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Regular Article |
a Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, follicle-stimulating hormone, puberty, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is presently unclear why adult levels of T can delay pubertal onset indefinitely in juveniles since adult males are reproductively competent under the influence of the same levels of circulating T. One possibility is that a decrease in the sensitivity to inhibitory steroids never occurs in these hamsters during pubertal transition. This could explain why these animals failed to overcome the inhibitory effects of exogenous T and E2 to reach puberty. In this case, other mechanisms are required to initiate puberty and support the continuing activity of the HPG axis despite the natural increase in circulating T [6]. A second possibility is that these animals, like other rodents studied, become less sensitive to inhibitory steroids as they approach puberty, allowing them to reproduce under high levels of circulating sex steroids. However, early exposure of prepubertal animals to adult levels of T and E2 in our previous study may have suppressed this transition toward sexual maturation. Regardless of the mechanisms involved, information on how these animals respond to inhibitory steroids during and after pubertal transition is critical to the understanding of changes in the HPG axis as the animal becomes sexually mature.
The goal of this study is twofold. First, we examined the ability of T and E2 to exert inhibitory effects on the HPG axis in peripubertal and adult Siberian hamsters. Our aim was to determine if sexual maturation in Siberian hamsters is accompanied by a decreased sensitivity to both steroids. Second, we investigated whether the actions of T and E2 on the HPG axis remain distinct at different ages. Our results indicate that the Siberian hamster remains very sensitive to the inhibitory effects of T but not of E2 after the onset of puberty. Further, the actions of T and E2 remain distinct throughout pubertal onset and adulthood.
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MATERIALS AND METHODS |
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Male Siberian hamsters were raised from birth under long-day photoperiod (16L:8D, lights-on at 0430 h) and weaned at 20 days of age. Animals were singly housed in shoebox cages and given water and rodent chow ad libitum. All experimental procedures are in accord with the animal protocols approved by the University of Colorado at Boulder Institutional Animal Care and Use Committee.
Hormone Treatments and Tissue Collection
Male Siberian hamsters at 35 days of age (peripubertal, n = 24) or 140–145 days of age (adult, n = 24) were subcutaneously implanted with a silastic capsule containing crystalline T, E2, or cholesterol (Ch, control) as described previously [5]. In brief, 1-mm-long silastic tubing was packed with undiluted T, E2, or Ch and sealed on each end with silicon glue. The capsules were allowed to equilibrate for at least 3 days in 0.9% saline until implanted. Animals were lightly anesthetized with halothane gas and the implant was inserted between the scapulae. An initial laparotomy was performed to ensure testes size was not significantly different among assigned groups before hormone implants. Retro-orbital blood samples were obtained for the measurement of circulating levels of FSH at 0, 15, and 30 days postimplant. Circulating T and E2 were also measured for 30-day-postimplant plasma samples. All animals were killed by cervical dislocation at 30 days postimplant and their pituitaries and testes removed for histological analysis and measurements of mass (testes) and hormone levels (pituitaries). All animals were inspected for the presence of a silastic capsule containing packed hormone.
An additional group of 28 untreated animals were separated into six groups of 4–5 animals. Each group was bled at one of the six specific ages (20, 25, 30, 35, 40, or 140 days of age) to obtain a normal plasma E2 profile.
Radioimmunoassays
FSH RIA Blood samples were collected into heparinized tubes, centrifuged at 3000 rpm for 8 min, and stored at -20°C until assayed for FSH by RIA. Detection of plasma and pituitary FSH by RIA was performed as described previously [5]. In brief, rFSH-I9, rFSH-RP2, and rFSH-S-11 from the NIH National Pituitary Program (obtained from Dr. A.F. Parlow) were used as the iodination stock, RIA standard, and antibody, respectively. The limit of detection was 1.0 ng/ml. Intra-assay and interassay coefficients of variation were 6.6 ± 2.4% and 8.9 ± 1.5%, respectively.
T and E2 RIAs Testosterone and E2 RIA kits were purchased from Diagnostic Systems (Webster, TX). Plasma samples were assayed according to manufacturer's directions. Assay limits of detection were 80 pg/ml and 6.5 pg/ml for T and E2, respectively. Both RIAs are highly specific and cross-react minimally with other forms of steroid hormones.
