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Regular Article |
a Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
ABSTRACT
Aging in the male human is accompanied by testicular atrophy, although relatively little is known about the mechanisms underlying germ cell loss. Testicular atrophy in the aged Brown Norway rat, an animal model for studies of aging in the human, has been attributed to a loss of spermatogonial stem cells. However, examination of testicular cross-sections from 27-mo-old Brown Norway rats indicated that approximately 14% of type A spermatogonia were stem cells. Furthermore, using bromodeoxyuridine labeling, we found that approximately 47% of these stem cells were actively dividing, with a cell cycle time of approximately 12.6 days. Both serum and testicular interstitial fluid testosterone levels were depressed in the aged rat. Therapy with the GnRH agonist, leuprolide, which has been empirically shown to reverse testicular atrophy in other models of germ cell loss, also partially restored spermatogenesis in the aged Brown Norway rat. The extent of testicular atrophy varied considerably, not only within the control and leuprolide-treatment groups but also between the left and right testes of the same animals. No significant difference was found between the mean percentage of populated tubules in 31-mo-old control animals (16.2 ± 28%, mean ± SD) and 31-mo-old leuprolide-treated animals (20.9 ± 19.8%), but categorical comparisons showed that significantly fewer leuprolide-treated animals and testes contained
1% populated tubules, indicating that GnRH agonist therapy stimulates differentiation of type A spermatogonia. An increase in the ratio of soluble to membrane stem cell factor mRNA levels was present in aged rats and partially reversed following leuprolide therapy.
aging, GnRH, spermatogenesis, testes
INTRODUCTION
Spermatogenesis is a dynamic, synchronized process taking place in the seminiferous epithelium of the testis, whereby spermatogonial stem cells proliferate and differentiate to produce mature haploid germ cells. This complex process is dependent upon paracrine-acting factors produced by supporting Sertoli cells, which line the seminiferous epithelium, as well as upon androgen produced by Leydig cells in the interstitium.
As the human life span is extended and the aging population increases, fertility issues become relevant at ever-increasing ages. Although controversial, gradual but steady declines in the number and quality of sperm [1–3] as well as levels of biologically available or free serum testosterone (T) [4, 5] have been reported with advancing age. Age-related differences in the human testis include reductions in volume or number of T-producing Leydig cells and changes in the morphology and number of germ-cell-supporting Sertoli cells, either of which may contribute to the age-related degeneration of germ cells during spermatogenesis [6–10]. These findings suggest that the mechanisms responsible for age-related declines in testicular function are intrinsic to the testis; however, changes in LH pulse interval and amplitude implicate defects in the hypothalamic-pituitary pole of the hypothalamic-pituitary-gonadal axis as well [11]. The mechanisms responsible for the decline in male reproductive function are poorly characterized, and little progress has been made toward reversing this aging process.
The Brown Norway rat, with its long, relatively disease-free life span, has been a useful model with which to study the process of male aging. The low incidence of both endocrine cancers and obesity in this strain allows the impact of aging to be dissociated from that of disease [12–14]. In the Brown Norway rat, atrophy of the seminiferous tubules begins focally and often unilaterally at approximately 18 mo of age; by 24 mo, testes contain either a mix of normal and regressed tubules or only regressed tubules [12, 13]. The progressive testicular atrophy that occurs in the aged rat has been attributed to a loss of spermatogonial stem cells; however, to our knowledge, spermatogonial stem cell population kinetics have not been characterized.
Testicular atrophy is not unique to the aging animal. Numerous agents, including environmental toxicants, radiation, and chemotherapeutics, also deplete germ cells, producing declines in spermatogenesis and infertility [15–19]. In the rat model, the failure of spermatogenic recovery persists long after treatment has ended, despite a residual population of proliferating spermatogonial stem cells [20, 21]. Chronic GnRH therapy has been demonstrated to reverse atrophy associated with many models of testicular injury in the rat [22–24]. The effects of prolonged GnRH agonist therapy are biphasic, consisting of an initial stimulatory phase, in which levels of serum FSH, LH, and T rise, followed by a down-regulation of LH and FSH secretion with a concomitant lowering of T [25]. The mechanism whereby GnRH agonist therapy stimulates spermatogenesis in models of injury, however, is poorly understood. It has been hypothesized that an elevation of intratesticular T (ITT), measured in whole-testis homogenates and reported as ng/g testis, contributes to the maintenance of testicular atrophy, and that agents reducing ITT levels stimulate the recovery of spermatogenesis [26]. Indeed, ITT levels were elevated as much as fourfold in the LBNF1 rat following radiation doses of 3.5 and 6 Gy, and GnRH agonist therapy not only effectively suppressed ITT levels but also stimulated a repopulation of atrophic tubules and restored fertility [22, 26].
