Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter's syndrome*

Yasuhisa Yamamoto1, Nikolaos Sofikitis1,2,4, Yasuyuki Mio3, Dimitrios Loutradis1, Apostolos Kaponis1 and Ikuo Miyagawa1

1 Department of Urology, Tottori University School of Medicine, Yonago, Japan, 2 Molecular Urology Laboratory, Department of Urology, Ionannina University School of Medicine, Ioannina, Greece and 3 MFC Clinic, Yonago, Japan


    Abstract
 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Klinefelter's syndrome is the most frequent chromosomal abnormality in infertile men. In this study, the chromosomes of round spermatids and spermatogonia/primary spermatocytes from men with non-mosaic Klinefelter's syndrome were examined, together with the Sertoli cell secretory function and sperm morphometry. METHODS: Twenty-four men with non-mosaic Klinefelter's syndrome and nine men with obstructive azoospermia underwent therapeutic testicular biopsy. When spermatozoa in the final filtrate were present, they were processed for sperm morphometry or ICSI. Sperm morphometry was evaluated by the maximal length and width of the sperm head, the length of the midpiece and the ratio of the acrosomal region to the total surface area of the head. When round spermatids were present, they were processed for fluorescent in-situ hybridization (FISH). FISH was also applied to fragments of seminiferous tubules. Sertoli cell secretory function was measured by the amount of androgen binding protein (ABP) secreted in vitro. RESULTS: More than 93% of the evaluated round spermatids were normal. The proportions of 24,XY and of 24,XX round spermatids to the total number were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Men with Klinefelter's syndrome who had spermatozoa in their testicular tissue (n = 12) were positive for both 46,XY and 47,XXY spermatogonia in their seminiferous tubules. In contrast, men with Klinefelter's syndrome without spermatozoa in their testicular tissue (n = 12) were positive for 47,XXY spermatogonia but negative for 46,XY spermatogonia in their seminiferous tubules. ABP profiles were significantly smaller in men with Klinefelter's syndrome who were negative for spermatozoa compared with men who were positive. Four pregnancies were achieved and five healthy babies were born. CONCLUSIONS: This study suggests that few 46,XXY spermatogonia undergo meiosis with an XX pairing and a Y univalent type of pairing. Hyperhaploid round spermatids (24,XY and 24,XX) may be produced by meiosis of 47,XXY spermatogonia. Men with Klinefelter's syndrome who are negative for testicular spermatozoa have a greater degree of Sertoli cell secretory dysfunction compared with men with Klinefelter's syndrome who are positive for spermatozoa. There are several defects in sperm morphometry with functional significance in men with Klinefelter's syndrome.

Key words: Klinefelter's syndrome/round spermatids/Sertoli cell/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
Klinefelter's syndrome represents the most commonly found human sex chromosomal abnormality. Furthermore, it is the most frequent chromosomal abnormality in infertile men (De Braekeleer and Dao, 1991Go; Bielanska et al., 2000Go). There is no natural fertility in the great majority of men with non-mosaic Klinefelter's syndrome and very few cases of naturally conceived offspring of proven paternity have been reported (Laron et al., 1982Go; Terzoli et al., 1992Go). Retrieval of spermatozoa from severely oligozoospermic semen or from the therapeutic testicular biopsy (TTB) of men with Klinefelter's syndrome has been reported (Sharara, 1998Go; Bielanska et al., 2000Go). Isolated spermatozoa from the semen or the TTB material of men with non-mosaic Klinefelter's syndrome have been used for ICSI with successful fertilization and delivery of offspring (Harari et al., 1995Go; Staessen et al., 1996Go; Tournaye et al., 1996Go; Bourne et al., 1997Go; Hinney et al., 1997Go; Nodar et al., 1998Go; Palermo et al., 1998Go; Reubinoff et al., 1998Go; Ron-El et al., 1999Go). Up to now the births of 14 healthy neonates have been reported following ICSI procedures with spermatozoa from patients with non-mosaic Klinefelter's syndrome (Bourne et al., 1997Go; Tournaye et al., 1997; Palermo et al., 1998Go; Reubinoff et al., 1998Go; Nodar et al., 1999; Ron-El et al., 1999Go, 2000Go). However, in one of the latter studies karyotype analysis from chorionic villous sampling revealed a 47,XXY chromosomal pattern in one fetus (Ron-El et al., 2000Go). The affected 47,XXY fetus was reduced on week 14 of gestation. In addition, fluorescence in-situ hybridization (FISH) of sex chromosomes in spare embryos of a Klinefelter's 46XY/47XXY male showed a high percentage of mosaic embryos with chaotic chromosome arrangements (Bielanska et al., 2000Go). Several studies have demonstrated an increased frequency of sex chromosome hyperhaploidy in spermatozoa of men with Klinefelter's syndrome. The abnormal embryos or fetuses derived by ICSI techniques using spermatozoa from mosaic or non-mosaic men with Klinefelter's syndrome and the increase in the frequency of sex chromosome hyperhaploidy in the spermatozoa of these patients, (i) indicates an increased risk of obtaining chromosomally abnormal newborns when spermatozoa are recovered from men with Klinefelter's syndrome and processed for assisted reproduction, and (ii) recommends the routine employment of preimplantation embryonic biopsy or amniocentesis.

