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Testis |
Population Council and The Rockefeller University,3 New York, New York 10021
Pediatric Surgical Research Laboratories,4 Massachusetts General Hospital, Boston, Massachusetts 02114
Pediatric Endocrine Division,5 Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-androstan-3,17ß-diol concentrations were much higher than that of testosterone. MIS-treated animals had fewer mesenchymal precursors on Day 15 and fewer differentiated Leydig cells on Day 35 with decreased numbers of BrdU+ nuclei. Apoptotic interstitial cells were observed only in the MIS-treated testes, not in the vehicle-treated group on Day 15. These data suggest that MIS inhibits regeneration of Leydig cells in EDS-treated rats by enhancing apoptotic cell death as well as by decreasing proliferative capacity.
Leydig cells, male reproductive tract, mechanisms of hormone action, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The process of Leydig cell regeneration after EDS ablation is controlled by both extratesticular hormones, such as luteinizing hormone (LH) [7, 9] and thyroid hormone [10], as well as local testicular factors, such as Kit ligand [11]. Other factors are also postulated to have a role in this process. One candidate is Müllerian inhibiting substance (MIS) or anti-Müllerian hormone, a homodimeric glycoprotein member of the transforming growth factor-ß superfamily [12]. MIS is a Sertoli cell product that is best known for its role in inducing regression of the Müllerian ducts in the male embryo. Recent studies suggest that MIS also acts as a paracrine hormone to inhibit Leydig cell proliferation and differentiation after birth [13–15]. Mice null for Mis [14, 16] or the receptor gene [17] develop Leydig cell hyperplasia and foci of Leydig cell tumors. In contrast, mice that overexpress human MIS have reduced Leydig cell numbers and serum testosterone concentrations [14, 18, 19]. Treatment of adult mice and rats with MIS induces transitory decreases in testosterone production [20, 21]. In addition, in vitro studies have shown that the MIS type II receptor is expressed in primary Leydig cells and that MIS can downregulate DNA synthesis and steroidogenesis in primary Leydig cell cultures [13, 15].
Although MIS inhibits DNA synthesis in proliferating Leydig cells from peripubertal rats [13, 15], their long doubling time in culture has hindered efforts to confirm a direct antiproliferative action of MIS. To investigate further the actions of MIS on Leydig cell proliferation and steroidogenesis, we employed the EDS model of Leydig cell regeneration. This study was designed to test whether MIS inhibits the proliferation and/or differentiation of regenerating Leydig cells after EDS ablation of mature Leydig cells. We asked whether MIS suppresses the recovery of Leydig cell numbers and alters steroidogenesis and whether these effects, if present, were transitory (present only immediately after MIS exposure) or sustained (still present 20 days beyond the period of exposure to MIS).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sixty-day-old adult male Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA) and housed in standard rat cages in a controlled light (14L:10D) and temperature (23°C) environment with ad libitum access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committees of both Rockefeller University (Protocol 91200-R2) and Duke University Medical Center (A433-00-09).
MIS and EDS
Recombinant human MIS was produced in Chinese hamster ovary cells transfected with the human MIS gene and purified by immunoaffinity chromatography in the Pediatric Surgical Research Laboratories at Massachusetts General Hospital [22]. The highly purified MIS was quantified by ELISA and its bioactivity verified in a urogenital ridge bioassay [23, 24]. Recombinant human MIS is bioactive across species, as demonstrated in the rat urogenital ridge assay [22], the mouse fetal ovary assay [25], and other in vitro systems [13, 14].
The EDS was a gift from Dr. L. Earl Gray, U.S. Environmental Protection Agency, Research Triangle Park, NC (purchased from Radion Co.).