Alterations in the production of testicular endocrine or paracrine factors have also been implicated in the failure of recovery of spermatogenesis [27]. One such paracrine-acting factor is stem cell factor (SCF; also known as c-kit ligand), a germ cell growth factor produced by Sertoli cells that interacts with the tyrosine kinase-receptor protein, c-kit, on spermatogonia and Leydig cells. The c-kit/SCF interaction, which directs spermatogonial proliferation, survival, and adhesion, is essential for normal development of spermatogonia [28, 29]. In the testis, SCF exists mainly in two forms, derived by alternate splicing of exon 6 of the Steel (Sl) locus. Exon 6 is removed in the membrane form of SCF (membrane SCF) but is retained in the soluble form (soluble SCF) [30]. Studies that have examined 2,5-hexanedione (2,5-HD)-induced testicular atrophy suggest that the irreversible nature of this lesion may be related to increases in the relative ratio of soluble to membrane SCF expression [23, 27]. By extension, alterations in testicular SCF expression may also contribute to the decline of spermatogenesis in the aged Brown Norway rat.
Mechanisms common to those observed in toxicant-induced testicular injury may produce the atrophy observed in the aged Brown Norway rat. The following studies were undertaken to examine spermatogonial stem cell kinetics in the aged rat and to establish the reversibility of testicular atrophy by GnRH agonist treatment. The involvement of ITT and SCF were explored as potential mediators for the decline of spermatogenesis in the aged Brown Norway rat.
MATERIALS AND METHODS
Chemicals
Leuprolide (Lupron Depot) was supplied by TAP Pharmaceuticals (Deerfield, IL). Leuprolide (3.75 mg of leuprolide acetate, 0.65 mg of purified gelatin, 33.1 mg of DL-lactic and glycolic acids copolymer, and 6.6 mg of D-mannitol) was reconstituted in 1.5 ml of sterile diluent (75 mg of D-mannitol, 7.5 mg of carboxymethylcellulose sodium, and 1.5 mg of polysorbate 80).
Animals
This study was conducted under federal guidelines for the use and care of laboratory animals [31] and was approved by the Brown University Animal Care and Use Committee. Male Brown Norway rats (6 or 27 mo old) were purchased from Harlan Sprague Dawley/National Institute of Aging (Indianapolis, IN) and allowed to acclimate for 7 days before the initiation of treatments. Rats were housed in groups of three per cage and were maintained at 21 ± 0.6°C, 35%–70% humidity, and a 12L:12D cycle. Food (Pro-Lab rodent chow no. 5001; Farmer's Exchange, Framingham, MA) and drinking water were provided ad libitum.
Germ Cell:Sertoli Cell Ratios
Testes from 27-mo-old Brown Norway rats (n = 5) were fixed in 10% (v/v) neutral buffered formalin; embedded in glycol methacrylate (Technovit 7100; Kulzer, Wehrheim, Germany); serially sectioned into thicknesses of 10, 5, 5, 3, 3, 3, 5, 5, and 10 µm; and then stained with periodic acid-Schiff (PAS) reagent and counterstained with hematoxylin (H). Sertoli cell nucleoli as well as type A, intermediate, and type B spermatogonia were counted in 50 atrophic tubules per testis using the central 3-µm section. Spermatogonial stem cells (defined as single and undifferentiated type A spermatogonia not within 25 µm of any other spermatogonia, adjacent to the peritubular cells, and having a visible cytoplasm) were first identified in the central 3-µm PAS/H section, then followed through serial sections as described elsewhere [21]. To correct for cell size, Abercrombie's correction factor was used, and all corrected cell counts were then normalized to the number of Sertoli cell nuclei with visible nucleoli, which was also corrected using Abercrombie's correction factor [32]. The testes from one animal were not included in the analysis, because only ghost-like outlines of the Sertoli cell nucleoli were present in many tubules, which presented a problem in normalizing the stem and type A spermatogonia numbers to Sertoli cell nuclei.