One of the objectives of the current study was to evaluate whether the hyperhaploid round spermatids or spermatozoa in men with Klinefelter's syndrome are produced by 47,XXY germ cells or by normal 46,XY testicular germ cells. Another objective was to measure the frequency of sex chromosomal abnormalities in round spermatids present in the TTB material of men with Klinefelter's syndrome. An additional purpose was to evaluate the morphometric characteristics of non-fixed/non-stained undisturbed testicular spermatozoa from men with Klinefelter's syndrome using a confocal scanning laser microscope–computer assisted system (CSLM–CAS) (Sofikitis et al., 1994Go). Another objective of the current study was to evaluate the Sertoli cell secretory function in men with Klinefelter's syndrome.


    Participants and methods
 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
Participants were 24 non-mosaic men with Klinefelter's syndrome. All these men had signed consent forms giving permission to process their biological fluids and tissues for several studies. Approximately 100 peripheral blood leukocytes had been analysed for chromosomes for each man by the Japanese Special Reference Laboratory (Matsue, Japan). All these men were negative for spermatozoa in the pellets of centrifuged semen samples. A unilateral TTB was performed on the same day that oocytes were recovered from the female partner. Transverse scrotal incision was performed under local anaesthesia, the testicular coverings were opened and a piece of testicular tissue (148–194 mg) was collected. TTB material was minced and filtered (via a 30–40 µm pore size filter). The filtrate was filtered again (via a 12 µm pore size filter; Whatman Co., NY, USA) as previously described (Yamanaka et al., 1997Go; Sofikitis et al., 1998aGo,bGo; Yamamoto et al., 1999aGo,bGo). Filtration via the 30–40 µm pore size filter and the 12 µm pore size filter removed the small pieces of seminiferous tubules and most of the Sertoli cell fragments, debris, tissue fragments, spermatogonia and primary spermatocytes from the final filtrate. Thus, in men with Klinefelter's syndrome positive for testicular foci of round spermatids and spermatozoa, the final (second) filtrate contained mainly spermatids and spermatozoa. Fragments of testicular tissue that did not pass through the first filter (i.e. seminiferous tubules) were processed to assess the ability of Sertoli cells to secrete androgen-binding protein (ABP) in vitro. Tissue FISH was performed in sections of seminiferous tubules fragments. Fractions of dispersed cells from each minced TTB sample that passed through the first or second filter were processed for centrifugation and microscopic observation of the pellet (to identify spermatozoa). When spermatozoa were identified, some of them were aspirated via a micropipette and processed for assisted reproduction. In addition, 60–120 spermatozoa per participant (from the final filtrate) were processed for assessment of sperm morphometry via CSLM–CAS microscopy. Additional spermatozoa from participants positive for spermatozoa were cryopreserved. Fractions of dispersed round germ cells in the final filtrate from each man were processed for CSLM–CAS microscopy and FISH.

FISH procedures for dispersed round germ cells
Due to the filtration techniques that had been performed, the final filtrates in the men with Klinefelter's syndrome with testicular foci of advanced spermatogenesis had been enriched in round spermatids and spermatozoa. Prior to FISH techniques, all glass slides had been observed via CSLM–CAS. CSLM–CAS is a relatively new approach in the field of microscopy. It provides a large magnification (>1000) and is capable of calculating various cellular and nuclear diameters, identifying intracellular organelles and subsequently recognizing primary spermatocytes, secondary spermatocytes and round spermatids (Sofikitis et al., 1994Go, 1998aGo,bGo,cGo; Yamanaka et al., 1997Go) (quantitative and qualitative criteria are applied; see below). The positions of cells considered to be spermatogonia/primary spermatocytes, secondary spermatocytes and round spermatids by CSLM–CAS microscopy were marked in a CSLM–CAS photo of the glass slide.

FISH techniques were applied to those glass slides that contained several round spermatids (additional round germ cells were occasionally present). In other words, smears containing several round spermatids were given a priority to process to FISH techniques. When for a given round germ cell, CSLM–CAS microscopy and FISH resulted in different characterizations for its spermatogenic (i.e. meiotic) stage, or when ambiguous signals were observed during fluorescent microscopy, the chromosomal patterns of these cells were excluded from the results. However, in >94% of the cells evaluated, both methods for germ cell identification (i.e. CSLM–CAS and FISH) resulted in consistent findings. Probes for chromosomes X (yellow colour), Y (red/pink) and 18 (blue/green) were obtained from Konishi Co. (Yonago, Japan). DAPI-K (Konishi) was used as a counterstain.

FISH for dispersed round germ cells was performed as previously described (Yamamoto et al., 1999aGo) with the assistance of the Japanese Special Reference Laboratory. Very briefly, after denaturation of probes and germ cell nuclei the three probes were co-hybridized in a hybridization mix (Chevret et al., 1996Go). The slides were then washed several times (Chevret et al., 1996Go). After the final wash, slides were air dried. Then an antifade solution (S-DP-K; Konishi) was applied to the slides (Chevret et al., 1996Go). The nuclei and fluorescence signals were observed using a fluorescence Olympus BX-K 60 microscope. The following filters were available/used: SMF/FITC/Texas red/DAPI-K and Aqua (Konishi). Only clear fluorescence signals of integral nuclei were analysed. Two signals of the same colour were considered to represent two individual chromosomes when the distance between these signals was larger than one signal's diameter. Spermatogonia/primary spermatocytes were 46,XY1818 or 47,XXY1818 germ cells (Yamamoto et al., 1999aGo). In addition, to characterize a nucleus as a nucleus of a spermatogonium or primary spermatocyte, each evaluated cell (prior to FISH) had to (i) show a regular shape, (ii) demonstrate one nucleus only, (iii) exhibit a maximal, minimal and oblique cellular diameter at least 15% larger than those characterizing secondary spermatocytes during observation, image analysis and morphometric analysis via CSLM–CAS (Sofikitis et al., 1998aGo,cGo), and (iv) show a nuclear diameter >=9 µm.

