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BRIEF REPORT |
Hospital for Children and Adolescents (A.M.W., L.D.), Helsinki University Central Hospital, University of Helsinki, FI-00029 Helsinki, Finland; University Department of Growth and Reproduction (C.E.H.-H., E.R.-D.M.), Rigshospitalet, DK-2100 Copenhagen, Denmark; and Department of Pediatrics (L.D.), Kuopio University Hospital, University of Kuopio, FI-70211 Kuopio, Finland
Address all correspondence and requests for reprints to: Dr. Anne M. Wikström, Helsinki University Central Hospital, Hospital for Children and Adolescents, P.O. Box 281, 00029 Helsinki, Finland. E-mail: anne.wikstrom{at}fimnet.fi.
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Abstract |
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Objective: The objective of this prospective clinical study was to characterize the testicular degeneration process during puberty in boys with KS by describing the immunoexpression of some developmentally regulated markers of testis maturation in relation to serum levels of reproductive hormones.
Setting: This study was conducted at a university central hospital pediatric referral endocrinology outpatient clinic.
Patients: Patients consisted of 14 boys with KS aged 10.1 to 14.0 yr.
Main Outcome Measures: Main outcome measures were immunoexpression of germ cell differentiation markers (AP-2
, CHK2, OCT-3/4, NY-ESO-1, MAGE-A4) and androgen action-related proteins [androgen receptor (AR), anti-Müllerian hormone (AMH), MIC2, inhibin B; 
- and ßB-subunits] in testicular biopsies of boys with KS in relation to serum reproductive hormone levels.
Results: In boys with KS, gonocytes differentiated to the spermatogonium stage, but no spermatocytes were visible. Despite this, down-regulation of AMH expression in the Sertoli cells occurred concomitantly with decreasing serum AMH levels. Expression of inhibin - and ßB-subunits appeared in the biopsies even when circulating inhibin B levels were undetectable. In the boys with KS compared with age-matched controls, the proportion of Sertoli cell nuclei expressing AR was smaller and cytoplasmic staining of Sertoli cells was constantly present.
Conclusions: We showed with several testis-specific markers in KS that gonocytes differentiate to spermatogonia and that the degeneration of the testes accelerates at the onset of puberty. Altered immunoexpression of AR indicates that a relative androgen deficiency, at least at the testicular level, develops in boys with KS during puberty.
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Introduction |
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The testicular degeneration process may start early in life but probably accelerates at the onset of puberty (1, 2). An arrest in spermatogenesis at the spermatogonium stage was apparent in our previous study of 14 10.1- to 14.0-yr-old boys with KS (2). Before onset of puberty, Sertoli cells’ function may be normal, because prepubertal boys with KS have normal serum levels of the Sertoli cell markers inhibin B and anti-Müllerian hormone (AMH) (2). Later, during puberty, the levels of these hormones rapidly decrease (2). In adolescent boys with KS, serum testosterone levels rise and remain throughout puberty within the low normal range for healthy boys (3). Concomitantly, however, a relative Leydig cell insufficiency develops as manifested by increasing serum LH levels, a leveling off in the increase of serum levels of the Leydig cell-specific insulin-like factor 3, and hyperplasia of the Leydig cells (2, 3, 4).
The aims of this study were to characterize the testicular degeneration process during puberty in boys with KS by describing the expression in the testis of several developmentally regulated proteins. We also correlated the results of protein expression with serum reproductive hormone levels.
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Subjects and Methods |
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Fourteen boys (subjects 1KS–14KS) with nonmosaic karyotype 47,XXY (lymphocytes) were enrolled from the Hospital for Children and Adolescents in Helsinki, Finland. All parents gave their informed consent for their son’s participation in this study approved by the research ethics committee of the Hospital District of Helsinki and Uusimaa. From each subject, an open testicular biopsy specimen was taken under general anesthesia; three of the boys (patients 8KS–10KS) had a second biopsy specimen taken from the contralateral testis after 1.0 to 1.6 yr. The major portion of the biopsies (n = 17) was cryopreserved for possible further use in assisted reproduction in adulthood, and a small remainder was fixed in formalin and embedded in paraffin. Histomorphometric analyses have already been published (2).
Thirteen identically prepared testicular specimens from the tissue archive of the same hospital served as controls (subjects 1C–13C). These biopsies had been performed as part of a routine clinical screening for lymphoblastic infiltration in boys previously treated for acute lymphoblastic leukemia/lymphoma. These specimens were judged as normal by morphological inspection. As adult controls, we used biopsies obtained during treatment from the histologically normal contralateral testes of 18- to 39-yr-old men diagnosed with testicular carcinoma in situ or with a testicular germ cell tumor (affected tissues were used as positive controls) at the University Hospital (Rigshospitalet) in Copenhagen, Denmark.
