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Testis |
Andrology Laboratory, ANZAC Research Institute, Concord Hospital, University of Sydney, Sydney, NSW 2139, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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leydig cells, luteinizing hormone, spermatogenesis, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although it has long been known that T is essential for the initiation and maintenance of spermatogenesis [5], recent genetic FSH-deficient models have shown that qualitatively normal mouse spermatogenesis can be initiated and maintained in the absence of FSH [6–9], although in each model spermatogenesis remains quantitatively subnormal. Any specific physiological role for such nonandrogenic Leydig cell products, however, remains to be established. Previous studies based upon inhibiting endogenous T secretion in mature rodents by gonadotropin withdrawal either by using testosterone and estradiol implants [10] or hypophysectomy [11] or by eliminating Leydig cells with ethane dimethanesulphonate (EDS) treatment [12] suggest that Leydig cell factors other than T are not required to restore spermatogenesis. These models, however, examined maintenance or reinitiation of spermatogenesis because germ cell development had been completed before experimental treatments. Furthermore, the uncertain influence of incomplete gonadotropin suppression, other pituitary hormones, and nonspecific effects of the cytotoxin EDS limits interpretation of these experiments. Whether Leydig cell factors other than T are essential for initiation of spermatogenesis has not been reported.
Hypogonadal (hpg) mice have a congenital deficiency of hypothalamic gonadotropin-releasing hormone [13] because of a naturally occurring mutation causing a major deletion of the GnRH gene [14]. Consequently, hpg mice undergo normal male sexual differentiation (because of gonadotropin-independent fetal Leydig cell function) but become androgen deficient from birth [15] while remaining fully androgen responsive. This contrasts with tfm mice or rats, which are functionally androgen deficient from conception and exhibit a similar spermatogenic block at pachytene spermatocyte stage [16]. However, because of their androgen resistance, the effects of androgen treatment cannot be evaluated. In hpg mice, Leydig cells remain sparse (
10% of wild type) and morphologically immature [17], resulting in gonads that do not undergo initiation of spermatogenesis [13]. Although initiation of qualitatively complete spermatogenesis in hpg mice can be achieved with exogenous T alone, testis weight and germ cell numbers remain 30%–40% of wild-type controls [6]. In contrast, after GnRH gene transfer [18] or transplantation of hypothalami containing GnRH neurons [19], hpg mice show restoration of full quantitative spermatogenesis. The present study was designed to determine whether restoration of LH activity (with hCG as a long-acting homolog of LH) leading to Leydig cell maturation and secretory activity could rectify the remaining quantitative deficiencies in testis development and germ cell numbers. This could also determine whether Leydig cell factors other than T are important in pubertal testis development and quantitative elaboration of germinal cell populations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The hpg colony was housed under standard conditions at the University of Sydney and ANZAC Research Institute. Genotypes (N/N, N/hpg, hpg/hpg) were assayed from tail DNA by polymerase chain reaction as described previously [6]. The Animal Ethics Committee of University of Sydney or CSAHS Animal Welfare Committee approved all experiments.
Experimental Design
Weanling (21-day old) hpg male mice (n = 5–15 per group) were either injected (i.p.) with hCG (Pregnyl, Organon Australia Pty Ltd, Sydney, Australia) at a range of doses (0.1U–100 IU/mouse) three times weekly for 6 weeks or given a subdermal implant of T (Sigma, Sydney, Australia) in a 1-cm silastic implant (id 1.47 mm, od 1.95 mm, ends sealed with Silastic adhesive, Dow-Corning, Sydney, Australia) for 6 weeks. This T treatment produces maximal stimulation of spermatogenesis [6]. In addition, controls included groups of untreated and saline (vehicle)-injected mice and untreated phenotypically normal littermates. All experiments were terminated when mice reached 9 weeks of age.
Tissue and Blood Collection
Terminal blood was collected under anesthesia via cardiac puncture and serum stored frozen at -20°C. Body weight and the wet weight of fresh (right) and fixed (left) testes and fresh (right) and fixed (left) epididymis and spleen were recorded. The right testis was quickly removed, snap frozen in liquid nitrogen, and stored at -80°C. The mouse was then perfused via the left ventricle with warm (37°C) heparin and saline solution followed by 30 ml of Bouins fixative. The left testis was immersed in fixative overnight and subsequently processed and embedded in methacrylate resin by using the Technovit 7100 embedding kit (Heraeus, Germany). Sections were cut with a Reichter Jung microtome. Thin (7 µm) sections stained with toluidine blue were used for photomicrographs and macroscopic histology. All procedures have been fully described previously [20].