We did not attempt to distinguish spermatogonia from primary spermatocytes (see Notes for Figures). These morphometric criteria for spermatogonia/primary spermatocytes are consistent with those previously proposed (Kimura and Yanagimachi, 1995Go) for the nuclei of human leptotene or pachytene primary spermatocytes (nuclear diameters: 9.46 or 11.47 µm respectively). Transmission electron microscopy (TEM) techniques performed in Tokyo TEM Laboratory (Tokyo, Japan) and the Japanese Special Reference Laboratory (Matsue, Japan) showed that >91% of the testicular cells satisfying the above CSLM–CAS-based criteria for spermatogonia/primary spermatocytes were indeed spermatogonia/primary spermatocytes.

The employment of CSLM–CAS to calculate cellular and nuclear diameters, observe intracellular organelles [i.e. acrosomal granule(s)] and subsequently recognize the germ cell type was originally proposed by Yamanaka et al. and Sofikitis and co-workers (Yamanaka et al., 1997Go; Sofikitis et al., 1998aGo,cGo). The majority of the cells characterized as spermatogonia/primary spermatocytes by the combination of CSLM–CAS microscopy and FISH techniques were 46,XY or 47,XXY germ cells exposing two signals or one large paired signal for the 18 chromosomes (late zygotene of pachytene stage of meiosis I). Sertoli cell nuclei or leukocytes were easily recognized (because of their shape and shape/size respectively) and excluded from the counting. Normal secondary spermatocytes were considered X-(2C) or Y-(2C) DNA cells (2C indicates a diploid amount of DNA) (Yamamoto et al., 1999aGo). In addition, to characterize a nucleus as a nucleus of a secondary spermatocyte, each evaluated cell (prior to FISH) had to exhibit a maximal, minimal and oblique diameter within the ranges of the morphometric criteria previously defined for secondary spermatocytes (observation and morphometric analysis via CSLM–CAS) (Sofikitis et al., 1998cGo). Normal round spermatids were characterized as X-(C) or Y-(C)-DNA cells (C indicates a haploid amount of DNA). To characterize a cell as a round spermatid, each evaluated cell (prior to FISH) had to exhibit an acrosomal granule attached to the nucleus (observation via CSLM–CAS) (Yamanaka et al., 1997Go). A large number of round spermatids (n = 190–210) were evaluated per participant (positive for testicular round spermatids). FISH analysis was stopped when 2400 round spermatid nuclei had been assessed in the 12 Klinefelter's men who were positive for haploid cells (see Results).

Secretion of ABP in vitro
Fragments of testicular tissue were incubated in Eagle's tissue culture medium based on Earle's salts (Antypas et al., 1994Go; Sofikitis et al., 1995Go) to which NaHCO3 (2 g/l), L-glutamine (2 mmol/l), penicillin (100 IU/ml) and streptomycin (50 µg/ml) were added. Incubations were performed at 32°C in a 5% carbon dioxide environment with constant shaking. After 4 h incubation, the tissue fragments were sedimented by centrifugation at 700 g for 8 min. The tissue fragments were resuspended in fresh medium and incubated for another 16 h. Preformed ABP in the testicular tissue was released into the medium during the initial preparation and the first 4 h of incubation (Ritzen et al., 1974Go; Antypas et al., 1994Go; Sofikitis et al., 1995Go). The ABP secretion rate was obtained by measuring the amount of ABP accumulating in the medium during the latter 16 h incubation period (Antypas et al., 1994Go; Sofikitis et al., 1995Go). After incubation, the tubes were centrifuged for 1 h and the supernatants were assayed for ABP as previously described (Ritzen et al., 1974Go; Antypas et al., 1994Go; Sofikitis et al., 1995Go). To measure ABP, the samples were pretreated as previously described (Ritzen et al., 1974Go). Finally, the samples were layered on gels containing radiolabelled dihydrotestosterone (H-DHT). Electrophoresis was carried out. Quantification of the ABP in each gel could be made when a steady state was achieved between dissociation and association of H-DHT with the ABP as it moved through the gel. Calculations of the amount of H-DHT bound to ABP under these steady state conditions were made using the formula of Ritzen and co-workers (Ritzen et al., 1974Go). The intra- and inter-assay coefficients of variation of the ABP assay were 7 and 12% respectively.

ABP was evaluated in eight men with Klinefelter's syndrome negative for spermatozoa and six men with Klinefelter's syndrome positive for spermatozoa.

FISH techniques in fragments of seminiferous tubules
FISH techniques were performed in sections of seminiferous tubules with the assistance of the Japanese Special Reference Laboratory. In brief, testicular tissue specimens were embedded in OC-PRF compound (SRL Co., Matsue, Japan). Sample sections were prepared and fixed on glass slides. OC-PRF compound was removed by placing the slides in CM jars (SRL) with xylene-MT (SRL) followed by incubation in 100% ethanol and air-drying. Tissue sections were pretreated in proteinase and washed as previously described (Huang et al., 1999). After serial ethanol dehydration incubations (Huang et al., 1999) slides were allowed to air dry. Slides were processed immediately for FISH when pretreatment was optimal (i.e. when nuclei appeared dark and flat). The previously described principles/steps (see above) for performance of FISH in dispersed germ cells were then applied to the slides.

