| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15261
Address all correspondence and requests for reprints to: Dr. Suresh Ramaswamy, Ph.D., Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S-831A, Scaife Hall, Pittsburgh, Pennsylvania 15260. E-mail: sramas{at}pitt.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Objective: This study examines the direct impact of elevated E2 levels on gonadotropin-induced pubertal testis development and function in the primate.
Design: Juvenile monkeys, which have characteristically little endogenous gonadotropin secretion, were treated with pulsatile infusions of recombinant monkey (rm) FSH (rmFSH) and LH (rmLH) in the presence (experiment 1, 
100 pg/ml for about 15–20 wk; experiment 2, 400 pg/ml for about 5 wk) or absence (control group) of elevated E2 in the circulation. Changes in circulating concentrations of E2, gonadotropins, testosterone (T), and inhibin B were monitored throughout the study. The number of Leydig cells per testis was determined after immunohistochemical staining for 3-ß hydroxysteroid dehydrogenase in experiment 2.
Results: Exogenous gonadotropin treatment produced physiological, episodic, and similar circulating concentrations of FSH and LH in both groups. Exposure to approximately 100 pg/ml of E2 appeared to blunt testicular T production. Exposure to approximately 400 pg/ml of E2 led to a significant (75%) inhibition of T production together with a marked (40%) decrease in Leydig cell numbers per testis and a notable inhibition in the growth of the testis. In contrast, E2 exposure had little effect on inhibin B production.
Conclusions: The direct testicular impact of elevated E2 is on Leydig cell number, T production, and testicular growth, but not on inhibin B production. This experimental paradigm provides a powerful primate model for the examination of the direct impact of E2 or other endocrine disrupters on pubertal testicular development.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ERß), respectively, in somatic (Sertoli and Leydig) and/or germ cells (1, 2, 3, 4). Evidence for a physiological role of E2 in male reproduction is provided by 1) studies of selective gene knockout (aromatase and ER) and knock-in (overexpression of aromatase) and 2) observations of men with mutations in aromatase or the ER gene. In these situations, defective or excessive E2 signaling could be associated with reproductive impairments including cryptorchidism, abnormal spermatogenesis, and infertility (5, 6, 7). Moreover, animal studies and epidemiological observations in humans have also indicated that environmental endocrine disrupters with estrogenic activity interfere with male reproductive potential (8, 9, 10, 11, 12, 13, 14). E2 and/or endocrine disrupters may harm the testis directly or indirectly, by affecting the hypothalamic-pituitary axis, throughout development. In higher primates, including man, puberty represents a distinct phase of postnatal testis development vulnerable to endocrine disruption (15, 16, 17, 18).
In primates, pubertal testicular development may be induced precociously in the juvenile by direct stimulation of the immature testes with exogenous gonadotropins (19). Using this approach, known as a testicular clamp, direct testicular effects of abnormal E2 milieu or endocrine disrupters may be examined without the confounding effects on feedback regulation of gonadotropin secretion. In the present study, the testes of juvenile monkeys were stimulated with intermittent iv infusions of homologous (monkey) gonadotropins in the presence or absence of elevated E2 levels. Here, the direct effects of elevated E2 levels on gonadotropin-induced pubertal augmentation in testicular T and inhibin B production, the number of Leydig cells, and testis growth are presented.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Eight juvenile male rhesus monkeys (Macaca mulatta, 13–16 months of age, 2.0–2.6 kg body weight) were used. In this species of macaque, pubertal reawakening of the hypothalamic-pituitary-testicular axis occurs at around 36 months of age (18). Body weight of animals was monitored weekly. The animals were maintained under a controlled photoperiod (lights on, 0700–1900 h), in accordance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee approved the experimental procedures. The initial activation (1–8 d) of Leydig cell T secretion by precocious gonadotropin stimulation in the control group of animals has been published (20).