Stem Cell Labeling Index
An additional group of aged male Brown Norway rats (27 mo old) were exposed to 0.3 M bromodeoxycytidine (BrdCyd) (Sigma Chemical Co., St. Louis, MO) via Alzet minipumps (Alza Corp., Palo Alto, CA) for 2–28 days (n = 3/time point) as described elsewhere [20]. At the end of the BrdCyd exposure, the animals were killed by exsanguination under CO2 anesthesia. Testes were fixed in 10% neutral buffered formalin, and a section of tissue (2- to 3-mm thick) was embedded in glycol methacrylate. The tissue was serially sectioned into thicknesses of 10, 5, 5, 3, 3, 3, 5, 5, and 10 µm, and the middle 3-µm section was immunohistochemically stained to detect bromodeoxyuridine (BrdU) as described elsewhere [20], then counterstained with PAS reagent. The remaining serial sections were stained with PAS/H.
The histological slides were coded, and the numbers of spermatogonial stem cells were determined in a double-blind manner using methods described elsewhere [20]. Once a stem cell was identified, the middle 3-µm section was examined to determine if the cell was positive or negative for BrdU staining. Labeling indices were determined in each animal and averaged for each time point. The percentage of BrdU-positive cells (i.e., labeling index) was plotted versus time (days) of BrdU exposure (Fig. 1A). The linear curve fit for the labeling index was performed with CA-Cricket Graph III software (Computer Associates, Islandia, NY).
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Leuprolide Recovery Study
Aged male Brown Norway rats (27 mo old) were randomly assigned to groups of equal body weight (BW). One group (n = 14, BW = 448 ± 10 g mean ± SEM) was designated as the control, and the other (n = 15, BW = 445 ± 12 g mean ± SEM) was administered the GnRH agonist leuprolide. Animals were administered three s.c. injections of leuprolide (1.5 mg/animal, 25 days apart) or sterile, filtered water. By 9 wk after the last leuprolide injection, 50% mortality had occurred in each group, and the study was terminated. Animals were killed by exsanguination under CO2 anesthesia. Terminal weights were measured for the following organs: brain, pituitary, testis, epididymis, prostate, and seminal vesicles (with coagulating glands, with and without seminal fluid).
Serum and ITT Measurements
Blood was collected by cardiac puncture, and the serum was separated and stored at -80°C for subsequent determination of T levels. Testicular interstitial fluid was collected from both testes according to methods described elsewhere [33]. Briefly, the pampiniform plexus was tied off with a silk suture, the testis removed from the body, and the tunica albuginea pierced at the opposite pole. A silk suture was then attached to the tunica with an autoclip, which was used to suspend the testis in the barrel of a 15-ml syringe. Interstitial fluid was collected into a preweighed, 1.5-ml, siliconized microcentrifuge tube by low-speed centrifugation (60 x g, 10 min, 4°C), and the fluid volume was recorded. To remove blood cells and cellular debris, the fluid was washed with 200 µl of 100 mM PBS, 1% BSA (w/v), and 0.01% thimerosol (w/v), followed by precipitation at 2500 x g (30 min, 4°C). The supernatant was transferred to a preweighed, siliconized tube. Volumes were then recorded, and samples were stored at -80°C for subsequent T determinations.
Measurements of T were made using an electrochemiluminescence immunoassay (Elecsys; Boehringer Mannheim, Indianapolis, IN) based on competitive binding of labeled T with magnetic microparticle separation. The assay analytical sensitivity (i.e., lower detection limit) was 0.020 ng/ml, with a functional sensitivity (i.e., smallest T concentration that can be reproducibly measured with an interassay coefficient of variation [CV] of 20%) of 0.12 ng/ml. The intra- and interassay CVs were less than 5% and 8%, respectively.