Cellular diameters had been measured via CSLM–CAS prior to the performance of FISH. Calculating the cellular diameters and observing the position of the germ cells within the seminiferous tubule facilitated the identification of germ cells (see above). The presence of 46,XY round germ cells lying near the basal membrane of the seminiferous tubule (normal spermatogonia) and the presence of 47,XXY round germ cells lying in the basal compartment of the seminiferous tubule (Klinefelter's spermatogonia) were evaluated. Sertoli cell nuclei were excluded from the analysis. The latter nuclei are easily recognized during observation via CSLM–CAS or after the performance of FISH because of their shape.

Confocal scanning laser microscopy of round germ cells or spermatozoa
Fractions of dispersed round germ cells with or without spermatozoa in the final filtrate were processed for observation via CSLM–CAS (Lasertec, Yokohama, Japan) prior to FISH. CSLM–CAS microscopy facilitated the identification of the spermatogenic stage of the observed round germ cells (see above). When spermatozoa were present in the dispersed testicular cells in the final filtrate, spermatozoal morphometric analysis was applied as previously described (Sofikitis et al., 1994Go, 1995Go). CSLM–CAS is a unique instrument in the field of microscopy because it allows observation of alive, undisturbed, non-fixed and non-stained cells under a large magnification (Sofikitis et al., 1994Go, 1998aGo). Therefore, the observed cells do not undergo any procedures that change their osmolarity and subsequently alter their shape and dimensions. The following morphometric parameters were evaluated (Sofikitis et al., 1994Go, 1995Go): the maximal length of the head (MLH), the maximal width of the head (MWH), the length of midpiece (LMP) and the ratio of the acrosomal region of the sperm head to the total surface area of the sperm head (100XARHS/TSH). The latter two morphometric parameters have been proven to have positive, strong and significant correlation with the sperm reproductive potential in IVF (Sofikitis et al., 1994Go).

ICSI procedures
When spermatozoa were found in testicular dispersed cells, they were analysed for quantitative motility (percentage of motile spermatozoa) and processed for ICSI. Details on the performance and the clinical outcome of ICSI procedures using spermatozoa from the participants of the current study have been presented in another communication (Yamamoto et al., 2001Go).

Obstructed azoospermic men
Nine men with obstructive azoospermia participating in an assisted reproduction trial underwent testicular biopsy at the day of oocyte retrieval. CSLM–CAS microscopy and FISH in dispersed testicular round spermatids were applied, together with morphometric analysis of the recovered spermatozoa. FISH analysis was stopped when a total of 902 round spermatid nuclei had been analysed.

Statistical analysis
Statistical analysis of sperm morphometric parameters and testicular ABP secretion rate was performed using Student's t-test when a normal distribution was present or Wilcoxon's test when a normal distribution was not present. Values were expressed as mean ± SD. A probability P < 0.05 was considered to be statistically significant. Differences in the proportions of round spermatids with a specific karyotype were analysed using {chi}2-test.


    Results
 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
Motility and morphometric parameters of recovered spermatozoa
Spermatozoa were found in the minced testicular samples of 12 men. The mean value of the percentage of motile spermatozoa was significantly smaller (P < 0.05) in the 12 men with Klinefelter's syndrome positive for spermatozoa than in obstructed azoospermic men (Table IGo). In eight men with Klinefelter's syndrome positive for testicular spermatozoa, all spermatozoa were immotile. There was no significant difference in the mean value of the MLH between men with obstructive azoospermia and men with Klinefelter's syndrome. However, the mean values of the 100XARHS/TSH ratio and the LMP were significantly smaller in men with Klinefelter's syndrome than in obstructed azoospermic men (Table IGo). In contrast, the mean value of the MWH was significantly larger in men with Klinefelter's syndrome (Table IGo).


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Table I. Motility and morphometric analysis of spermatozoa from men with Klinefelter's syndrome (KS) positive for testicular foci of haploid cells and from men with obstructive azoospermia (OA)
 
ICSI outcome
Outcome/details on ICSI techniques for the 12 men with Klinefelter's syndrome with testicular spermatozoa have been extensively described in another investigation (Yamamoto et al., 2001Go). The fertilization rate was 49%. Four cycles resulted in clinical pregnancies. One twin pregnancy and three single pregnancies were achieved. Two XY male and three XX female healthy newborns were delivered (Yamamoto et al., 2001Go).

Karyotypes of round spermatids (FISH in dispersed round spermatids)
Percentages of haploid, diploid and disomic round spermatids in men with Klinefelter's syndrome are shown in Table IIGo. The percentage of (23,X plus 23,Y)-haploid round spermatids in men with Klinefelter's syndrome was >93% (Table IIGo; Figure 3Go). The proportions of diploid (46,XX or 46,YY) round spermatids, or disomic-24,YY-round spermatids, or hypohaploid-(22,0)-round spermatids (nullisomic for sex chromosomes) to the total number of evaluated round spermatids in men with Klinefelter's syndrome were not significantly different compared with the group of obstructed azoospermic men. However, the ratio of hyperhaploid-24,XY-round spermatids to the total number of evaluated round spermatids and the ratio of hyperhaploid-24,XX-round spermatids to the total number of evaluated round spermatids were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Furthermore, the ratio of diploid-46,XY-round spermatids to the total number of evaluated round spermatids was significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. In the group of men with Klinefelter's syndrome, the observed ratio of 23,X-round spermatids to the number of haploid and normal round spermatids (1247/2245) was significantly larger than the expected ratio (1/2).