Immunohistochemistry
The protein expression pattern was analyzed by immunohistochemistry with several antibodies (Table 1
). The staining was performed by a standard indirect peroxidase method as previously reported (5, 6, 7, 8, 9, 10, 11, 12). Staining intensity of the cells or tubules was systematically scored, and the proportion of stained cells or tubules in each section was semiquantitatively evaluated as a percentage of positive cells/tubules.
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Blood samples for hormone assays were drawn from the KS subjects concurrently with the testicular biopsy taking and during follow-up (2, 3, 4). Serum hormone levels were determined as previously described (2).
Statistics
Descriptive data are reported as median and range or as mean ± standard deviation. The Mann-Whitney U test was used for comparisons between groups; P < 0.05 was considered significant.
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Results |
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The expression of AP-2 and OCT-3/4 was analyzed in the seven youngest patients with KS (1KS–7KS) and in the youngest controls (1C and 2C). None of the markers showed any expression in these sections, whereas the positive control sections of testicular carcinoma in situ were, as expected, strongly positive.
MAGE-A4 was expressed in all spermatogonia in both KS and control specimens (Table 2). In contrast, only a subset of spermatogonia was positive for CHK2 (40–80%) and NY-ESO-1 (0–50%). MAGE-A4 staining helped to detect spermatogonia even in samples (patients 9KS, 10KS, and 12KS) in which no germ cells were seen by morphological inspection of serial sections. Furthermore, some tubules with no apparent germ cells showed nonspecific expression of MAGE-A4 (Fig. 1A). The clearly focal degeneration process in the KS testis was evident in all stainings; one or a few tubules containing spermatogonia were surrounded by Sertoli cell-only (SCO) tubules (Fig. 1A). No difference between the KS and control samples was visible in expression intensity of any of the studied X-linked markers MAGE-A4, NY-ESO-1, and MIC2.
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Down-regulation with age of AMH expression in Sertoli cells occurred at an older age in KS than in controls (Table 2
). A more heterogeneous staining of the tubules appeared in both groups with decreased expression (Fig. 1B). Older boys with KS showed weaker immunoexpression of the inhibin -subunit in Sertoli cells than did younger ones (Table 2). The majority of the KS samples (10 of 17), especially those from the older boys, showed clear nuclear staining that was absent from all the control samples. The Sertoli cells in tubules containing spermatogonia were less intensively stained with the -subunit than were those in the SCO tubules (Fig. 1C), a pattern also seen in controls but one not as marked. Expression of inhibin ßB-subunit expression in Sertoli cells remained stable in all KS specimens regardless of age (Table 2).
Expression of the androgen receptor (AR)
Both in KS and controls, the number of Sertoli cell nuclei expressing AR increased with age, but the proportion of stained nuclei was greater in the age-matched controls (Table 2). Expression was weaker in tubules containing not yet fully mature Sertoli cells; no consistent differences in intensity were evident between tubules with and without germ cells. Weak cytoplasmic staining was detectable in Sertoli cells of all KS samples, but this was seen only in the youngest control patients (Table 2). Nuclear expression of AR in Leydig cells and interstitial cells in the young patients in both groups was similar (Table 2). In contrast to the controls, the older patients with KS showed very strong AR expression both in the nuclei and cytoplasm of Leydig cells (Table 2 and Fig. 1, E and F).
Correlations to serum hormone levels
The 10 boys with KS with spermatogonia in their first testicular biopsy had lower serum testosterone (24.8 ± 31.9 vs. 208.4 ± 202.3 ng/dl; P = 0.03), FSH (2.7 ± 5.4 vs. 20.2 ± 18.5 IU/liter; P = 0.04), and LH (0.9 ± 2.1 vs. 5.7 ± 5.7 IU/liter; P = 0.02) levels than did the four boys with KS with no spermatogonia present, but no differences were detectable in serum inhibin B (87.8 ± 38.9 vs. 87.0 ± 142.7 pg/ml; P = nonsignificant) and AMH (104.5 ± 51.1 vs. 99.8 ± 181.7 ng/ml; P = nonsignificant) levels. Serum AMH level decreased (2) concomitantly with the immunoexpression. Serum inhibin B levels decreased (2), but expression of both subunits remained detectable in the testis tissue even when circulating inhibin B became undetectable.