Surgical procedures
Surgeries were performed under sterile conditions. For iv catheterization and castration, the animals were first sedated with ketamine hydrochloride (10–20 mg/kg body weight, im; Ketaject, Phoenix Scientific, Inc., St Joseph, MO, or Vetlar Parke-Davis, Morris Plains, NJ) and subsequently maintained under anesthesia with isoflurane (1–2.5% in oxygen; Abbott Animal House, North Chicago, IL). Empty or E2-filled (Steraloids Inc., Newport, RI) SILASTIC-brand capsules (Dow Corning Corp., Midland, MI) were implanted sc while under sedation. Each animal received 2 iv catheters (internal jugular and femoral vein), as described previously (21). The postoperative care of animals in remote sampling cages has also been described previously (21).
Collection of blood samples
Before SILASTIC capsule implantation and catheterization, single blood samples (5–10 ml) were collected by femoral venipuncture under ketamine sedation to harvest serum (for use in preparation of gonadotropin infusates) or plasma (to establish baseline hormone concentrations). Collection of single blood samples (2–3 ml) was continued after iv catheterization on a weekly basis via an indwelling catheter. Additionally, series of sequential blood samples (2–3 ml/time) were collected during selected 3-h intervals, at about 10 min before and at 5, 20, 40, 60, 80, 120, and 170 min after a gonadotropin pulse (see Experimental design). Blood cells from plasma were diluted in sterile saline and returned to the respective animal. Serum or plasma was stored at –20 C.
Testicular clamp with gonadotropins
Stock solutions of recombinant monkey (rm) FSH (rec moFSH-I-1, AFP6940A) and rmLH (rec moLH-I-1, Bio, AFP6936A), obtained from Dr. A. F. Parlow (National Hormone and Peptide Program), were prepared at 1 µg/10 µl in sterile Dulbecco’s PBS (Dulbecco’s PBS without CaCl2 and MgSO4; Life Technologies, Inc., Grand Island, NY) and stored at –80 C.
Custom infusate of rmFSH and rmLH was prepared for each animal in sterile Dulbecco’s PBS containing 2 mg/ml kefzol and 1% serum from the respective monkey. The doses of rmFSH and rmLH used were 30 and 600 ng/kg per pulse, respectively. These were selected to mimic the physiological ranges of the normal adult male (22, 23). Gonadotropin infusates were made in batches, stored in aliquots at 4 C, and used over a period of 2–3 wk. The doses of gonadotropins were readjusted to body weight when a new batch of infusate was prepared. The frequency of gonadotropin infusion was set at 1 pulse every 3 h, a frequency in the range of endogenous LH and T secretion in the adult male monkey (24). The infusate was filtered (0.22-µm filter; Fisher Scientific, Pittsburgh, PA) before use in animals.
Experimental design
The overall design of the experiment is schematically presented in Fig. 1
. Within the framework of this design, two experiments were performed.
|
Seven to 12 wk later, the animals were implanted with indwelling venous catheters. About 1 wk later, a series of blood samples was collected over a 3-h period to obtain baseline values for circulating gonadotropins, T, and inhibin B. After 2–5 d, the testicular clamp with exogenous gonadotropin was initiated (d 0). Circulating concentrations of infused gonadotropins and the testicular T response were monitored in a series of frequent blood samples collected during selected 3-h interpulse intervals on a regular basis. Changes in circulating concentrations of inhibin B were examined after exposure to E2 alone and at selected times after gonadotropin stimulation. Because inhibin B secretion is largely apulsatile (26, 27), single samples collected at 60 min after a gonadotropin pulse were selected for assay.
It should be noted that three animals (one E2 treated and two control) inadvertently received incorrect doses of rmFSH and rmLH (25 and 480 ng/kg per pulse, respectively) during wk 6 and 7 of gonadotropin treatment. However, no notable differences were seen in hormone levels during this period, suggesting minimal impact of this deviation from the protocol. In one E2-treated animal, the patency of the gonadotropin infusion line had to be restored by recatheterization.
