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Regular Article |
a Institut für Humangenetik, Universität Würzburg, Biozentrum, Am Hubland, 97074 Würzburg, Germany
b Klinik und Poliklinik für Dermatologie und Allergologie, 80802 München, Germany
c Institut für Humangenetik, Universität Göttingen, 37073 Göttingen, Germany
ABSTRACT
In order to evaluate a possible paternal age effect, testicular sperm cells from three men aged 81, 82, and 83 yr were analyzed by two-color- and three-color-fluorescence in situ hybridization for disomy rates of chromosomes 1, 17, 18, X, and Y as well as for diploidy frequencies. A minimum of 1500 sperm cells per donor and probe was evaluated due to the low number of spermatozoa in the preparations. Diploidy and disomy frequencies were in the same range as found in men aged <30 yr, a slight increase only being noticed for XY nuclei.
aging, meiosis, sperm
INTRODUCTION
Aneuploidy is the most frequently detected chromosomal abnormality in human liveborns and in spontaneous abortions. In the vast majority (>90%) of autosomal trisomies 13, 18, and 21 as well as for the 47,XXX condition, a maternal origin of the additional chromosome can be shown. In contrast, about 50% of 47,XXY cases and all 47,XYY cases are due to paternal meiotic nondisjunction [1–3]. While it is well known that the risk for trisomic offspring increases with advanced maternal age, a paternal age effect has been discussed controversially [4–8]. Using fluorescence in situ hybridization (FISH), several studies addressed the topic of a paternal age effect by analysis and comparison of disomy rates in ejaculated spermatozoa from young (<30 yr) and older donors (50 to >60 yr) [9–13]. Assuming a possible age effect to be more pronounced in even older men, in the present manuscript for the first time we analyzed testicular sperm cells from men aged over 80 yr for disomy rates of chromosomes 1, 17, 18, X, and Y as well as for diploidy.
MATERIALS AND METHODS
Testis Biopsies
Testicular tissue was obtained from three men aged 81, 82, and 83 yr. The testes were extirpated in the course of treatment for prostate carcinoma and showed normal testicular histology. All men gave their informed consent prior to the study. The testes were cut into pieces and fixed in methanol:glacial acetic acid (3:1).
Testis Preparations
Very small pieces of the fixed testes were transferred into a drop of 50% acetic acid placed on a slide on a heating plate at 45°C. By pipetting up and down, cells were dissociated from the tubules and fixed on the slide. Under phase-contrast optics, the slides were controlled for the presence of adequate numbers of spermatozoa.
Probes for DNA
The DNA probes pUC1.77 [14] and alphasatellite 17 (biotinylated; Appligene ONCOR, Illkirch, France) were used to detect chromosome 1q12 and chromosome 17cen, respectively. The probe pUC1.77 was labeled by nick translation with digoxigenin-16-dUTP. For simultaneous detection of chromosomes 18, X, and Y, probe mixture 1 (AneuScreen-Kit; Vysis, Stuttgart, Germany), containing the fluorochrome-labeled 
-satellite DNA probes Spectrum Green CEP X, Spectrum Orange CEP Y, and Spectrum Aqua CEP 18, was applied.
In Situ Hybridization
Prior to in situ hybridization, the testis preparations were treated for 5–10 min with pepsin (250 µg/100 ml 0.01 M HCl) at 37°C. Subsequently, the slides were washed in 1x PBS and passed through an alcohol series (70%, 85%, and 100% ethanol). After air drying, the preparations were denatured in 70% formamide/2x SSC (saline-sodium citrate; 1x SSC is 0.15 M NaCl, 0.015 M sodium citrate) at 70°C for 2–5 min or, alternatively, in 2 N NaOH at room temperature. For two-color FISH (chromosomes 1/17), probe preparation, hybridization, and detection with avidin-tetramethylrhodamine isothiocyanate (TRITC) and mouse-anti-digoxigenin-fluorescein isothiocyanate (FITC) were performed as described previously [15]. Three-color FISH (chromosomes 18/X/Y) was carried out as recommended by Vysis.
Scoring of Sperm Cells and Analysis
Slides were screened on a Zeiss Axiophot equipped with an FITC/rhodamine double bandpass filter, an aqua filter, and a 4',6-diamidino-2-phenylindole (DAPI) filter. In double hybridizations chromosome 1 yielded a green hybridization signal and chromosome 17 a red one. Triple hybridizations resulted in a green signal for chromosome X, a red signal for the Y chromosome, and a blue one for chromosome 18. Analysis was restricted to well-delineated spermatozoa. Sperm nuclei with indistinct margins or those with indistinct, diffuse signals were excluded from examination. Due to the low number of spermatozoa present in the preparations, the minimum number of cells to be evaluated was set at 1500. Sperm nuclei were classified as disomic for a specific chromosome if hybridization resulted in two equal-sized signals, separated from each other by a distance of at least one signal diameter. Diploid sperm cells displayed two signals for each autosome or two gonosomal and two autosomal signals.
RESULTS
Testis preparations of the three patients exhibited low but sufficient numbers of spermatozoa to warrant hybridization. As compared to FISH on ejaculated sperm nuclei, hybridization efficiency in testicular sperm cells was reduced (about 80% vs. 95–99%). Changes in denaturation time and/or pretreatment of slides did not increase the efficiency consistently. Thus, only positively hybridized, well-delineated spermatozoa were included in the analysis (Fig. 1). In total, 12 586 spermatozoa from the three individuals were evaluated (Table 1). The average diploidy frequency was 0.1% ranging from 0.06% to 0.11%. Average disomy 1 and 17 determined in 6940 nuclei were 0.1% and 0.07%, respectively. Disomy frequencies of the sex chromosomes and chromosome 18 established in 5646 spermatozoa yielded rates of 0.11% XX, 0.11% YY, 0.18% XY, and 0.12% disomy 18.