Evaluation of Spermatogenesis
After collection of interstitial fluid, half of each testis was immersion-fixed in 10% neutral buffered formalin, and the other half was detunicated and snap-frozen in liquid nitrogen for mRNA analysis. Formalin-fixed cross-sections (2- to 3-mm thick) were embedded in glycol methacrylate, sectioned (3.0 µm), and stained with PAS/H. To evaluate spermatogenesis, one tubule cross-section from each testis was divided into nine grids, and each tubule lying within a grid was scored as atrophic (i.e., no spermatogonia later than type A) or populated (i.e., 
3 germ cells that had reached type B or more advanced stages). A minimum of 200 tubules were scored per testicular cross-section. Due to the extreme within-animal variability, the number of testes containing less than 1% populated tubules was compared both on a per-testis and per-animal basis using the Fischer Exact Test.
Reverse Transcription-Polymerase Chain Reaction
Expression of the soluble and membrane splice forms of SCF was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Total RNA (2 µg), isolated as described elsewhere [24], was reverse transcribed into cDNA in a 20-µl reaction of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol containing 125 µM 2'-deoxynucleoside 5'-triphosphate mix (dNTP mix; 125 µM each of dATP, dGTP, dCTP, and dTTP), 1 µl (3 µg) of random hexamers, and 200 U (1 µl) of Superscript RNase H- reverse transcriptase (Gibco BRL, Gaithersburg, MD). Synthesis of cDNA was conducted at 42°C for 50 min, followed by 70°C for 15 min.
The primers used for PCR quantification of the membrane:soluble SCF mRNA ratio were sense primer 1 ACT-TGG-ATT-ATC-ACT-TGC-ATT-TAT-C and antisense primer 2 CTT-CCA-GTA-TAA-GGC-TCC-AAA-AGC. These primers correspond to exon 2 (base pairs [bp] 200–224) and exon 7 (bp 896–874) of the SCF cDNA sequence reported by Martin et al. [34]. Differential splicing occurs at exon 6, generating two PCR fragments: one of approximately 605 bp corresponding to membrane SCF mRNA, and one of approximately 698 bp corresponding to soluble SCF mRNA. The PCR reaction was conducted with 2 µl of cDNA and consisted of a 100-µl reaction mixture of 20 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 200 µM dNTP mix, and 20 pmoles each of sense and antisense primers. The PCR reaction was initiated by the addition of 2.5 U Taq polymerase (Gibco BRL) 1 min after samples had reached 94°C. One PCR cycle consisted of 1 min each at 94, 53, and 72°C, and PCR product was found in the exponential phase between cycles 24 and 32. Aliquots (10 µl) of the reaction were taken for each sample from cycles 26 through 32 (7 cycles/sample), then loaded onto a 1% agarose gel and separated by electrophoresis (130 V, 1 h). In each gel, a 100-bp molecular weight marker was included to identify the SCF products. The relative ratios of soluble to membrane SCF mRNA expression were determined by estimating the densities of PCR-generated DNA fragments using National Institutes of Health (NIH) image 1.61 (Bethesda, MD). For each sample, density was measured for seven cycles, and the mean of a minimum of three cycles, within the linear range, was used for each testis. Each treatment group consisted of seven to eight animals, with two testes analyzed per animal.
Statistical Analyses
Statistical analyses were performed using StatView software (Abacus Concepts, Berkeley, CA). Organ weight data, T levels, and SCF ratios were analyzed by one-way ANOVA, followed by multiple pair-wise comparisons using the Fisher protected least significant difference test. The number of testes and animals with 1% or fewer populated seminiferous tubules were compared in control and leuprolide-treatment groups using the Fisher exact test. For all analyses, the criterion of significance was set at P < 0.05.
RESULTS
Assessment of the Spermatogonial Stem Cell Population in the Aged Rat
To address the role of spermatogonial stem cells in failure of spermatogenesis in the aged Brown Norway rat, spermatogonial stem cell content and kinetics were examined in atrophic tubules of testes from 27-mo-old rats. When examined by light microscopy, the testes of these rats (n = 6) displayed varying degrees of atrophy, both between animals and within the left and right testes of the same animal. Of the 12 testes examined, 7 contained 1% or fewer populated tubules. Two animals contained only atrophic tubules in both testes. Three animals had one testis that contained only atrophic tubules and one that contained a mix of normal and atrophic tubules, and one animal had one normal testis and one that contained only atrophic tubules.