Histology
Testes were immersion fixed in Bouin fixative overnight and then stored in 70% ethanol. Testes were dehydrated through a standard series of increased ethanol concentrations and were embedded in paraffin wax. Ten-micrometer sections were cut, mounted on poly-L-lysine coated slides, and stained with hematoxylin and eosin. For morphometric analysis, 6–8 fields per section from a total of 15 sections per animal were scored. A minimum of three animals per treatment group was measured. Sections were all from the center of the testis, and only tubules cut in a cross-section that appeared round were measured. General characteristics of cell types and appearance, seminiferous tubule diameter, and lumen diameter were recorded. Measurements were made using bright-field microscopy with a calibrated micrometer.
Statistical Analysis
Differences among groups were tested using one-way ANOVA followed by Tukey honestly significant difference post hoc analysis. For analysis of all hormone levels by RIA, animals with undetectable levels were assigned a value of 0.00 ng/ml and included in the mean calculation. Hence, some of the mean values are below the limits of assay detectability. Differences were considered significant when P < 0.05. All data are presented as mean ± SEM.
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RESULTS |
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Testosterone treatment markedly affected testes morphology and spermatogenesis in both peripubertal and adult animals. In both age groups, the lumen of T-treated animals was either completely closed or very small (<5 µm; Figs. 2A, 3A, and 4A). Testosterone treatment also significantly reduced seminiferous tubule diameter in both peripubertal and adult animals compared with Ch-treated controls (Fig. 2B). In T-treated peripubertal animals, the seminiferous tubules were tightly packed together with very little interstitial space (Fig. 3A). Consequently, the Leydig cells appeared to be few, and many were shrunken or irregularly shaped, as seen in pyknotic cells, suggesting that T treatment may have compromised the health of these cells (Fig. 3A). However, in T-treated adult animals, the interstitial space appeared normal and Leydig cells were more numerous, yet many Leydig cells still appeared pyknotic (Fig. 4A). In both age groups, primary and secondary spermatocytes were present, but spermatids were scarce and mature sperm absent, suggesting spermatogenesis was adversely affected by T treatment (Figs. 3A and 4A).
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Consistent with our previous findings in prepubertal animals [5], E2 actions on testes morphology and spermatogenesis were distinct from T in both age groups studied. In peripubertal animals, E2 treatment significantly reduced seminiferous tubule and lumen diameters compared with controls (Fig. 2). In adult animals, however, E2 treatment had no effect on seminiferous tubule or lumen diameters (Figs. 2 and 4B). In both peripubertal and adult E2-treated animals, the interstitial space and Leydig cell populations appeared similar to Ch-treated controls (Fig. 3, B and C; Fig. 4, B and C). Treatment with E2 affected spermatogenesis in peripubertal but not in adult animals. In peripubertal animals, there were numerous primary and secondary spermatocytes, but fewer spermatids were present and mature sperm absent (Fig. 3B). On the other hand, E2 treatment did not appear to affect spermatogenesis in the adults when compared with controls, as primary spermatocytes, secondary spermatocytes, spermatids, and mature sperm were present (Fig. 4B).
Testosterone and E2 had no effect on plasma FSH levels in peripubertal or adult animals (Table 1). Several animals in each treatment group had undetectable levels of plasma FSH; therefore, the calculated group mean is below the limit of detectability for the assay (1.0 ng/ml). The number of animals with detectable plasma FSH within the treatment group is indicated in Table 1. Consistent with our previous finding in prepubertal animals [5], E2 treatment significantly reduced FSH concentration in the pituitary in both peripubertal and adult animals (Table 1). T-treated animals, however, were not different from control animals in either age group (Table 1).
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The dose of T administered during this study is sufficient to significantly elevate circulating T in peripubertal animals to levels similar to, but never exceeding, those in intact adult males (Table 1) [5]. However, E2 implants did not result in significantly higher plasma E2 compared with controls in either age group (Table 1). Plasma E2 profiles of animals during and after pubertal transition revealed circulating E2 was highest immediately before pubertal onset (20 days of age; Fig. 5). This level was significantly higher than all other time points studied.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our observation that circulating E2 is high in prepubertal males and steadily declines thereafter (Fig. 5) raises the novel possibility that E2 may play a physiological role in inhibiting the activity of the HPG axis prior to, but not after, the attainment of sexual maturity in male Siberian hamsters. This possibility is further supported by a potent inhibitory effect of E2 on testicular development in peripubertal, but not adult, hamsters. This study, however, investigated the progression of puberty and not the mechanisms controlling pubertal onset. Future studies using estrogen agonists and antagonists are required to establish the specific role of estrogen in the initiation of pubertal onset in these hamsters. In other species studied, the role of estrogen in pubertal onset has not been clearly established. For example, a previous study reported active immunization against estrogen in prepubertal lambs failed to advance the onset of puberty [7].