Stereology
Thick (25 µm) sections were prepared for stereology from upper, lower, and middle sections of the testis and stained with periodic acid-Schiff. Sertoli and spermatogenic cell types were classified according to Russell et al [21]. Sections were evaluated by using the optical disector technique and CAST software (Olympus, Copenhagen, Denmark) as described previously [20].
Hormone Assays
Frozen testis was homogenized in phosphosaline and TritonX-100 buffer and extracted with hexane:ethyl acetate (3:2). Serum and testicular T were assayed by radioimmunoassay as described previously [22]. Undetectable T levels were assigned the detection limit of the assay for analysis. Human FSH was measured with a two-site immunoradiometric assay (DELFIA, Wallac, Turku, Finland) as described previously [22]. This FSH assay detects epitopoes on both subunits to measures intact, dimeric human FSH but does not cross-react with hCG (106 IU hCG is measured as <0.3 IU/l FSH).
Antibodies to hCG were detected by specific binding of iodinated hCG. Briefly, hCG (Pregnyl, Organon Australia Pty Ltd) was iodinated with chloramine T (Sigma) and separated from free iodine with Con A Sepharose (Pharmacia, Sydney, Australia). Serum (100 µl, diluted 1:100 in assay buffer) was incubated with either buffer or excess hCG (20 µg per tube) and 10 000 CPM I125 hCG overnight (18 h) at room temperature with bound and free tracer separated by 20% polyethylene glycol precipitation.
Statistics
For analysis, testis and epididymis weight were corrected for body weight to adjust for differences in body size caused by litter size or treatment. Organ weights and T levels were log transformed prior to analysis to maintain equality of variance. Quantitative data from stereological analysis are presented as cells per testis or per SC. In some analyses, organ weights and hormone data that did not differ between untreated and saline-treated hpg mice were combined for subsequent data analysis. Data were analyzed by one-way ANOVA with suitable linear contrasts and Fishers LSD test for posthoc comparisons by using NCSS 2001 software (NCSS, Kaysville, UT). All data were expressed as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testis weight (per body weight) was increased by all hCG doses in a dose-dependent fashion compared with untreated or saline-treated hpg mice (0.10 ± 0.01 [n = 25] and 0.043 ± 0.004 [n = 11], respectively, for pooled data) (Fig. 1). An hCG dose of 1 IU produced plateau stimulation of testis weight. Testis weight after hCG doses between 1 and 100 IU did not differ significantly from the effects of T treatment. After treatment with 1U hCG or T, however, testes remained at 31% and 24%, respectively, of phenotypically normal littermate controls. Neither body nor spleen weight were significantly different between groups treated with hCG or T (data not shown).
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Testosterone
Human CG produced dose-dependent increases in serum and testicular T compared with untreated or saline-treated hpg mice (Fig. 1). For testicular T, plateau effects were achieved with 1 IU of hCG, and maximal hCG effects at doses of 1 IU or higher were similar to phenotypically normal littermate controls but significantly higher than the effects of T treatment. Serum T was stimulated by 1 IU hCG and higher doses with plateau effects produced by a dose of 1 IU hCG. All hCG doses of 1 IU or higher produced serum T similar to those after T treatment.
Human CG Antibodies
Antibodies to hCG were detected in serum of all hpg mice treated with 10 IU (median 8.9% specific tracer binding, range 1.2%–19%, n = 7) and 100 IU (median 5.8% specific tracer binding, range 4.7%–7.6%, n = 7) of hCG but not in any untreated hpg mice (n = 6) or hpg mice treated with saline (n = 6), 0.1 (n = 8), 0.3 (n = 5), or 1 (n = 6) IU or in phenotypically normal littermate control (n = 5) mice. There was no significant difference between hCG antibody titres in the 10- and 100-IU treatment groups. To avoid possible confounding effects of hCG immunoneutralization, stereological analyses of hCG effects were restricted to the 1 IU and lower doses.
Follicle-Stimulating Hormone
Pregnyl from the same batches used in the experiments contained no detectable FSH, with 1 IU hCG having less than 2 x 10-6 IU human FSH immunoreactivity.
Histology
Control testis (Fig. 2A) demonstrated complete spermatogenesis with all tubules showing well-developed lumina compared with hpg testis (Fig. 2B), where spermatogenesis is arrested at the pachytene spermatocyte stage and seminiferous tubules have no lumen.
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Both 1 U hCG (Fig. 2C) and T (Fig. 2D) induced complete spermatogenesis. Tubules with well-formed lumen and similar tubular diameters were observed with both treatments, but they remained smaller than phenotypically normal controls. Human CG treatment produced hypertrophy and hyperplasia of Leydig cells in the interstitium (Fig. 2E), whereas with T treatment, Leydig cells remained immature and spindle shaped as indicated by arrows (Fig. 2F). Both hCG (Fig. 2G) and T (Fig. 2H) treatment led to maturation of the germinal epithelium, which was spatially organized into identifiable stages with mature basal SC nuclei (stars) and mature haploid sperm (arrows).