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Discussion |
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and OCT-3/4 but positive for the spermatogonia markers CHK2, MAGE-A4, and NY-ESO-1. Some SCO tubules expressed MAGE-A4 unspecifically, probably as a result of leakage of antigens from degenerating apoptotic spermatogonia. Despite careful reviewing of serial sections, we detected no primary spermatocytes, neither pachytene nor earlier stages of leptotene spermatocytes. These results indicate that in KS, germ cell differentiation is, at least partially, blocked at the spermatogonium or early primary spermatocyte stage. It seems that in KS, spermatogonia have difficulty entering meiosis; instead, they proceed to apoptosis at puberty. However, evidence suggests that many adult patients with KS have foci of complete spermatogenesis (1). Studying the expression of MAGE-A4, NY-ESO-1, and MIC2 was of particular interest because these genes are located on the X chromosome. Staining intensity was identical in KS and controls, which may indicate that one of these X-linked gene loci is inactivated at this developmental stage.
At puberty, AMH expression in Sertoli cells is down-regulated concomitantly with the appearance of meiotic germ cells and up-regulation of androgen sensitivity of the Sertoli cell, both features of the final maturation of the Sertoli cells (6, 13, 14, 15). The absence of germ cells did not prevent down-regulation of AMH during puberty, but the boys with KS showed a delay in disappearance of AMH expression that may be explained in part by the lack of influence of meiotically dividing germ cells and in part by hypoandrogenism.
Inhibin -subunit was weaker in older than in younger patients with KS, probably as a result of the accelerated degeneration of the Sertoli cells. A sign of this degeneration process may also be the aberrant nuclear expression of the -subunit in the Sertoli cells. It is evident that inhibin B synthesis is altered in subjects with KS because both subunits were expressed in the Sertoli cells even when serum inhibin B was unmeasurable.
In normal males, AR expression first appears in Sertoli cell nuclei just before the onset of puberty but before final maturation of the Sertoli cells concomitantly with rising concentrations of FSH and testosterone (13). In the absence of androgens, AR is located in the cytoplasm (16). A study of one patient with KS showed AR expression in nuclei of Sertoli and peritubular cells, but in contrast to healthy men, his Leydig-cell AR was located in the cytoplasm (17). In the XXY mouse, immunoexpression has been nearly absent from the Sertoli cells and localized to the cytoplasm of the Leydig cells (18). In our boys with KS, AR expression in Sertoli cell cytoplasm and the smaller proportion of Sertoli cell nuclei expressing AR may be the result of lower testosterone levels. However, the alteration in AR expression may also reflect some primary dysfunction of the Sertoli cells or Sertoli-germ cell interactions. In agreement with earlier studies (17, 18), cytoplasmic staining of Leydig cells in our older boys with KS may be a sign of impaired function of their hypertrophied Leydig cells as also indicated by their high serum LH levels and leveling off in increase in insulin-like factor 3 levels (2, 3, 4). No difference appeared, however, between our boys with KS and their controls in AR immunoexpression in Leydig cell nuclei, nor were any differences evident between tubules containing spermatogonia and SCO tubules, a finding demonstrated in a study of cryptorchid men (19).
In conclusion, we demonstrate an altered immunoexpression of AR in pubertal boys with KS, which may indicate development of a relative androgen deficiency, at least at the testicular level. Serum reproductive hormone levels correlated with the presence of germ cells most accurately detected by MAGE-A4 immunostaining. We also provide additional evidence that KS gonocytes differentiate into mature spermatogonia without significant delay. Germ cells in KS subjects were eliminated most likely at the onset of meiosis, perhaps already as spermatogonia B or preleptotene spermatocytes. This process may begin in a few cells in midchildhood, but it most probably accelerates rapidly at the onset of puberty.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: A.M.W., C.E.H.-H., and E.R.-D.M. have nothing to declare. L.D. consults for Novartis and Pfizer and has received lecture fees from Ferring.
First Published Online December 5, 2006
1 A.M.W. and C.E.H.-H. have contributed equally to this study. 
Abbreviations: AMH, Anti-Müllerian hormone; AR, androgen receptor; KS, Klinefelter syndrome; SCO, Sertoli cell-only.
Received August 28, 2006.
Accepted November 27, 2006.
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is a developmentally regulated marker of testicular carcinoma in situ and germ cell tumors. Clin Cancer Res 10:8521–8530This article has been cited by other articles:
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  R.B. Sciurano, C.V. Luna Hisano, M.I. Rahn, S. Brugo Olmedo, G. Rey Valzacchi, R. Coco, and A.J. Solari Focal spermatogenesis originates in euploid germ cells in classical Klinefelter patients Hum. Reprod., May 14, 2009; (2009) dep180v1. [Abstract] [Full Text] [PDF]
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 H. E. Chemes, R. A. Rey, M. Nistal, J. Regadera, M. Musse, P. Gonzalez-Peramato, and A. Serrano Physiological Androgen Insensitivity of the Fetal, Neonatal, and Early Infantile Testis Is Explained by the Ontogeny of the Androgen Receptor Expression in Sertoli Cells J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4408 - 4412. [Abstract] [Full Text] [PDF]
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