Experimental Protocol
Intraperitoneal injections were administered after sedation with isofluorane anesthesia. Twenty rats received 75 mg/kg body weight (BW) EDS by i.p. injection on Day 1. On Days 11, 12, 13, and 14, 10 rats received daily intratesticular injections of 75 µg human recombinant MIS in 80 µl and 10 rats received vehicle (0.1 M PBS, pH 7.4,) in a volume of 80 µl. The pharmacologic dose of MIS was chosen based on a prior study that demonstrated the efficacy of this dose to modulate Leydig cell steroidogenesis [20]. In that study, intratesticular injection of 75 µg MIS caused a transient elevation of serum and intratesticular fluid MIS concentrations for 48 h. Five rats in each group were killed 1 day after completing the daily MIS injections (Day 15) and the remaining five were killed 20 days later (Day 35). One hour before being killed, the rats were weighed and given 5-bromo-2'-deoxyuridine (BrdU; Sigma, St. Louis, MO) at 100 mg/kg BW by i.p. injection. The rats were anesthetized with 100 mg/kg BW of pentobarbital i.p., then one testis was removed, weighed, flash-frozen, and stored at -80°C. Blood was collected by cardiac puncture, then separated and the serum stored at -20°C until assay. The rats were then perfused via the left ventricle with Bouin solution for 30–45 min [26]. The perfused testes were removed, weighed, postfixed in Bouin solution overnight at 4°C, and stored in 70% ethanol at 4°C until further processing for stereology and immunohistochemistry.
Stereology
The numbers of Leydig cells and their mesenchymal-like precursors were measured by stereology using the fractionator method [27–31]. Fixed testes were sequentially fractionated into small blocks representing a known fraction of the whole testis. Selected blocks were embedded in paraffin and exhaustively sectioned at 4 µm. Contiguous pairs of sections were mounted onto glass slides, which were stored at room temperature. Each slide represented a known fraction of the original paraffin block, which could be extrapolated to a known fraction of the whole testis. In every fractionation step, the selection of a set of blocks or of 4-µm-thick sections was performed with systematic random sampling.
Leydig cells were cytologically identified by positive staining for cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) while mesenchymal-like precursor cells were defined as spindle-shaped interstitial cells with minimal cytoplasm and an elongated nucleus. The mesenchymal cells did not express P450scc by immunohistochemistry and were located within the interstitial spaces but not in direct contact with the seminiferous tubules (as were peritubular myoid cells) or blood vessels (as were pericytes). Leydig cells and mesenchymal-like cells were counted in randomly selected areas using a planapochromatic 60x objective. Following the physical dissector method, nuclei in the test area of a section were not counted if they were also observed in the corresponding reference area in the contiguous serial section. The counting rule stipulated that nuclei visualized partially at the lower or right edge of the test area were included, whereas those located at the upper or left edges were excluded. At least 100 cells were scored for each sample.
The estimated total numbers of Leydig cells and mesenchymal cells per testis were calculated by multiplying the number of cells scored by the product of the reciprocals of each of the sampling fractions. The total number of BrdU-positive cells per testis was determined based on the percentage of proliferative cells and the total number of cells per testis calculated by stereology.
Hormonal Assays
To determine the effect of MIS treatment on Leydig cell steroidogenic function, the concentrations of serum testosterone and LH and intratesticular testosterone and 5-androstan-3,17ß-diol (3-diol) were assessed by RIA using 3H-testosterone, 125I-LH or 3H-3-diol, respectively, as the tracers [32]. The frozen testicular fragments were homogenized in 70% methanol and ether-extracted before the intratesticular steroid assays were performed [33]. A known amount of tritiated testosterone was added to the homogenate during the extraction procedure to calculate the percentage recovery. Results were corrected accordingly.
TdT-Mediated dUTP-Biotin Nick End Labeling (TUNEL)
To determine whether MIS induces cell death during the early stages of Leydig cell regeneration (Day 15), in situ end labeling was performed on samples from rats treated with MIS or vehicle and killed 24 h after the last injection. Labeling was performed following a previously described procedure [34]. Briefly, sections were dewaxed and rehydrated, incubated with proteinase K, washed in 100 mM PBS, then fixed in 4% paraformaldehyde. Endogenous peroxidase activity was quenched with 0.1% H2O2 in PBS. Sections were then washed and incubated in TdT buffer (0.03 M Tris, pH 7.2, 0.014 M sodium cacodylate, 0.001 M cobalt chloride) for 20 min, followed by incubation in TdT buffer containing TdT and biotin-dUTP for 1 h at 37°C. After stopping the reaction with two washes in saline sodium citrate and a 2% BSA PBS blocking incubation, the colored reaction product was visualized using the Vectastain Elite ABC Kit (Vector Labs, Burlingame, CA). Sections were counterstained with Mayer hematoxylin, dehydrated, and mounted.