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Table II. Chromosomal patterns of round spermatids from men with Klinefelter's syndrome (KS) and obstructive azoospermia (OA)
 


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Figure 3. FISH in a seminiferous tubule from a man with non-mosaic Klinefelter's syndrome negative for haploid cells in minced testicular tissue. Only 47,XXY spermatogonia (type II seminiferous tubule; see Discussion) were observed. Pink/red, yellow or blue/green signals are characterized as p, y and b respectively. Prior to FISH techniques, all sections had been observed via CSLM–CAS and the maximal, minimal and oblique diameter of each germ cell were measured (Sofikitis et al., 1998aGo,cGo). CSLM–CAS microscopy had pre-identified: (i) spermatogonia/primary spermatocytes from their diameters/morphometry and their positions within the seminiferous tubule; and (ii) Sertoli cell nuclei from the presence of the characteristic Sertoli cell nucleus shape (Sofikitis et al., 1998aGo,cGo).


Number of cell Signals Presumed karyotype Comments

1 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus
2 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus
3 p,y,y,b,b 47,XXY1818 47,XXY Sertoli cell nucleus
4 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium/primary spermatocyte nucleus
5 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium/primary spermatocyte nucleus
6 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium/primary spermatocyte nucleus
7 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus

 
FISH in seminiferous tubules
Both 46,XY and 47,XXY spermatogonia were seen in the seminiferous tubules of the remaining 12 men with Klinefelter's syndrome who were positive for round spermatids/spermatozoa in the minced testicular samples (Figures 1 and 2GoGo). However, the number of 46,XY spermatogonia was always smaller than the number of 47,XXY spermatogonia in each evaluated seminiferous tubule of the latter 12 men. In all seminiferous tubule sections of the 24 men with Klinefelter's syndrome, all the Sertoli cell nuclei had karyotype 47,XXY. No 46,XY spermatogonia were found in the seminiferous tubule sections of the 12 men with Klinefelter's syndrome who were negative for round spermatids and spermatozoa in the minced testicular samples. In the seminiferous tubules of the latter men, only 47,XXY Sertoli cells and 47,XXY germ cells were seen (Figure 3Go). Few seminiferous tubules without both Sertoli cells and germ cells were found in the testicular fragments of each of the 24 participants (Figure 4Go).



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Figure 1. FISH in a seminiferous tubule from a man with non-mosaic Klinefelter's syndrome positive for haploid cells in minced testicular tissue. Both 47,XXY and 46,XY spermatogonia are present (type III seminiferous tubule; see Discussion). Pink/red, yellow or blue/green signals are characterized as p, y and b respectively. Prior to FISH techniques, all sections had been observed via CSLM–CAS and the maximal, minimal and oblique diameter of each germ cell were measured (Sofikitis et al., 1998aGo,cGo). CSLM–CAS microscopy had pre-identified: (i) spermatogonia/primary spermatocytes from their diameters/morphometry and their positions within the seminiferous tubule; and (ii) round spermatids and Sertoli cell nuclei from the presence of the round spermatid acrosomal granule and the characteristic Sertoli cell nucleus shape respectively (Yamanaka et al., 1997Go; Sofikitis et al., 1998aGo,cGo).


Number of cell Signals Presumed karyotype Comments

 1 p,y,y,b,b 47,XXY1818 Sertoli cell nucleus of 47,XXY karyotype
 2 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus
 3 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus
 4 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus
 5 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium nucleus
 6 p,y,b,b 46,XY1818 46,XY spermatogonium nucleus
 7 p,y,y,b,b 47,XXY1818 47,XXY primary spermatocyte nucleus
 8 p,y,y,b,b 47,XXY1818 47,XXY primary spermatocyte nucleus
 9 p,y,y,b,b 47,XXY1818 47,XXY primary spermatocyte nucleus
10 p,y,y,b,b 47,XXY1818 47,XXY Sertoli cell nucleus
11 p,b 23,Y18 23,Y18 haploid round spermatid nucleus
12 p,y,b 24,XY18 24XY hyperhaploid round spermatid nucleus

 


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Figure 2. FISH in dispersed testicular cells from a man with non-mosaic Klinefelter's syndrome positive for haploid cells in minced testicular tissue. Pink/red, yellow or blue/green signals are characterized as p, y and b respectively. Prior to FISH, all samples had been observed via CSLM–CAS. CSLM–CAS microscopy had pre-identified round spermatids from the presence of the round spermatid acrosomal granule and spermatogonia/primary spermatocytes from their diameters, i.e. morphometry (Yamanaka et al., 1997Go; Sofikitis et al., 1998aGo,cGo).


Number of cell Signals Presumed karyotype Comments

1 p,y,b 24,XY18 24,XY18 hyperhaploid round spermatid nucleus
2 p,b 23,Y18 23,Y18 haploid round spermatid nucleus
3 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium/primary spermatocyte nucleus
4 p,y,y,b,b 47,XXY1818 47,XXY spermatogonium/primary spermatocyte nucleus
5 p,b 23,Y18 23,Y18 haploid round spermatid nucleus
6 y,b 23,X18 23,X18 haploid round spermatid nucleus
7 y,b 23,X18 23,X18 haploid round spermatid nucleus
8 p,b 23,Y18 23,Y18 haploid round spermatid nucleus

 


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Figure 4. Empty seminiferous tubule (type I). Neither Sertoli cells nor germ cells are present.

 
Notes for Figures 1–3GoGoGo
It should be emphasized that the colours in the printed Figures 1, 2 and 3GoGoGo are weaker than those originally observed via the fluorescent microscope due to technical difficulties in printing small spots of different colours in photos. In addition, occasionally the colour spots (i.e. signals of chromosomes) in the printed figures have become larger or smaller than those originally observed during fluorescent microscopy.