At the end of this experiment, the animals were subjected to unilateral orchidectomy (UO). The weight of the testis (first) was recorded, and tissue was variously fixed or frozen for later use. At this time, the animals were assigned to experiment 2.
Experiment 2. Effect of exposure to approximately 400 pg/ml circulating E2 on testis growth and function. Immediately after removal of the first testis, the animals in the E2 group received five additional E2-filled capsules (4 cm long), while the control animals received matching empty capsules. This marked d 0 of the second experiment. Circulating concentrations of E2, infused gonadotropins, and the testicular T and inhibin B responses continued to be monitored, as described for experiment 1.
About 5 wk later, the second testis was removed, the weight recorded, and tissue processed as described for the first testis.
The number of Leydig cells per testis was enumerated in the second testes collected at the end of the study. Leydig cells were identified by immunohistochemical staining for 3-ß hydroxysteroid dehydrogenase (3-ß HSD, rabbit polyclonal antibody raised against recombinant human type II 3-ß HSD). The choice of 3-ß HSD as Leydig cell marker was based on the observation that E2 has little effect on this enzyme activity (28, 29). Unless otherwise stated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Bouin’s-fixed, paraffin-embedded testis sections (5 µm thick) were dewaxed, rehydrated, incubated for 30 min in 3% (vol/vol) hydrogen peroxide to block endogenous peroxidase activity, washed in Tris-buffered saline (TBS, 0.05 M Tris and 0.85% NaCl, pH 7.6), treated with normal goat serum (1:1 dilution in TBS containing 0.1% BSA), and incubated at 4 C overnight with the primary antibody (3-ß HSD, 1:1000 in TBS/BSA). Negative control sections were incubated without primary antibody. Sections were washed in TBS, incubated for 60 min with the secondary antibody conjugated to biotin (1:100 goat antirabbit IgG biotin conjugate), washed again with TBS, and incubated for 60 min with streptavidin-peroxidase conjugate (1:200). After a final TBS wash, staining was visualized by the diaminobenzidine method, and the reaction was terminated by washing the slides in distilled water. Sections were counterstained with hematoxylin (Gills no. 3), dehydrated, cleared in xyline, and mounted using Protocol mounting medium (Fisher Scientific, Pittsburgh, PA). The 3-ß HSD staining was performed at least twice.
The volume fraction of 3-ß HSD positive Leydig cells per testis was determined by the point-counting method as described previously (30). A grid of intersecting lines (20 x 20 eyepiece graticule) was superimposed over a section, a total of 25 randomly chosen fields were examined (x40 magnification), and the number of test points falling over the nuclei of 3-ß HSD-positive cells were counted and converted to a percentage of the total points per animal. The mean diameter of 25 3-ß HSD-positive Leydig cell nuclei per animal was calculated from the average of two perpendicular measurements per nucleus. Finally, Leydig cell number per testis was determined using the values for Leydig cell nuclear volume and the testis volume, the latter obtained by dividing the testis weight by the standard value for specific gravity (1.04).
Extraction of testicular steroids
A portion of frozen testis tissue from each animal was homogenized in 1 ml PBS gel (0.1%) buffer and, after adding [1,2,6,7-3H]testosterone (2000 cpm), the homogenate was extracted twice in 5 ml diethyl ether, as described previously (31). After evaporation of ether, the extract was reconstituted in 1 ml of PBS gel buffer and stored at –20 C until required for assay. The mean (±SEM) recovery for T was 85 ± 11%. Recovery for E2 was not determined.
Assays
Gonadotropins. Plasma levels of rmFSH and rmLH were determined using homologous RIAs described previously (22, 23). The mean sensitivity of the FSH and LH assay was 0.09 and 0.12 ng/ml, respectively. Intra- and interassay coefficients of variation were no more than 9.1 and 8.9% for FSH and no more than 7.1 and 7.07% for LH, respectively.