Immunohistochemistry
Testis blocks were fixed in Bouin, then embedded in paraffin for sectioning at a thickness of 5 µm. Leydig cells were identified on the basis of positive immunohistochemical staining for P450scc using a rabbit anti-P450scc antibody (Cat #RDI-P450SCCabr; Research Diagnostics, Inc., Flanders, NJ). Spindle-shaped interstitial cells lacking P450scc staining were classified as mesenchymal-like precursors. Proliferative activity was assessed in the same sections using an anti-BrdU antibody (Cat #RPN 202; Amersham Pharmacia Biotech, Piscataway, NJ).
The sections were first dewaxed, then microwaved at maximum power (1380 W) for 13 min in 100 mM citrate, pH 6.0, for antigen retrieval. Endogenous peroxidase activity was quenched by incubation in 0.3% H2O2 in methanol for 18 min. The slides were washed with 0.1% Tween 20 in 10 mM PBS, incubated for 20 min in blocking serum (10% normal goat serum PBS), then exposed to the monoclonal anti-BrdU antibody at room temperature for 1 h in a humidified chamber. After washing, the slides were incubated with a biotin-goat anti-mouse antibody (Zymed Labs, San Francisco, CA) diluted 1:200 in 5% normal goat serum (NGS) for 20 min at room temperature. Immunoreactive cells were visualized using the Vectastain Elite ABC Kit (Vector Labs) with 3'3 diaminobenzidine tetrachloride (Boehringer Mannheim, Roche Applied Science, Indianapolis, IN) as the chromogen. To identify Leydig cells, the same sections were subsequently washed well, blocked with 10% normal goat serum, and incubated with an antibody to P450scc for 1 h. The sections were then incubated with a biotin-goat anti-rabbit antibody 1:200 in 5% NGS in PBS for 30 min. After a wash step, immunoreactive cells were visualized using alkaline phosphatase (Vectastain ABC-AP) and Vector Red AP Substrate (Vector Labs). Sections processed with PBS instead of the primary antibody were used as negative immunohistochemistry controls.
Immunohistochemistry was performed on alternate sections using a rabbit polyclonal antibody to 5-reductase, type 1, at 1:100 (gift of Dr. Bernard Robaire, McGill University, Montreal, PQ, Canada) [35], a commercial rabbit anti-P450scc antibody at 1:3000 (Cat #RDI-P450SCCabr, Research Diagnostics, Inc.), and a rabbit anti-porcine P450c17 antibody at 1:6000 (gift of Dr. Dale Buchanan Hales, University of Illinois, Chicago, IL) with the Vectastain Elite ABC Kit (Vector Labs) as described above. All sections were counterstained with hematoxylin.
Data Analysis
All data were evaluated at two time points, on Day 15 post-EDS, 24 h after the last bilateral injection of MIS or vehicle, and on Day 35 post-EDS, 21 days after the last injection of MIS or vehicle. The results are expressed as mean ± SEM. Differences between paired means were assessed using the Student t-test analysis or the Mann-Whitney U-test. Differences were regarded as significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body and testis weights were equivalent in MIS- and vehicle-treated rats at both time points (Days 15 and 35 after EDS) (Table 1).
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Cell Numbers
To evaluate the effect of MIS on Leydig cell regeneration after EDS-induced depletion, the total number of mesenchymal-like precursor cells and Leydig cells per testis and their proliferative indexes were determined at both time points. On Day 15, fewer mesenchymal cells were observed in the MIS-treated group than in the vehicle-treated group, 4.8 ± 0.7 (x106 cells per testis, mean ± SEM) versus 9.0 ± 1.3 (x 106, mean ± SEM), P < 0.05 (Fig. 1). By Day 35, the number of mesenchymal cells was no different between the MIS- and vehicle-treated groups (Fig. 1).