Comparing CSLM–CAS images of seminiferous tubules prior to FISH with images of the same seminiferous tubules taken after FISH, we noticed that occasionally the position of the cells (with regard to the basal membrane) within a seminiferous tubule changes (in a small degree) when FISH is applied. Primary spermatocytes are considered to arise from spermatogonia that lose their contact with the basal membrane. Considering that the position of germ cells within the seminiferous tubule changed to a small degree when FISH was applied, we did not attempt to distinguish between spermatogonia and primary spermatocytes in most of the cases in the current study. However, when a 46,XY or 47,XXY germ cell nucleus was obviously away from the basal membrane and the respective cell satisfied the criteria for spermatogonia/primary spermatocytes (see Materials and methods), we gave the characterization of the primary spermatocyte nucleus. In contrast, when a 46,XY or 47,XXY germ cell was in contact with (or near) the basal membrane and the respective cell satisfied the criteria for spermatogonia/primary spermatocytes, it was characterized as spermatogonium.

In general, most primary spermatocytes are considered to be at the pachytene stage. During the pachytene stage of meiosis I of normal 46,XY spermatogonia, two closed signals for X and Y chromosomes and one large (paired) signal for the 18 chromosome bivalent are usually anticipated. In some photos that we have not exhibited in the current version of the manuscript, the above pattern of signals was found for normal primary spermatocytes (i.e. late zygotene and pachytene stage of first meiosis). We have chosen Figures 1–3GoGoGo for presentation because, in these figures, all 46,XY and 47,XXY primary spermatocytes expose two different signals for the autosomal 18 chromosomes, indicating that it is not rare for the majority of primary spermatocytes within a seminiferous tubule section of a man with Klinefelter's syndrome to not be in the pachytene stage.

Hormonal profiles
Mean peripheral serum levels of FSH in men with Klinefelter's syndrome were 14–56 IU/l. Normal values of FSH for males of reproductive age in our facilities are considered to range from 3–11 IU/l. There were no significant differences in peripheral serum FSH profiles between the 12 men with Klinefelter's syndrome positive for spermatozoa and the 12 men with Klinefelter's syndrome negative for spermatozoa.

ABP secretion from testicular tissue in vitro
ABP secretion from testicular tissue was evaluated in eight of the 12 men with Klinefelter's syndrome who were negative for haploid cells and in six men from the 12 with Klinefelter's syndrome who were positive for haploid cells. ABP secretion profiles were significantly smaller (Table IIIGo) in men with Klinefelter's syndrome negative for haploid cells in the minced testicular tissue than in men with Klinefelter's syndrome positive for haploid cells. All eight men with Klinefelter's syndrome who were negative for haploid cells and evaluated for ABP secretion had smaller ABP profiles than each of the six men positive for spermatozoa. In other words, among 14 men with Klinefelter's syndrome, those eight with the greater degree of Sertoli cell secretory dysfunction were negative for spermatozoa.


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Table III. Secretion of androgen-binding protein (ABP) in vitro by testicular tissue of men with Klinefelter's syndrome (KS)
 

    Discussion
 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
Gonotypes of offspring fathered by men with Klinefelter's syndrome
The presence of, (i) large proportions of hyperhaploid 24,XY or 24,XX round spermatids and (ii) increased frequency of a 46,XY chromosomal pattern in round spermatids and subsequently in spermatozoa of men with Klinefelter's syndrome raise the probability of generation of chromosomally abnormal embryos when spermatozoa are processed for ICSI. On the other hand, all the 14 neonates that have been reported in the literature by other investigators following ICSI using spermatozoa from patients with non-mosaic Klinefelter's syndrome are normal (Bourne et al., 1997Go; Tournaye et al., 1997; Palermo et al. 1998Go; Reubinoff et al., 1998Go; Nodar et al., 1999; Ron-El et al., 1999Go, 2000Go). In addition, the five offspring in the current study were normal. Only one fetus conceived by spermatozoa recovered from a man with non-mosaic Klinefelter's syndrome was of 47,XXY karyotype in another study (Ron-El et al., 2000Go). The latter embryo was reduced at week 14 (Ron-El et al., 2000Go). The high proportion of normal fetuses that develop from the implantation of embryos generated by the fertilization of oocytes by spermatozoa recovered from men with Klinefelter's syndrome may be due to, (i) the large percentage of normal (23,X or 23,Y) haploid cells (among a population of 2400 testicular round spermatids in the current study >93% had a normal haploid karyotype; these spermatids are expected to produce normal haploid spermatozoa) and (ii) the presence of an endometrial filter impeding the implantation of chromosomally abnormal embryos.

Do sex chromosomal non-disjunctions occur during meiosis I or II of 46,XY primary spermatocytes of men with Klinefelter's syndrome?
The current study is consistent with previous studies (Chevret et al., 1996Go; Guttenbach et al., 1997Go) showing an increased incidence of 24,XY spermatozoa in men with Klinefelter's syndrome compared with chromosomally normal men. Increased frequency of 24,XY composition was found in round spermatids from men with Klinefelter's syndrome compared with obstructed azoospermic men. The incidence of 24,XX round spermatids in men with Klinefelter's syndrome was also significantly higher than expected. These increases in the frequencies of 24,XY and 24,XX round spermatids surely cannot be explained by meiotic non-disjunction of sex chromosomes in normal testicular 46,XY primary spermatocytes. Non-disjunction during meiosis I should produce equal numbers of 24,XY round spermatids and round spermatids nullisomic for the sex chromosomes (Chevret et al., 1996Go; review). The findings presented in Table IIGo are inconsistent with similar increases in 24,XY round spermatids and round spermatids nullisomic for the sex chromosomes (hypohaploid) and subsequently do not support non-disjunction of sex chromosomes during meiosis I of normal 46,XY primary spermatocytes.