To the best of our knowledge, this is the first report of E2 treatment increasing testes mass in adult animals. In fact, our data contradict a previous report that E2 administration (>100 µg kg-1 day-1) for 60 days to adult male rats significantly reduced testes mass, sperm motility, and serum FSH and LH levels [8]. Our results were unexpected, but recent findings suggest several possible mechanisms for the stimulatory action of E2 on the testes. In one study, when prepubertal rats were given both E2 and FSH, E2 enhanced the stimulatory effect of FSH on spermatogenesis, resulting in a significant increase in the number of spermatogonia compared with rats given FSH alone [9]. In that study, however, mean testes mass was not different between the two groups, suggesting that an increase in total number of spermatogonia alone may not be sufficient for the observed increased testes mass in our study.
Another possible mechanism for E2-induced increase in testicular mass is that E2 may disrupt the normal fluid balance in the testes, resulting in fluid accumulation and increased testes mass. Studies have shown that E2 is an important regulator of fluid balance in the efferent ductules of the testes (see [10] for a review). For instance, in estrogen receptor alpha-null (
ERKO) mice, the efferent ductules are severely dilated due to their inability to reabsorb testicular fluid [11]. As a consequence, fluid accumulates in the testes and results in dilated lumens and reduced germinal epithelium in the seminiferous tubules [12]. This effect begins in the caudal portion of the testes and propagates to other regions of the testes as the animal ages. Similarly, adult rats and mice given an antiestrogen, ICI182,780, also showed dilation and increased fluid retention in the efferent ductules [13, 14]. While mild testicular fluid accumulation is not always apparent by conventional histological observations, fluid that accounted for a 30% increase in the testicular mass (Fig. 1) should visibly distort testicular morphology. In fact, most cases of severe testicular fluid accumulation resulted in gross morphological alterations that are readily visible [12–14]. Our histologic analysis revealed no difference in the overall sizes of the lumen and seminiferous tubule diameters between E2-treated and control animals. Again, the influence of E2 on testicular fluid balance, if any, may not be the sole contributor of increased testicular mass.
A third possible mechanism for E2-stimulated increase in testicular mass is that E2 may stimulate FSH or LH secretion, resulting in excessive stimulation of Sertoli, Leydig, and germ cells in the testes [15, 16]. Our results failed to support a stimulatory effect of E2 on circulating FSH (Table 1), and the lack of a LH RIA sensitive enough to measure circulating LH in the Siberian hamster precluded our ability to assess E2 effects on LH secretion.
At present, the precise mechanism responsible for E2 stimulation of testicular mass is not known. However, it is clear that adult Siberian hamsters differ markedly from other rodent species investigated in their ability to respond to E2 with increased gonadal growth. Unlike laboratory rats and mice, Siberian hamsters are a photoperiodic species that undergoes seasonal gonadal regression and spontaneous recrudescence. Whether or not this plastic nature of gonadal growth allows for the observed increased testes mass in response to E2 treatment remains to be investigated. It should also be noted that, because the animals in this study were gonadally intact, unidentified gonadal factors might influence the progress of puberty and steroid responsiveness of the testes.
Testosterone or E2 treatments in peripubertal animals did not significantly alter circulating FSH compared with controls (Table 1). This lack of effect, however, could be partially attributed to the very low circulating FSH levels in control peripubertal Siberian hamsters, making any further drop in circulating FSH difficult to detect with the sensitivity of our existing RIA. In adult animals, we also did not find any significant difference in plasma FSH among treatment groups (Table 1). Again, this lack of effect could be a consequence of the limited RIA sensitivity. Our previous study in prepubertal hamsters [5] reported circulating FSH levels that were several times higher than those in the older hamsters used in this study. This discrepancy is due to the presence of a prominent peak in circulating FSH during the early pubertal phase that drops off rapidly in late puberty [6]. Our previous study measured plasma FSH levels at the time of this pubertal FSH peak, whereas our current study measured FSH levels at the tail end the peak [5]. Thus, our current FSH levels are lower than our previous report. However, it is unclear why our FSH levels are several orders of magnitude lower than earlier reports [6, 17] on circulating FSH in Siberian hamsters at various stages of sexual maturation. The most likely explanation is the difference in standards, antibody, and iodination stock used in some earlier studies, as a more recent report showed circulating FSH levels consistent with our data using the same RIA reagents [18].