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During the early phase of Leydig cell regeneration at Day 15, the number of Leydig cells was similar between MIS- and vehicle-treated animals (Fig. 2). By the later time point, Day 35 after EDS (21 days after completing the MIS treatment), the MIS-treated testes had fewer Leydig cells than the vehicle-treated testes, 14.0 ± 1.7 versus 21.2 ± 2.3 (x106) cells per testis, P < 0.05 (Fig. 2). Thus, compared with vehicle treatment, MIS-treated animals had 47% fewer mesenchymal cells at the early time point and 34% fewer Leydig cells at the later time point. These results demonstrate that MIS inhibited the recruitment and/or proliferation of Leydig cell progenitors and limited the recovery in numbers of mature differentiated Leydig cells after EDS-induced depletion.
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Proliferative Index
To determine whether the observed differences in cell number with MIS administration were due to effects of MIS on cellular proliferation, the percentage of BrdU-labeled mesenchymal-like precursor cells was determined in the same testis samples that had been used for stereology. The results, expressed as the total number of BrdU+ nuclei per testis, provide an index of active proliferation (Fig. 3). On Day 15, the MIS-treated group had twice as many BrdU+ cells on testicular sections than the control group (P < 0.05). In contrast, on Day 35, the testicular sections from MIS-treated animals revealed 4.5-fold fewer BrdU+ cells than those from the vehicle-treated group (P < 0.05).
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TUNEL Labeling
To determine if the reduction in the number of mesenchymal cells on Day 15 is due to a proapoptotic effect of MIS on regenerating Leydig cell precursors, TUNEL was performed to identify cells undergoing apoptotic cell death. No TUNEL-labeled nuclei were observed in the interstitial cells of any sections from vehicle-treated testes while scattered positive nuclei were observed in the MIS-treated group. The TUNEL-positive nuclei displayed the characteristic DNA fragmentation observed in cells undergoing apoptosis (Fig. 4). This suggested that the reduction in the number of mesenchymal cell precursors in the MIS-treated animals on Day 15 was due, at least in part, to a proapoptotic effect of MIS on these cells during the regeneration process after EDS ablation.
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Hormonal Measurements
Serum concentrations of testosterone and LH were not statistically different in MIS-treated and vehicle-treated animals at either time point. In both groups, the serum testosterone concentrations were low but detectable on Day 15 and within the range of normal adult values by Day 35 (Fig. 5). LH concentrations followed an inverse pattern, being higher on Day 15 and normal on Day 35. Similarly, the intratesticular concentrations of testosterone and 3-diol were low on Day 15 and higher on Day 35. In addition, on Day 35, the concentration of intratesticular testosterone was significantly elevated in the MIS-treated group compared with the control group (P < 0.05) (Fig. 6). When calculated as the testosterone production rate, the MIS-treated animals produced 15.9 ng testosterone/106 Leydig cells versus 2.8 ng/106 Leydig cells in vehicle-treated controls (P < 0.05). At both time points, the intratesticular concentrations of 3-diol exceeded that of testosterone by 3- to 6-fold (P < 0.01). These data showed that, although the steroidogenic capacity of the regenerating Leydig cells was increased by MIS treatment (higher testosterone production rate and intratesticular androgen concentrations), serum androgen concentrations were not statistically different. This may reflect the small number of animals studied and the inherent wide fluctuations of serum androgen concentrations or the markedly reduced numbers of Leydig cells.