Non-disjunction during meiosis II of normal 23,X-(diploid DNA)-secondary spermatocytes should produce equally increased frequencies of 24,XX round spermatids and round spermatids nullisomic for the sex chromosomes. Non-disjunction during meiosis II of normal 23,Y-(diploid DNA)-secondary spermatocytes should produce equally increased frequencies of 24,YY round spermatids and round spermatids nullisomic for the sex chromosomes (Guttenbach et al., 1997Go; Bielanska et al., 2000Go). Thus, when sex chromosomal non-disjunction occurs during both second meiotic divisions of one normal 46,XY primary spermatocyte (formation of four round spermatids from two secondary spermatocytes), one 24,XX round spermatid, one 24,YY round spermatid and two hypohaploid (nullisomic for sex chromosomes) round spermatids are produced. The findings in Table IIGo do not support non-disjunction during meiosis II of normal 46,XY primary spermatocytes. The proportion of hypohaploid round spermatids to the total number of evaluated round spermatids was significantly smaller than the proportion of 24,XX round spermatids plus 24,YY round spermatids to the total number of evaluated round spermatids. It is suggested that it is more likely that the hyperhaploid 24,XY round spermatids and 24,XX round spermatids are produced by regular meiosis of the 47,XXY primary spermatocytes.

Controversy on the karyotypes of spermatogonia in men with Klinefelter's syndrome
FISH performed on seminiferous tubules showed that in men with Klinefelter's syndrome, who are positive for round spermatids and spermatozoa in minced testicular tissue, the number of 47,XXY spermatogonia is larger than the number of 46,XY spermatogonia in the seminiferous tubule. However, the vast majority of round spermatids in the minced testicular tissue are haploid and normal. Thus, it appears that only a few 47,XXY spermatogonia undergo meiosis. Alternatively, it may be suggested that spermatocytes or spermatids derived from the latter cells undergo apoptosis. Another interesting finding is that in each man with Klinefelter's syndrome without round spermatids and spermatozoa in the minced testicular tissue, 46,XY spermatogonia were not present in the seminiferous tubule sections evaluated (i.e. only 47,XXY spermatogonia were present). It appears that the lack of 46,XY spermatogonia in the seminiferous tubule has a negative prognosis for the presence of round spermatids and spermatozoa in the minced testicular tissue. The latter thesis is consistent with the hypothesis that most of the 47,XXY spermatogonia do not undergo meiosis and subsequently, if 46,XY spermatogonia are absent in the seminiferous tubule, there may be only a small chance of finding foci of 47,XXY cells undergoing meiosis/advanced spermatogenesis and spermiogenesis in the testicle. Although, in the current study, all 12 men (with Klinefelter's syndrome) with seminiferous tubules negative for 46,XY spermatogonia were negative for spermatids/spermatozoa in the minced testicular tissue, the probability that, in general, few men with Klinefelter's syndrome who exhibit only 47,XXY spermatogonia in their seminiferous tubules are positive for testicular foci of spermatozoa cannot be ruled out.

It should be mentioned that the international literature provides both evidence for and against the presence of XXY spermatogonia in men with Klinefelter's syndrome (Mroz et al., 1998Go; review). To solve the controversy on the above dilemma, we applied FISH techniques to fragments of seminiferous tubules. This technique vividly showed that XXY spermatogonia exist in seminiferous tubules from men with non-mosaic Klinefelter's syndrome and that their number is larger than the number of 46,XY spermatogonia within each seminiferous tubule section. In fact, it appears that there are three types of seminiferous tubules in men with Klinefelter's syndrome: type I seminiferous tubules are empty (both Sertoli cells and germ cells are absent; Figure 4Go); type II seminiferous tubules contain Sertoli cells and are positive for 47,XXY spermatogonia and negative for 46,XY spermatogonia (Figure 3Go); type III seminiferous tubules contain Sertoli cells and are positive for both 47,XXY spermatogonia and 46,XY spermatogonia (Figure 1Go).

Type of pairing of sex chromosomes in 47,XXY primary spermatocytes
Considering that our findings do not indicate sex chromosome non-disjunctions during meiosis I or II of 46,XY primary spermatocytes in men with non-mosaic Klinefelter's syndrome (see above) and subsequently similar numbers of 23,X round spermatids and 23,Y round spermatids are expected to have been produced from the meiosis of the normal 46,XY primary spermatocytes, (i) the excess of hyperhaploid 24,XY round spermatids and (ii) the increased proportion of 23,X round spermatids compared with the 23,Y round spermatids in men with Klinefelter's syndrome in our study may be attributable to a regular meiosis of some of the 47,XXY primary spermatocytes with an XX pairing and a univalent Y.