Testosterone (T). Plasma and testicular T levels were determined using either a previously described RIA employing antiserum T3-125 (Endocrine Sciences, Calabasas Hills, CA; see Ref.24) or a commercially available solid-phase RIA kit (Total T; Diagnostic Products Corp., Los Angeles, CA). Although the cross-reactivity of dihydrotestosterone (DHT) with the two T antibodies was approximately 44 and 4%, respectively, DHT constitutes only approximately 1–2% of testicular androgens (32) and, as plasma T levels measured by both assays were very similar (Simorangkir, D. R., and T. M. Plant, unpublished results), it is unlikely that DHT contributed in a major fashion to the T values. The mean sensitivity of the two assays was approximately 0.07 and 0.03 ng/ml, respectively. Intra- and interassay coefficients of variation for the former method were no more than 9.9 and 9.8%, respectively, and for the kit assay were no more than 9.4 and 7.7%, respectively.
E2. Plasma and testicular E2 levels were measured using a double antibody RIA kit (Diagnostic Products Corp., CA). The mean sensitivity of the assay was approximately 2.4 pg/ml, and intra- and interassay coefficients of variation were no more than 12.7 and 13.3%, respectively.
Inhibin B. Circulating inhibin B concentrations were determined using an inhibin B ELISA kit (Diagnostic Systems Laboratories, Inc., Webster, TX) as described previously (33). The sensitivity of the assay was 7 pg/ml, and intra- and interassay coefficients of variation were no more than 5.6 and 7.6%, respectively.
Statistical analysis
The significance of differences between mean values of body weight, testis weight, hormone concentrations, and the number of Leydig cells was determined by multifactor ANOVA, with repeated measures followed by Fisher’s least significant difference test or Student’s t test, as appropriate, using the GB STAT statistical program (version 6.5.6 Pro; Dynamic Microsystems Inc., Silver Spring, MD). Hormone concentrations below the sensitivity of the assays were assigned a value equivalent to the minimum detectable concentration. Because testicular E2 content in some samples was undetectable, testicular E2 data are presented without statistical analysis. Statistical significance was accepted at P 
0.05. All data are expressed as mean ± SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Circulating concentrations of infused gonadotropins
Before initiation of gonadotropin infusion, mean circulating concentrations of endogenous gonadotropins in both groups were around the detectable limit of respective assays. Intermittent infusion of exogenous gonadotropins produced similar episodic patterns and significant increases in their circulating concentrations in both groups (Fig. 2 and Table 1). The recombinant gonadotropin infusate was stable when stored at 4 C over 2–3 wk (Table 1).
|
|
2.4 pg/ml) of the assay. Implantation of a 3-cm E2-filled capsule increased the circulating levels of this steroid significantly, to a range of 89–101 pg/ml, which was sustained during the experiment (Fig. 3).
|
and Table 2). By wk 7, in the E2-treated group, a suggestion of blunting in T production was noted (Fig. 4 and Table 2).
|
|
Experiment 2. Effect of exposure to approximately 400 pg/ml circulating E2 on testicular T response and Leydig cell number.
Implantation of additional E2-filled capsules resulted in a significant, approximately 4-fold increase in blood E2 levels that was sustained during the experiment (Fig. 3
).
As one testis was removed at the end of experiment 1, the testicular T and inhibin B responses in this experiment are a reflection of these hormones produced by the testis remaining in both groups.
Accordingly, in the control group, the mean and peak T concentrations in circulation before UO were 3.73 ± 0.91 and 8.30 ± 2.41 ng/ml, which decreased significantly after UO to 1.45 ± 0.21 and 3.40 ± 0.64 ng/ml, respectively. These levels were sustained for the remaining duration of the experiment (Table 2).