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Expression of Steroidogenic Enzymes
Immunohistochemistry was performed on testes collected on Days 15 and 35 to examine the effect of MIS on P450scc, P450c17, and 5-reductase protein expression in regenerating Leydig cells. Fifteen days after EDS ablation, the expression of all three enzymes was low and similar between MIS- and vehicle-treated testes (not shown). At 35 days, immunoreactivity of each of the three enzymes was more intense per cell in MIS-treated than vehicle-treated animals (Fig. 7), but there appeared to be fewer Leydig cells per microscopic field of MIS-treated testes, consistent with the stereology and hormone data. The reduced numbers of Leydig cells and higher concentration of intratesticular testosterone in the MIS-treated animals compared with control suggest that androgen secretory capacity and steroidogenic enzyme expression per cell is higher after MIS administration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The inhibitory effects of MIS on Leydig cell precursors are more potent than that of thyroid hormone, which has also been studied in the EDS depletion model [10]. Thyroid hormone decreased the numbers of mesenchymal-like cells by 8% on Day 14 and 26% on Day 21 while causing a near doubling of mature Leydig cells, suggesting that thyroid hormone stimulates the differentiation of progenitor cells to mature Leydig cells. The EDS depletion model was also used to evaluate the effects of the Kit ligand (also known as stem cell factor) on Leydig cells [11]. Kit ligand was observed to promote the regeneration of Leydig cells, most notably during the first 2 wk after EDS treatment. Kit ligand and MIS are both synthesized by Sertoli cells, indicating that regeneration and, by extension, pubertal development of Leydig cells are under local Sertoli cell paracrine control, with a balance between MIS-mediated inhibition and Kit-mediated stimulation determining the rates of these processes.
It has been reported that adult transgenic mice overexpressing MIS have 50% fewer immature and 80% fewer mature Leydig cells than wild-type littermates [14], whereas recombinant mice lacking MIS [16] or the MIS type II receptor [17] develop Leydig cell hyperplasia. As the adult complement of Leydig cells is generated during pubertal maturation of the testis, these findings suggest that MIS action during postnatal testicular development is essential to achieve normal Leydig cell numbers. We have previously demonstrated that primary Leydig cells express the MIS type II receptor and are responsive to MIS in vitro [13]. Although the secretion of MIS by Sertoli cells gradually declines after birth, it remains detectable in a stage-specific pattern even in the adult animal [37]. Moreover, the expression of the MIS type II receptor on Leydig cells increases from Day 9 to Day 14 and remains abundant in the pubertal and adult animal [13, 37]. With this developmental increase in receptor expression and presumably corresponding increase in responsiveness during the period of Leydig cell proliferation, we speculate that physiologic concentrations of MIS are sufficient to modulate Leydig cell numbers. Indeed, it is conceivable that the expression of MIS must be balanced between a minimal level that avoids Leydig cell hyperplasia and a maximal level to permit normal proliferation.
Our data demonstrated apoptotic interstitial cells in the MIS-treated testes whereas no apoptotic cells were observed in the vehicle-treated testes on Day 15. The finding of apoptosis in the interstitial cells was validated by our observation of TUNEL-positive germ cells, indicating apoptosis of germ cells due to testosterone deficiency after EDS treatment, as previously demonstrated [38]. Although TUNEL provides only preliminary evidence of apoptotic cell death in the Leydig cell precursors in this model of Leydig cell regeneration, MIS is known to indirectly induce apoptosis of ductal epithelial cells during Müllerian duct regression [39–41] and causes apoptosis in the fetal lung [42]. Thus, it is conceivable that MIS employs a similar mechanism to inhibit the expansion in Leydig cell numbers after EDS ablation. We hypothesize that MIS modulates the numbers of mesenchymal Leydig cell precursors, in part, by inducing apoptosis, thereby using another mechanism to regulate Leydig cell regeneration in addition to its suppression of proliferative activity. This mechanism of action may also be at play during the regulation of normal peripubertal development.
Although serum testosterone and LH concentrations were similar between MIS-treated and control groups, the intratesticular androgen levels were higher in MIS-treated testes on Day 35, suggesting an increase in steroidogenic capacity, which would compensate for the decrease in Leydig cell numbers. This was supported by immunohistochemistry showing more intense steroidogenic enzyme expression in Leydig cells from MIS-treated testes. With MIS treatment, the Leydig cells may have a greater capacity for steroidogenesis due to higher expression of the LH receptor and thereby higher responsiveness to LH. The MIS treatment may induce a delay in the maturational changes in LH receptor expression that normally occur during puberty. We have previously observed that steady-state LH receptor mRNA levels are higher in immature compared with adult Leydig cells [43]. Thus, the regenerating Leydig cells in MIS-treated animals at 35 days post-EDS may have higher expression of the LH receptor, similar to that observed in the immature stage of Leydig cell development.