Stereology
SC SC numbers (Fig. 3) were significantly increased by maximal hCG or T treatment compared with untreated hpg mice (1.5 ± 0.1 million per testis). There was no significant difference between maximal effects of hCG and T treatments (2.1 ± 0.1 million per testis vs. 2.0 ± 0.1 million per testis), whereas SC numbers remained lower than in phenotypically normal littermate controls (55% and 52%, respectively).
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Germ cells Spermatogonial (SG) numbers (Fig. 4) were increased by 1 IU hCG treatment, but lower doses (0.1, 0.3 IU) and T treatment had no effect compared with untreated controls. Maximal hCG effects produced significantly fewer SG than phenotypically normal littermate controls (2.7 ± 0.3 vs. 5.5 ± 0.6 million per testis).
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For spermatocyte (Sc), round (RS) and elongated (ES), maximal hCG effect (1 IU) increased germ cell numbers to the same extent as T treatment (Fig. 4). Nevertheless, for both hCG and T treatment, each germ cell population remained lower than phenotypically normal littermate controls.
Lower hCG doses produced small, nonsignificant increases in haploid cells (RS, ES), which were much lower than the 1 IU hCG dose.
Germ cell:SC ratio
The mean number of germ cells supported by each SC was evaluated by the germ cell:SC ratio for each germ cell type. For SG, the SG:SC ratio was significantly increased (2-fold) by 1 IU hCG treatment to levels significantly greater than those for T treatment (Fig. 5).
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For Sc, the ratio was increased by both 1 IU hCG and T treatment to a similar extent (7.5-fold). For the haploid germ cells, both RS (Fig. 5) and ES (Fig. 5) were undetectable in the untreated hpg mice but were increased to similar germ cell ratios by 1 IU hCG and T treatment.
Lower hCG doses (<1 IU) had lesser effects on all germ cell ratios. All ratios remained significantly lower than in phenotypically normal littermate controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and -ß; cytokines IL-1 and IL-6; and vasoactive factors endothelin-1 and angiotensin-II [25, 26]. Such nonandrogenic Leydig cell products might act upon peritubular, Sertoli, or interstitial vascular or neural cells, but no clear physiological mechanisms or specific intratesticular role of such local nonandrogenic Leydig cell products has been identified [3]. Although paracrine modulators of Leydig cell function are readily detected by the sensitive LH/hCG bioassay of Leydig cell testosterone production, functional bioassays for Leydig cell products influencing spermatogenesis are not available because of the complexity of cellular functions and interactions. A useful paradigm to evaluate putative nonandrogenic Leydig cell factors is whether spermatogenesis can be induced or maintained in the absence of functional Leydig cells. Hence, previous studies inactivating Leydig cell function by either LH deprivation via hypophysectomy [11], GnRH immunoneutralisation [27], or EDS-induced Leydig cell destruction [12] have suggested that T accounts for most or all LH-mediated actions. However, each of these model systems involved only the mature testis, so the evaluation was restricted to the maintenance or reinitiation of previously established spermatogenesis [10, 28]. In contrast, this study has found that hCG can induce qualitatively complete spermatogenesis in hpg mice, which are gonadotropin and androgen deficient from birth [15]. The maximal hCG effects on testis growth as well as absolute and relative germinal cell populations were very similar to those produced by T alone. Although there was a significantly greater effect of hCG than T on spermatogonial populations, all more advanced germ cell populations were affected similarly by hCG and T treatment. This implies that the main, or the only important, Leydig cell product for initiation of murine spermatogenesis is T. Caveats of this conclusion are that differences in germ cell dynamics may be overlooked, and that this pharmacological dosage cannot fully exclude other local effects having secondary modulatory effects. The differences in spermatogonial numbers may reflect similar observations of hCG effects on spermatogonial proliferation in the absence of FSH and not replicated fully by testosterone previously reported as due to Leydig cell secretions acting directly on spermatogonia [29] or via paracrine effects on SC secretions [30–32]. Conversely, in the present experiments the failure of hCG to fully restore quantitatively normal spermatogenesis any more than T can achieve indicates that either non-Leydig cell factors or Leydig cell factors at times before weaning might be important in explaining the persisting deficit in testis growth and germinal development when either hCG or T are supplied from weaning onwards. The immunogenicity of higher hCG doses prevented an examination of their efficacy.