Atrophic seminiferous tubules contained predominately Sertoli cells and occasional germ cells. Most germ cells remaining in atrophic tubules were type A spermatogonia (3.74 ± 0.26 per 100 Sertoli cells), of which a significant proportion (
14%) were stem cells (0.51 ± 0.14 per 100 Sertoli cells). Intermediate and type B spermatogonia were observed much less frequently (0.13 ± 0.06 per 100 Sertoli cells).
Having determined that stem cells make up a significant subset of type A spermatogonia, stem cell kinetics were assessed by histological analysis. The continuous labeling index (Fig. 1A) rose rapidly through Day 7, then reached a plateau. The intersection of the rapidly rising (Days 2–7) and plateau (Days 10–28) phases of the curve was used to calculate the growth fraction (GF) or percentage of actively dividing cells, as well as the cell cycle time minus the cell synthesis time (Tc - Ts). The Tc was estimated using the following equation [35]: slope of the rapidly rising portion of the curve = 2[ln (1 + GF)]/Tc. The GF of spermatogonial stem cells was determined to be 47%, with the remaining 53% of stem cells being slowly cycling or quiescent. The Tc was calculated as 12.6 days. A representative spermatogonial stem cell, positive for BrdU staining, is shown (Fig. 1, B and C).
Leuprolide Studies
General observations Leuprolide treatment was initiated in 27-mo-old rats (n = 14–15/group) with the intention that animals would be killed and tissues harvested 12 wk after the last injection. This time point was chosen based on previous studies [23] that indicated an increase in testicular weights and corresponding repopulation of atrophic tubules 12 wk after leuprolide treatment. However, in our studies with the aged Brown Norway rat, 50% mortality occurred in both sham-treated control (7/14) and leuprolide-treated (7/15) groups by 9 wk after the last injection, and the study was terminated early. This mortality rate is consistent with historical data, which indicates that approximately 50% of Brown Norway rats are expected to live between 30 and 32 mo of age [36].
Whereas aged rats had significantly higher BWs than 6-mo-old control animals, treatment with leuprolide resulted in slight (8%) yet significant decreases in BW (Table 1). Testicular and epididymal weights were both significantly decreased in aged rats, and these weights were further depressed by leuprolide treatment (Table 1). The decreases in testicular size with age were accompanied by an increase in testicular interstitial fluid volume, which was partially reversed in aged rats administered leuprolide (Table 1). The accessory sex organs (i.e., prostate and seminal vesicles) did not exhibit any age-related changes in weight; however, following leuprolide treatment, both the prostate and seminal vesicles were decreased in size.
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Histopathological analysis The extent of spermatogenesis varied considerably, not only within the control and leuprolide-treatment groups but also between the left and right testes of the same animal (Fig. 2, A and B). In the 31-mo-old, sham-treated control group (n = 7), 10 of the 14 testes contained 1% or fewer populated tubules. Of the seven sham-treated control animals, four had two completely atrophic testes, containing primarily Sertoli cells and a few undifferentiated, type A spermatogonia in the atrophic tubules (Fig. 3A). Of the remaining control animals, two had one completely atrophic testis, with the contralateral testis containing a mix of atrophic and populated tubules, and one had two testes containing a mix of atrophic and populated tubules. In animals that were administered leuprolide beginning at 27 mo of age and then killed at 31 mo (n = 8), only 3 of 16 testes contained 1% or fewer populated tubules, and no animals in this group had two completely atrophic testes. Three of the leuprolide-treated rats had one atrophic testis and a contralateral testis containing a mix of atrophic and populated tubules; the testes of the remaining leuprolide-treated rats contained a mix of atrophic and populated tubules in both testes. Seminiferous tubules of leuprolide-treated animals contained a mixture of germ cell types. The most mature germ cell types observed were step 9 spermatids (Fig. 3B).