The temporal profile of androgens during recovery after EDS has been shown to be similar to that observed during normal pubertal maturation [44], confirming the utility of this model for the study of Leydig cell development. The speculation that MIS may induce a maturational delay in Leydig cell development is supported by the characteristic ratio of intratesticular androgens in the MIS-treated animals. During male reproductive development in Sprague-Dawley rats, 3-diol is the predominant steroid in circulation during Days 15–40 postpartum [45], and the major intratesticular androgen in 2- to 4-wk old rats [46]. In contrast, testosterone production is predominant in sexually mature animals. The higher intratesticular concentration of 3-diol as compared with testosterone in the MIS-treated animals is typical of immature pubertal animals and suggests a relative immaturity of the regenerating Leydig cells.
We conclude that MIS has a regulatory effect on adult Leydig cell regeneration after EDS-mediated depletion. MIS inhibits the proliferation of Leydig cell precursors and may promote their apoptotic cell death. This inhibitory effect exceeds the effect observed with thyroid hormone and is sufficient to reduce Leydig cell numbers, despite the short duration of the exposure in this experimental model. From the steroid data, we infer that the steroidogenic enzymes are maintained in a more immature profile by the exposure to MIS during recovery of Leydig cell numbers and differentiation of mesenchymal-like precursor cells to a steroidogenically mature Leydig cell. The developmental decline in expression of MIS in the prepubertal testis may permit the peripubertal proliferation of the Leydig cell progenitor cells to generate the full adult complement of Leydig cells. These findings, in conjunction with the Leydig cell phenotypes of mice with alterations in MIS expression, support a physiologic role for MIS in the regulation of adult Leydig cell numbers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Mary M. Lee, Division of Pediatric Endocrinology, 308 Bell Building, Box 3080, Bell Research Drive, Duke University Medical Center, Durham, NC 27710. FAX: 919 684 8613; Lee00140{at}mc.duke.edu
Received: 25 July 2003.
First decision: 30 August 2003.
Accepted: 22 October 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone, 5-androstane-3, 17ß-diol, 5-androstane-3ß-, 17ß-diol, and 17ß-estradiol from male beagles with spontaneous or induced benign prostatic hyperplasia. Invest Urol 1981 19:142-147[Medline]This article has been cited by other articles:
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K. De Gendt, N. Atanassova, K. A. L. Tan, L. R. de Franca, G. G. Parreira, C. McKinnell, R. M. Sharpe, P. T. K. Saunders, J. I. Mason, S. Hartung, et al. Development and Function of the Adult Generation of Leydig Cells in Mice with Sertoli Cell-Selective or Total Ablation of the Androgen Receptor Endocrinology, September 1, 2005; 146(9): 4117 - 4126. [Abstract] [Full Text] [PDF]
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X. Wu, R. Arumugam, S. P. Baker, and M. M. Lee Pubertal and Adult Leydig Cell Function in Mullerian Inhibiting Substance-Deficient Mice Endocrinology, February 1, 2005; 146(2): 589 - 595. [Abstract] [Full Text] [PDF]
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T. R. Kumar Divide and Differentiate: Ghrelin Instructs the Leydig Cells Endocrinology, November 1, 2004; 145(11): 4822 - 4824. [Full Text] [PDF]
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M. L. Barreiro, F. Gaytan, J. M. Castellano, J. S. Suominen, J. Roa, M. Gaytan, E. Aguilar, C. Dieguez, J. Toppari, and M. Tena-Sempere Ghrelin Inhibits the Proliferative Activity of Immature Leydig Cells in Vivo and Regulates Stem Cell Factor Messenger Ribonucleic Acid Expression in Rat Testis Endocrinology, November 1, 2004; 145(11): 4825 - 4834. [Abstract] [Full Text] [PDF]
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