In the E2-treated group, the mean and peak T concentrations in circulation before UO and the addition of E2-filled capsules were 2.76 ± 0.71 and 5.28 ± 1.15 ng/ml, which also decreased significantly after UO to 1.47 ± 0.86 and 2.23 ± 0.83 ng/ml, respectively. Within a week, however, the mean and peak T values further declined significantly to a range of 0.52–0.63 and 1.03–1.30 ng/ml, respectively (Fig. 5 and Table 2), in an inverse relation to the robust increase in E2 levels (see Fig. 3).
|
In tandem with the decreased T production, the mean number of 3-ß HSD positive Leydig cells in the second testis of the E2-treated group was approximately 40% less (57 ± 13 x 106) compared with that in the control group (93 ± 18 x 106; Fig. 6), although this difference was statistically not significant.
|
).
|
Mean baseline circulating inhibin B concentrations in control and E2-treated groups were 362 ± 79 and 413 ± 60 pg/ml, respectively. Treatment with E2, either alone or during gonadotropin infusion, had little impact on inhibin B secretion. The changes in blood levels of this hormone after gonadotropin stimulation were very similar in both groups (Fig. 8).
|
50%) decline in the mean circulating inhibin B concentrations in both groups (Fig. 8). A further elevation in circulating E2 levels did not affect inhibin B production by the remaining testis. No significant differences were noted between groups. Effects of exposure to approximately 100 and 400 pg/ml of E2 in the circulation on testis weight
In the control group, the mean weight of second testis (1.81 ± 0.48 g) was significantly greater (P = 0.0473) than that of first testis (1.23 ± 0.26 g), whereas in the E2-treated group, the corresponding values (1.33 ± 0.24 and 1.23 ± 0.16 g) were statistically not different (P = 0.590). Although the mean weight of second testis (1.81 ± 0.48 g) in the control group was notably greater than that in the E2-treated group (1.33 ± 0.24 g), this difference was statistically not significant.
At the end of experiment 1, the testes of E2-treated animals were inguinal and those in control animals were inguinal (n = 3) or scrotal (n = 1). At the end of experiment 2, the location of the remaining (second) testis in the control group was either scrotal (n = 2) or could be moved to the scrotum (n = 2), while those in the E2-treated group of animals continued to be inguinal.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The immediate effect of increasing the E2 milieu was the dramatic inhibition of T production during gonadotropin-induced testicular puberty, particularly after the high level of E2 exposure. It is likely that the foregoing effect results from a direct impact of E2 on Leydig cells and their steroidogenic machinery, as these interstitial cells, which possess estrogen receptors (1), are the only source of LH-mediated T synthesis and secretion. Indeed, in hypophysectomized, human chorionic gonadotropin/LH-treated rats and in primary cultures of Leydig cells, diethylstilbestrol, E2, or endocrine disrupters are shown to inhibit T production (29, 34, 35, 36, 37, 38). An in vitro study of human testicular tissue has noted that long-term E2 treatment is associated with irreversible changes in the Leydig cells and T production (39). Studies of withdrawal of E2 treatment using the present model should provide conclusive in vivo evidence to this effect.
At the cellular and molecular level, several possibilities may account for the E2-mediated inhibition of Leydig cell T production (40, 41, 42, 43). In this study, the number of Leydig cells was markedly lower in E2-treated animals, indicating an impairment in the establishment of the pubertal complement of this steroidogenic cell type, thereby leading to decreased T production. In this regard, E2 exposure has been shown to 1) decrease Leydig cell volume and inhibit repopulation of Leydig cells after ethylene dimethanesulfonate treatment in the rat (29, 34) and 2) impair levels of mRNA and/or protein of important regulators/enzymes of steroidogenesis (28, 44, 45). E2 may also impair Leydig cells and their steroidogenic function indirectly by affecting Sertoli cell factors, such as stem cell factor and anti-Müllerian hormone, regulating Leydig cell development and function (46, 47, 48). Collectively, these data suggest that E2-mediated inhibition in testicular T production may occur at multiple stages and via many pathways of Leydig cell development and function.
In the present study, testicular E2 and T contents did not reflect those in the circulation, most likely due to the great variation encountered in measuring testicular steroid content, the reliability of which has been debated (49). The foregoing discrepancy, however, does not detract the conclusion that E2 exerts a direct effect on Leydig cell number and T production.