In this study, treatment with E2 significantly lowered total pituitary FSH concentration in both age groups (Table 1). This is in agreement with our previous results in prepubertal animals [5]. The ability of E2 to significantly decrease pituitary FSH in prepubertal, peripubertal, and adult hamsters but not testicular mass in the latter suggests that the efficacy of E2 inhibitory action does not decline equally at all sites of the HPG axis as the animal becomes sexually mature. Clearly, adults are able to compensate for the deficiency in pituitary FSH and still secrete sufficient FSH to maintain spermatogenesis. Our results also showed T treatment, unlike E2, did not affect pituitary FSH concentration at either age, further demonstrating the distinct nature of T and E2 actions (Table 1).
In this study, circulating T and E2 levels in hormone-implanted animals were all within the physiological limit for this species (Table 1). Surprisingly, T and E2 implants did not significantly elevate plasma T and E2 in adults (Table 1). This result was unexpected because we anticipated hormones coming from the capsules to add to endogenous hormones produced by the testes, elevating circulating T and E2 levels above those of controls. It is possible that Leydig cell function and endogenous steroid hormone synthesis were compromised in the steroid-treated group, leaving the capsule as the primary source of gonadal steroid.
At present, the precise role of high E2 levels in prepubertal male hamsters is unclear (Fig. 5). A similar observation has been made previously only once in rats by Döhler and Wuttke [19], who first reported high levels of E2 in prepubertal male rats between Postnatal Days 9 and 19. It is possible that E2 plays a preparatory role in the initiation of spermatogenesis at the time of pubertal onset. Recent studies in ERKO mice have shown that an estrogen receptor-dependent process is required for normal spermatogenesis (see [20] for a review). Despite normal plasma gonadotropin and androgen levels, ERKO mice are infertile because of the lack of functional mature sperm. E2 has also been shown to be a germ cell survival factor in humans, reinforcing the idea that E2 is required for male fertility [21]. Another possibility is that E2 serves as a primary suppressor of the HPG axis until the time of puberty, after which the decline in circulating level and activity diminishes its role in HPG regulation (Fig. 5). This idea parallels the gonadostat theory, which proposed a decrease in sensitivity to the inhibitory actions of gonadal steroid hormones as a mechanism for pubertal onset [1]. Although this theory has been refuted in several species (see [22] for a review), the decreased inhibitory actions of E2 could still play a notable role in the pubertal initiation of male Siberian hamsters.
Taken together, our data implicate E2 as a major regulator of the HPG axis in male Siberian hamsters. High circulating E2 prior to pubertal onset as well as a decline in sensitivity to the inhibitory effects of E2 after puberty strongly suggest the changing role of E2 during pubertal progression. In addition, contrary to what has been observed in the rat [1], an age-dependent decline in sensitivity to the inhibitory effects of T does not occur in the Siberian hamster. The ability of Siberian hamsters to respond persistently to the inhibitory effects of T at all ages contrasts sharply with their changing responsiveness to E2, further affirming the distinct roles of these two steroids in the regulation of the reproductive function in the male Siberian hamster.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NSF IBN-9996398 to P.-S.T. and a Research Grant from the University of Colorado to G.R.L. 
2 Correspondence: Toni R. Pak, Department of Environmental, Population, and Organismic Biology, University of Colorado, Campus Box 334, Boulder, CO 80309. FAX: 303 492 8699; toni.pak{at}colorado.edu
Accepted: March 22, 2002.
Received: January 11, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  T. R. Pak, G. R. Lynch, D. M. Ziegler, J. B. Lunden, and P.-S. Tsai Disruption of pubertal onset by exogenous testosterone and estrogen in two species of rodents Am J Physiol Endocrinol Metab, January 1, 2003; 284(1): E206 - E212. [Abstract] [Full Text] [PDF]
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