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Because of this variability, we evaluated the extent of spermatogenesis by two methods: one based on the mean percentage of populated tubules, and one based on the number of animals or testes with a low population index. No significant difference was found between the percentage of populated tubules in 31-mo-old control (16.2% ± 28%, mean ± SD) and 31-mo-old leuprolide-treated (20.9% ± 19.8%) rats when compared by the Student t-test. However, given the generally bimodal distribution (Fig. 2, A and B), comparisons based on the mean percentage of populated tubules may not be informative. Therefore, additional statistical analyses using Fisher exact test were performed, in which we determined whether treatment altered the proportion of animals and testes considered to be severely atrophic (defined as having 1% or fewer populated tubules). Following leuprolide treatment, significantly fewer animals and testes contained 1% or fewer populated tubules (Table 2). A cutoff of less than 5% populated tubules also showed significant differences between 31-mo-old control and GnRH agonist-treatment groups on both a per-animal and per-testis basis (P < 0.026). When the cutoff was raised to 10%, the P value was 0.055 for the comparison of individual testes and 0.196 for comparisons of individual animals. Therefore, statistical significance in the categorical comparison does not depend upon any single, arbitrarily selected cutoff.
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The nuclei of Sertoli cells in atrophic tubules of control aged rats were heterochromatic, exhibited a shrunken appearance with a convoluted nuclear profile, and were located throughout the tubule (Fig. 4A). These findings are consistent with those of previous studies, which also described alterations of Sertoli cell morphology in aged rats [13, 37]. In contrast, Sertoli cells of leuprolide-treated rats had larger, rounded nuclei with increased euchromatin. Adjacent Sertoli cells lined the basal surface of the tubule lumen of both atrophic and populated tubules (Fig. 4B).
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Serum T and ITT levels
It has been hypothesized that elevated ITT levels contribute to the maintenance of testicular atrophy induced by cytotoxic therapy [22]. Leuprolide, given as a slow-release depot over an extended period of time, produces a transient increase followed by a sustained suppression of the pituitary-gonadal axis [25], and its effects on the seminiferous epithelium are believed to be mediated by a suppression of ITT levels [22, 26]. Therefore, serum T and testicular interstitial fluid T (i.e., ITT) levels were measured in aged control and leuprolide-treated animals to test the hypothesis that elevations in ITT may also explain testicular atrophy that arises in the setting of aging, and that hormonal suppression may stimulate recovery. The results indicate that ITT is not elevated in the aged rat testis (Table 1). The ITT levels were significantly depressed (P 0.05) in 31-mo-old control (57.9 ± 8.3 ng/ml) and leuprolide-treated (63.4 ± 10.4 ng/ml) animals relative to 6-mo-old control animals (134.0 ± 42.6 ng/ml) (Table 1); however, no difference was found in ITT levels between 31-mo-old control and 31-mo-old leuprolide-treated animals. Total serum T levels were also significantly decreased (P 0.05) in leuprolide-treated animals (0.6 ± 0.2 ng/ml) compared with 6-mo-old (3.1 ± 1.6 ng/ml) control animals, but not in age-matched, sham-treated controls (1.2 ± 0.3 ng/ml) (Table 1).
SCF expression The relative ratios of soluble and membrane splice forms of SCF were significantly different (P < 0.01) in testes of 31-mo-old compared with 6-mo-old Brown Norway rats (Fig. 5). In testes of 6-mo-old rats, the membrane form of SCF predominated, comprising 77% ± 1% of total SCF. Expression of the soluble form of SCF predominated in 31-mo-old Brown Norway rats, and the membrane form of SCF comprised only 49% ± 2.4% of total SCF. Treatment of aged rats with leuprolide significantly increased the proportion of membrane SCF to 56% ± 2.2% of total SCF mRNA. The percentage of populated tubules within a testis correlated positively with membrane SCF levels (P < 0.01, r = 0.698, df = 28) among aged rats (data not shown, includes both 31-mo-old control and leuprolide-exposed rats).
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DISCUSSION
Aging in the Brown Norway rat is accompanied by testicular atrophy. It has been hypothesized that germ cell loss in the aged testis is due to a failure in stem cell proliferation [37]; however, to our knowledge, previous studies have failed to examine the stem cell population.
We report here the presence of an actively dividing stem cell compartment in the aged, atrophic testis, with apparently normal kinetics. Approximately 47% of the spermatogonial stem cell population were actively dividing, with a Tc of 12.6 days. These observations are compatible with spermatogonial stem cell population kinetics predicted for normal testes [38–41]. Our findings indicate that the testicular atrophy observed in the aged Brown Norway rat does not result from a failure in stem cell proliferation. Instead, testicular atrophy may result from a defect in the production of viability-maintaining factors or from the overproduction of death-inducing factors in the local paracrine environment. The existence of an active stem cell compartment suggests that if the population of committed progenitor cells could be maintained, then recovery might be possible.
An interesting finding is that germ cell content and stem cell kinetics in the aged rat approximate those previously observed when our laboratory examined the persistent atrophy that follows toxicant-induced testicular injury [20, 21]. The similarity in stem cell kinetics between aged Brown Norway rats and 2,5-HD-treated rats led us to hypothesize that similar mechanisms may establish and maintain atrophy in the aged rat testis. By extension, we hypothesized that GnRH agonist therapy, which reverses testicular atrophy induced by 2,5-HD, may also rescue spermatogenesis in the aged rat testis.
As predicted, treatment with the GnRH agonist leuprolide stimulated a modest increase in spermatogenesis in the aged rat testis. The distribution of populated tubules in leuprolide-treated animals was significantly different from that in both 27-mo-old and 31-mo-old, sham-treated controls and was characterized by fewer testes containing 1% or fewer populated tubules. Whereas 58% of 27-mo-old testes and 71% of 31-mo-old, sham-treated control testes contained 1% or fewer populated tubules, only 19% of 31-mo-old, leuprolide-treated testes contained 1% or fewer populated tubules, indicating a partial recovery of spermatogenesis by leuprolide therapy. The most mature germ cells present after GnRH agonist treatment were step 9 spermatids, despite the presence of some tubules with completely normal spermatogenesis before the initiation of leuprolide therapy in 27-mo-old rats. This suggests that the kinetics of suppression may be different in the aged rat, such that the release from suppression did not happen until just before the 9-wk time point at which spermatogenesis was examined. Alternatively, the aged rat may be more sensitive to GnRH agonist suppression. More dramatic increases in spermatogenesis likely would be effected by optimizing the timing of leuprolide administration relative to the onset of atrophy, because previous studies that have examined repopulation of atrophic tubules by GnRH agonist therapy have demonstrated greatest recovery when leuprolide therapy is initiated immediately after radiation or toxicant exposure [22, 23].
Previous studies that have examined testicular atrophy, which develops after exposure to radiation and treatment with chemotherapeutics, suggest that the persistent nature of the injury is related to an increase in ITT concentrations, which were measured in whole-testis homogenates and reported as ng/g testis. Leuprolide, given as a slow-release depot over an extended period of time, produces a transient increase followed by a sustained suppression of the pituitary-gonadal axis [25]. Whereas decreases in serum T, LH, and FSH accompany leuprolide treatment [25], its effects on seminiferous tubule repopulation are believed to be mediated by a suppression of ITT levels [22, 26]. It was unclear to us whether this was also the case in the aged rat testis, because ITT values in the aged, regressed testis have been reported to either increase or decrease depending on whether ITT levels were measured in whole-testis homogenates or testicular fluid and whether ITT values were reported on a ng/g testis, ng/testis, or ng/ml basis. For example, ITT concentrations were reported to be elevated in regressed, aged rat testes when measured on a ng/g testis basis in whole-tissue homogenates [12, 42], which is consistent with what is seen in other models of testicular atrophy. However, other studies have reported a decrease in seminiferous tubule fluid T levels when measured on a ng/ml basis [14]. That ITT levels are actually depressed in the aged rat testis is supported by studies that indicate decreases in T production by both Leydig cells and aged testes when perfused in vitro with maximally stimulating LH [14, 43, 44] as well as declines in serum T [12, 14, 43] with age. Likewise, our studies demonstrated decreases in serum T as well as ITT when measured on a ng/ml basis in testicular interstitial fluid of the aged rat. Together, these studies provide evidence that elevations in ITT are not required for the maintenance of testicular atrophy in the aged rat.
In our studies, serum T and ITT levels were not measured during leuprolide therapy. However, treatment of aged rats with leuprolide did produce reductions in testicular and epididymal weights as well as decreases in sex accessory tissue weights relative to those of 31-mo-old control rats, suggesting that ITT levels were depressed during leuprolide therapy. By 9 wk after the last leuprolide injection, ITT levels had returned to aged control levels. However, the restoration of ITT levels in leuprolide-treated rats to those of aged controls may not have occurred soon enough to allow for the differentiation of round to elongate spermatids. This may partly explain the reduced testicular weights in leuprolide-treated animals compared with control animals, which did contain elongate spermatids in some tubules.
It is possible that a decrease in ITT levels below some threshold value mediates the repopulation of seminiferous tubules. Paracrine-acting factors produced by the Leydig cell, other than T, may also be crucial for seminiferous tubule repopulation. On-going studies in our laboratory, which have examined testicular atrophy induced by the environmental toxicant 2,5-HD, indicate that leuprolide re-establishes spermatogenesis in this model, but not if T-producing Leydig cells are ablated with the Leydig cell toxicant ethane dimethane sulphonate (unpublished results). Overall, the mechanism by which sustained leuprolide treatment rescues spermatogenesis is poorly understood.
One paracrine-acting factor of interest is SCF. Expression of the two mRNAs coding for the two different forms of SCF are developmentally regulated in both rat [23] and mouse [45] testis, with a preferential expression of the membrane form occurring during reproductive maturation and being maintained through adulthood. Preferential expression of the membrane form in Sertoli cells may have a role in mediating adhesion of c-kit-expressing germ cells to Sertoli cells [45]. Indeed, Sertoli cells from Sl/Sld mutant mice, which have a deletion that results in loss of expression of the membrane form of SCF, cannot bind germ cells. These Sl/Sld mutant mice are infertile, despite retained expression of the soluble form of SCF [45]. In the aged rat testis, the soluble form of SCF was preferentially expressed, and this relative increase in soluble SCF mRNA levels may be detrimental to Sertoli/germ cell interactions and contribute to the development of testicular atrophy. Why levels of soluble SCF are preferentially expressed in the atrophic testis and what involvement, if any, alterations in T levels play in this process are currently not known. Treatment of aged rats with leuprolide resulted in increased relative expression of membrane versus soluble SCF and stimulation of spermatogenesis. Additional time-course studies are required to determine whether the change in soluble SCF levels contribute to, or result from, the higher levels of spermatogenesis.
The decrease in germ cell numbers in the aged rat testis is not due to an absence of germ cells or a failure in germ cell proliferation, because our studies indicate apparently normal stem cell kinetics in the aged rat testis. Studies of reproductive aging in the Brown Norway rat suggest that germ cell loss is due to an increase in germ cell apoptosis [42]. Sertoli cells support germ cell growth and differentiation [46] as well as apoptosis [47] through production of a number of paracrine factors, and it has been proposed that age-related declines in testicular function may be attributed to age-related changes in Sertoli cell function [13]. Consistent with this hypothesis, age-related changes in the Sertoli cell transcripts CP-2/cathepsin L and transferrin have been reported in testes from 24-mo-old Brown Norway rats [13]. Our findings of alterations in SCF expression also point toward a Sertoli cell defect; however, it is difficult to draw conclusions without a clear understanding of the factors that regulate SCF RNA processing. Hypothetically, sustained treatment with leuprolide alters the production of hormonal and paracrine-acting factors by Leydig cells, resulting in increased release of viability-maintaining and/or apoptosis-inhibiting factors by Sertoli cells. The mechanisms involved in seminiferous tubule repopulation by leuprolide are not well understood, but our results indicate that transient GnRH therapy, followed by a period of posttreatment recovery, partially restores spermatogenesis in the aging rat model.
ACKNOWLEDGMENTS
The authors wish to thank Terry Law of Children's Hospital (Boston, MA) for T measurements and Elizabeth Shipp for help with BrdU labeling in the stem cell kinetic studies. Statistical review was provided by Lucy Hannah of the Center for Statistical Sciences at Brown University.
FOOTNOTES
First decision: 25 September 2000.
1 Supported in part by NIEHS grant RO1-ES05033 and by the Burroughs Wellcome Fund. H.S. is supported by NIEHS training grant ES07272-08. 
2 Correspondence: Kim Boekelheide, Department of Pathology and Laboratory Medicine, Brown University, Box G-B518, Providence, RI 02912. FAX: 401 863 9008; kim_boekelheide{at}brown.edu
Accepted: November 29, 2000.
Received: August 17, 2000.
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