The direct impact of elevated E2 on Sertoli cell inhibin B production was also examined in the present study. Ethynyl estradiol or diethylstilbestrol treatment of neonatal rats (during the proliferative phase of Sertoli cell development) resulted in significant and persistent decreases in inhibin B levels in association with marked changes in the circulating FSH levels and Sertoli cell number (50, 51, 52). In contrast, in the present study, inhibin B levels were similar between control and E2-treated animals, indicating that this aspect of Sertoli cell function during puberty is not directly affected by exposure to elevated E2 levels. Moreover, as inhibin B levels are positively correlated to Sertoli cell number in the adult rat and monkey (53, 54), the absence of a difference in inhibin B levels between control and E2-treated animals in the present study suggests that E2 may not affect the establishment of pubertal Sertoli cell number.
In the present study, despite a sustained gonadotropin input, the growth of the second testis in the E2-treated group was inhibited. Moreover, testes of the E2-treated animals were inguinal compared with the mostly scrotal location of gonads in the control group. The foregoing observations suggest two possibilities. First, E2 might directly impair the initiation of spermatogenesis, thereby inhibiting testis growth. Second, inhibition of testis growth by E2 may be mediated by the blunting in testicular T production. In this regard, the persistent, cryptorchid location of the testes of E2-treated animals may be due to the significant decrease in T production as T regulates trans-inguinal testis descent (55). Morphometric analyses of the testes from the present study and future experiments designed to replace the loss of T by exogenous means in the presence of elevated E2 levels should clarify these issues.
The testes are innervated, and, during puberty, testicular neuronal systems exhibit a marked degree of plasticity (56, 57, 58). Interestingly, in the present study, UO did not elicit compensation in either T or inhibin B production by the remaining testis. In contrast, in the testicular clamped adult monkey, compensation in the production of these hormones occurred after UO (33). Thus, neuronal or other gonadotropin-independent mechanisms of hormone compensation are likely to be established at a later stage of pubertal testes growth.
The E2-treated animals were relatively heavier, but an increase in food consumption was not apparent in these animals. It may be that the high degree of E2-mediated sex skin swelling of abdominal and scrotal areas contributed to the increased body weight in this group.
In summary, the testicular-clamped juvenile monkey provides a powerful experimental model to study the direct actions of endocrine disrupters on pubertal testicular development. The present study focused on the direct testicular effects of elevated E2 levels during gonadotropin-induced testicular puberty. The results demonstrate that E2 selectively affected pubertal augmentation in Leydig cell number and T production and in the growth of the testes, but not Sertoli cell inhibin B secretion.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
A preliminary report of this study was presented at the 36th Annual Meeting of the Society for the Study of Reproduction, July 19–22, 2003, Cincinnati, OH (Abstract 454).
First Published Online July 19, 2005
Abbreviations: DHT, Dihydrotestosterone; E2, 17ß-estradiol; 3-ß HSD, 3-ß hydroxysteroid dehydrogenase; rm, recombinant monkey; T, testosterone; UO, unilateral orchidectomy.
Received January 14, 2005.
Accepted July 7, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß proteins in the testis and male reproductive system of human and non-human primates. Mol Hum Reprod 7:227–236 hydroxylase/C17–20 lyase in the fetal rat testis is reduced by maternal exposure to estrogens. Endocrinology 137:1063–1070[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  L. Aksglaede, A. Juul, H. Leffers, N. E. Skakkebaek, and A.-M. Andersson The sensitivity of the child to sex steroids: possible impact of exogenous estrogens Hum. Reprod. Update, July 1, 2006; 12(4): 341 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Plant, S. Ramaswamy, and M. J. DiPietro Repetitive Activation of Hypothalamic G Protein-Coupled Receptor 54 with Intravenous Pulses of Kisspeptin in the Juvenile Monkey (Macaca mulatta) Elicits a Sustained Train of Gonadotropin-Releasing Hormone Discharges Endocrinology, February 1, 2006; 147(2): 1007 - 1013. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology |
