis and epididymis. Gradual increase in expression of 80-kDa HSA after day 40 suggests that it is possibly regulated by androgen. To study the androgen-regulated expression of 80-kDa, adult male rats were treated with 75 mg/kg body weight of ethylene dimethane sulfonate (EDS), which selectively destroys Leydig cells and thus induces complete androgen withdrawal. It was observed that the staining intensity decreased following EDS treatment in rat testis as well as epididymis, and it was regained after supplementation with dihydrotestosterone. Increased expression during sexual maturation at the time of testosterone surge and its regulation by antiandrogen/androgen treatment suggest androgen-dependent expression of 80-kDa HSA in rat testis and epididymis.

Histological changes of the testis and epididymis in adult rats as a result of Ley dig cell destruction after ethane dimethane sulfonate treatment: a morphometric study

To quantitatively study the histological changes of the testis and epididymis as a result of a drastic reduction of testosterone secretion. Fourteen adult Sprague-Dawley rats were injected intraperitoneally with ethane dimethane sulfonate (EDS, 75 mg/kg) and the same number of animals were injected with normal saline as a control. At days 7 and 12 (after treatment), respectively, half of the animals from each group were killed. The testes and epididymides were removed and tissue blocks embedded in methacrylate resin. The cell number per testis was estimated using the stereological optical disector and some other parameters were obtained using other morphometric methods. The EDS treatment resulted in an almost complete elimination of Leydig cells but had no effect on the numbers of Sertoli cells per testis. At day 7 after EDS treatment, many elongated spermatids were retained in the seminiferous epithelium and many round spermatids could be seen in the epididymal ducts. At day 12, a looser arrangement of spermatids and spermatocytes became evident, with apparent narrow empty spaces being formed between germ cells in an approximately radial direction towards the tubule lumen; the numbers (per testis) of non-type B spermatogonia and spermatocytes were similar to controls, whereas that of type B spermatogonia increased by 59%, and that of early round, elongating and late elongated spermatids decreased by 37%, 72% and 52%, respectively. The primary spermatogenic lesions following EDS administration were (i) spermiation failure and (ii) detachment of spermatids and spermatocytes associated with impairment in spermiogenesis and meiosis.