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DISCUSSION
While advanced maternal age represents a well-documented risk for increased maternal meiotic nondisjunction, an age effect on paternal aneuploid germ cell production has been discussed controversially [3]. Because the paternal contribution to autosomal trisomies is low and early selection against unbalanced conceptions occurs, it is difficult to evaluate the actual correlation between paternal age and meiotic nondisjunction by screening aneuploid offspring.
Thus, in the past few years several studies set out to look for a possible age effect in meiotic nondisjunction by direct analysis of human mature spermatozoa. Analyzing human sperm chromosome complements after in vitro fertilization of golden hamster eggs, Martin and Rademaker [16] observed a highly significant increase of structural chromosome abnormalities with advanced donor age but found a significantly negative correlation between donor age and hyperhaploidy. These data, however, were based on relatively few sperm chromosome complements per individual (average 53). In three subsequent studies of this group [9, 11, 12] the question of a paternal age effect was addressed by FISH analysis of ejaculated spermatozoa. Martin et al. [9] determined diploidy and disomy frequencies of chromosomes 1, 12, X, and Y in over 225 000 sperm cells from 10 probands aged 21 to 52 yr and observed a significant increase in disomy 1 and Y with advanced donor age. The significantly increased rate of YY spermatozoa was confirmed by Kinakin et al. [11].
Again no correlation between proband age and XX or XY disomy nor diploidy was found. Spermatozoa from the same donors were analyzed by McInnes et al. [12] for disomy 1, 13, and 21. In contrast to the earlier study of Martin et al. [9], no increase of disomy 1 with donor age was observed. Disomy frequencies for chromosomes 13 and 21 were not found to be age related in this study. Rousseaux et al. [13], on the other hand, discuss a possible age effect on nondisjunction rates of chromosome 21, because two of their three probands aged >60 yr tested for chromosomes 14 and 21 exhibited higher disomy 21 frequencies as compared to men <30 yr. Similarly, Griffin et al. [10] demonstrated the highest frequencies of chromosome 18 disomy in their oldest donors (50–60 yr). However, neither the incidence of disomy 18 nor of diploidy were significantly increased. The XX and XY disomy were found in significantly higher rates in old donors as compared to young ones. In contrast to the data of Kinakin et al. [11] for YY disomy, only a borderline effect was observed. In the present investigation due to the scarcity of spermatozoa in the testes preparations, rather few cells (minimum 1500) could be analyzed. Furthermore, the hybridization efficiency in these testicular sperm nuclei did not exceed 
80%, although different treatments were tested in order to improve this rate. Joseph et al. [17] also reported reduced hybridization levels in testicular spermatozoa. As possible reasons the different maturation stages and the accessibility of the hybridization probe to sperm chromatin were discussed.
For all three males examined in the present study, autosomal disomy frequencies as well as diploidy rates were within the same range as those observed in ejaculated spermatozoa from donors below the age of 30 [18, 19]. Although in the above-mentioned studies somewhat elevated disomy frequencies were observed for some of the autosomes analyzed in older donors, the rates also did not reach statistical significance.
For the sex chromosomes, the data are somewhat controversial: while Kinakin et al. [11] found an age effect of YY disomy only, Griffin et al. [10] observed an increase especially of XX and XY spermatozoa. From our data a slight increase might only be inferred for XY disomy, with XX and YY disomy rates equaling those of young men. However, this might at least be partially explained by the low number of analyzable sperm cells and/or lower hybridization efficiency in our preparations. Overall, the differences observed for the sex chromosomes in the different studies might as well be due to interindividual variation.
Taken together, the data suggest that the paternal age effect on meiotic nondisjunction appears to be a minor one, affecting preferentially the sex chromosomes. Nevertheless, investigation of more old donors and larger numbers of spermatozoa is indicated before drawing any final conclusions.
FOOTNOTES
First decision: 8 November 1999.
1 Correspondence: Michael Schmid, Department of Human Genetics, University of Würzburg, Biozentrum, Am Hubland, 97074 Würzburg, Germany. FAX: 49 931 8884069; m.schmid{at}biozentrum.uni-wuerzburg.de 
Accepted: July 20, 2000.
Received: October 7, 1999.
REFERENCES
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  S.S. Young, B. Eskenazi, F.M. Marchetti, G. Block, and A.J. Wyrobek The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men Hum. Reprod., May 1, 2008; 23(5): 1014 - 1022. [Abstract] [Full Text] [PDF]
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 B. Kuhnert and E. Nieschlag Reproductive functions of the ageing male Hum. Reprod. Update, July 1, 2004; 10(4): 327 - 339. [Abstract] [Full Text] [PDF]
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 N Rives, G Langlois, A Bordes, N Simeon, and B Mace Cytogenetic analysis of spermatozoa from males aged between 47 and 71 years J. Med. Genet., October 1, 2002; 39(10): e63 - 63. [Full Text] [PDF]
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B. Eskenazi, A.J. Wyrobek, S.A. Kidd, X. Lowe, D. Moore II, K. Weisiger, and M. Aylstock Sperm aneuploidy in fathers of children with paternally and maternally inherited Klinefelter syndrome Hum. Reprod., March 1, 2002; 17(3): 576 - 583. [Abstract] [Full Text] [PDF]
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