This study utilized hCG as a long-acting, structurally homologous analog of LH. It binds to and activates the same LH receptor and exerts similar effects on Leydig cell maturation and steroidogenesis [2]. Human CG stimulates increased serum T concentrations within 2 h after administration and remains elevated for several days [33], allowing administration at longer intervals than daily. Prolonged hCG treatment can induce hyperplasia and hypertrophy of mature [34] and immature [35] Leydig cells, as well as those reappearing after EDS treatment [36]. In hpg mice, LH stimulates steroidogenesis and proliferation [17, 37–39]. One drawback of hCG is its immunogenicity as a foreign protein. In this study we observed that lower doses (
1 IU) of hCG did not produce anti-hCG antibodies, whereas doses of 10 or 100 IU injected i.p. three times weekly for 6 weeks (total 18 doses) cause development of anti-hCG antibodies. This is consistent with a previous study that used doses of 10 or 100 IU and found that neutralizing anti-hCG antibodies developed consistently after 14 or more days but not after 7 daily injections [40]. Although the reason for the dose-dependent immunogenicity of hCG in mice is unclear, it has permitted an evaluation of hCG effects at doses up to 1 IU three times weekly for 6 weeks without interference by anti-hCG antibodies. Although Pregnyl is an extract of human urine, it is unlikely that contamination with FSH [41] or any intrinsic FSH-like bioactivity of hCG [42] would have influenced the experimental findings, because the FSH content of the highest dose (100 IU) of Pregnyl used contained <2 x 10-4 IU FSH. By contrast, daily treatment with 1 IU of pure recombinant human FSH has no effect on testis weight [43].
The present study observed that, despite hCG-induced maturation of Leydig cell function as indicated by their morphology and intratesticular and serum T concentrations, there was no further increase in testis weight or haploid germ cell number compared with effects of T alone. Hence, hCG-stimulated Leydig cell maturation and secretory activity commencing at weaning still failed to restore testis weight or spermatogenesis to levels comparable with phenotypically normal control mice. The restoration of normal testis development in hpg mice after GnRH gene transfer [18] and fetal hypothalamic transplants [19] proves the defect in testis growth in hpg mice is not caused by any genetic limitation intrinsic to the testis. There are a number of possible explanations for the failure to rectify this deficit. First, other non-Leydig cell factors such as FSH or paracrine factors influencing SCs may be necessary. The most important role of FSH for initiation of spermatogenesis may be the control of SC proliferation in the perinatal period [44–46]. Because each SC provides functional and structural support for a finite number of developing germ cells, the size of the SC population determines the final germ cell carrying capacity of the adult testis [46]. Transgenic mouse models expressing human FSH [20, 22] or an activating mutation of the human FSH receptor [47] in mouse SCs on an hpg background support the possibility that the functional deficiency of FSH is partly responsible. In these models, the addition of FSH effects on SCs, in conjunction with T, further rectifies the deficit in testis growth and spermatogenesis, though the net effect still falls short of wild-type mice. Despite a profusion of paracrine factors possibly involved in regulation of spermatogenesis, none have an established physiological role [25, 26]. A second caveat is that the timing or pattern of LH replacement may have important effects, which our experiment did not replicate. For example, postnatal LH effects before the time of weaning [48], when our experiments commenced, or effects that depend on the pulsatility of endogenous LH secretion [49, 50] were not replicated in our experiments. Finally, the hCG dose, limited by its immunogenicity, may have been insufficient to fully replicate LH effects. Both the early onset and the dose limitation of chronic LH stimulation could be addressed by constructing a transgenic model from the transgenic hCG mouse model as described elsewhere [51, 52] transferred onto the hpg background, similar to those we have used to characterize FSH effects [20, 22, 47]. Future studies of hCG effects in our FSH transgenic models may determine the magnitude of this interaction in the initiation of spermatogenesis.
We conclude that hCG treatment can initiate qualitatively complete spermatogenesis in the gonadotropin-deficient hpg mouse. With the exception of spermatogonia, the testicular effects of the maximal hCG doses were identical to the maximal effects of T treatment on testis weight, SC numbers, and absolute and relative (to SC) germ cell populations, whereas hCG alone also stimulated Leydig cell maturation and normalized intratesticular T content. Yet, the functional maturation of Leydig cells or the contribution of any nonandrogenic Leydig cell factor(s) caused by hCG treatment cannot rectify the remaining quantitative deficit of spermatogenesis in the androgen or hCG-treated hpg mouse. This remaining deficit may be because of a lack of other regulators of spermatogenesis including FSH, paracrine intratesticular factors, or failure to fully replicate physiological patterns of LH secretion by hCG treatment commencing at weaning. Because T is both necessary and sufficient to induce spermatogenesis, and because it replicates all hCG effects, it is likely that T accounts for all hCG effects in the induction of spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 61 2 9767 9101; djh{at}anzac.edu.au
Received: 15 May 2003.
First decision: 7 June 2003.
Accepted: 18 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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