XX pairing and a univalent Y type of pairing in 47,XXY primary spermatocytes is expected to result in increased proportions of 23,X round spermatids/spermatozoa and 24,XY round spermatids/spermatozoa (post-meiosis) in the testicles of men with Klinefelter's syndrome (Chevret et al., 1996Go; Mroz et al., 1998Go). This model is consistent with the findings of the current study. In contrast, if an XY sex vesicle and a univalent X type of pairing had been present in 47,XXY primary spermatocytes, regular segregation of the sex chromosomes would have resulted in increased proportions of 23,Y round spermatids/spermatozoa and 24,XX round spermatids/spermatozoa (post-meiosis) in the testicles of men with Klinefelter's syndrome in our study (Chevret et al., 1996Go; review). This model cannot explain the decreased proportion of 23,Y-round spermatids compared with 23,X-round spermatids in our study. In addition, this model cannot explain the results published by Chevret and co-workers for a man with a mosaic Klinefelter's syndrome (Chevret et al., 1996Go). Furthermore, no such pairing has been observed in dispersed cells from testicular biopsy material of men with Klinefelter's syndrome (Skakkebaek et al., 1969Go; Luciani et al., 1970Go; Vidal et al., 1984Go). However, the possibility that, within the population of 47,XXY primary spermatocytes that undergo meiosis, a small subpopulation exposes an XY sex vesicle and a free extra X chromosome cannot be ruled out. An attractive speculation is that an XX pairing and a univalent Y chromosome type of pairing occurs in the great majority of 47,XXY primary spermatocytes that undergo meiosis, whereas an XY pairing and a univalent X chromosome type of pairing occurs in the minority of 47,XXY primary spermatocytes that undergo meiosis. This speculation can explain, (i) the increased proportions of XY and XX round spermatids in men with Klinefelter's syndrome compared with obstructed azoospermic men; (ii) the increased proportion of XY round spermatids compared with XX round spermatids in men with Klinefelter's syndrome; and (iii) the larger proportion of X round spermatids compared with Y round spermatids in men with Klinefelter's syndrome. The preferential pairing of homologous sex chromosomes in primary spermatocytes with three gonosomes has previously been proposed by Hulten and Tettenborn and co-workers by meiotic I chromosome analysis of 47,XYY men (Tettenborn et al., 1970Go; Hulten, 1979Go).

Sperm morphometry
In the current study, we evaluated four morphometric parameters of spermatozoa from men with Klinefelter's syndrome. Some of these parameters have positive, strong and significant correlations with sperm function. LMP is known to correlate positively, significantly and strongly with sperm motility, and the 100XARHS/TSH ratio correlates positively, significantly and strongly with the sperm acrosin content (Sofikitis et al., 1994Go, 1995Go). In contrast, MWH is considered to have a significant and negative correlation with the sperm fertilizing capacity (Sofikitis et al., 1994Go). The significantly smaller values of LMP and 100XARHS/TSH ratio and the significantly larger values of MWH in spermatozoa of men with Klinefelter's syndrome compared with obstructed azoospermic men indicate defects in the sperm organelles and morphometry and subsequently in sperm function. Although the latter defects in sperm functional characteristics, (i) would have been of clinical importance only if IVF methods had been applied (using ejaculated spermatozoa) and (ii) may not affect ICSI outcome, they definitely indicate that the development of sperm acrosome, head and midpiece do not proceed normally in men with Klinefelter's syndrome.

Sertoli cell secretory function
The ABP secretion in the 14 men with Klinefelter's syndrome in whom an ABP assay was performed was much smaller compared with the results previously reported for a group of healthy non-smokers with inguinal hernia and a group of smokers with inguinal hernia (Sofikitis et al., 1995Go). The current study indicates that there is a decrease in testicular ABP secretion in vitro and subsequently a Sertoli cell secretory dysfunction in men with non-mosaic Klinefelter's syndrome. In addition, men with Klinefelter's syndrome who were negative for spermatozoa had a significant decrease in their ABP profiles compared with men with Klinefelter's syndrome who were positive for spermatozoa (Table IIIGo). Since it is known that optimal Sertoli cell secretory function is important for the process of spermatogenesis, defects in Sertoli cell secretory function may further impede focal spermatogenesis in some men with non-mosaic Klinefelter's syndrome. The fact that among 14 men with Klinefelter's syndrome, those eight with the greater degree of Sertoli cell secretory dysfunction were negative for testicular spermatids and spermatozoa in the minced testicular tissue and did not exhibit 46,XY spermatogonia in their seminiferous tubules may suggest that the absence of normal 46,XY spermatogonia has a negative effect on Sertoli cell function. Germs cells are known to regulate Sertoli cell function (Sharpe, 1989Go). Thus, the current study reveals two mechanisms probably contributing to the presence or absence of foci of spermatozoa in subpopulations of men with Klinefelter's syndrome: (i) men who exhibit both 46,XY and 47,XXY spermatogonia in their seminiferous tubule have a greater probability for testicular foci of spermatozoa than men who exhibit only 47,XXY spermatogonia type in their seminiferous tubule; and (ii) among men with non-mosaic Klinefelter's syndrome, those with a high degree of Sertoli cell secretory dysfunction have a poor prognosis for having testicular foci of spermatozoa.


    Acknowledgements
 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank the Japanese Special Reference Laboratory for assisting us in the evaluation of ABP profiles and the performance of FISH in a large number of samples. In addition, we would like to thank Dr Nikolaos Kalinderis for statistically analysing our data.


    Notes
 
* A part of this study was presented at The 95th Annual Meeting of The American Urological Association in Atlanta, Georgia, USA from April 29, to May 4, 2000 Back

4 To whom correspondence should be addressed at: the Department of Urology, Tottori University School of Medicine,36 Nishimachi, Yonago 683, Japan. E-mail: akrosnin{at}hotmail.com Back


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 Top
 Abstract
 Introduction
 Participants and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on April 2, 2001; accepted on November 19, 2001.