Hum Genet (1994) 93 : 32-34 human ..

genetics �9 Springer-Verlag 1994

Achievement of meiosis in XXY germ cells: study of 543 sperm karyotypes from an XY/XXY mosaic patient

J. Cozzi, E. Chevret, S. Rousseaux, R. Pelletier, V. Benitz, H. Jalbert, B. S~le

Cytogenetic and Reproductive Biology Laboratory, Grenoble University Medical School, F-38043 Grenoble, France

Received: 20 November 1992 / Revised: 1 June 1993

Abstract. Human sperm chromosomes from a 46,XY/ 47,XXY male were obtained using the technique of in vitro penetration of zona-free hamster eggs. The analysis of 543 sperm complements shows a significantly increas- ed incidence (0.9%) of hyperhaploid gonosomal 24,XY sets, with a lack of the expected corresponding gonosomal hypohaploidies, and a normal rate of autosomal non-dis- junctions. These results support the suggestion that 47,XXY cells are able to go through meiosis and to form spermatozoa. Only 24,XY sperm chromosomal constitu- tions were observed suggesting a preferential pairing of homologous sex chromosomes in 47,XXY spermatocytes.

Introduction

Klinefelter syndrome is one of the most common sex chromosomal abnormalities resulting in various degrees of dysfunction of spermatogenesis and infertility. Karyo- type 47,XXY and a mosaic constitution are usually asso- ciated with azoospermia or severe oligozoospermia. The gonadal defect in Klinefelter syndrome patients seems to be related to the chromosome constitution of germinal cells. The few meiotic studies of these patients (Kjessler 1966; Skakkebaek et al. 1969; Luciani et al. 1970; Dutril- laux et al. 1971; Laurent et al. 1973; Vidal et al. 1984) have shown the arrest of meiosis in the rare spermatocytes produced. Meiotic studies in patients with mosaic consti- tutions 46,XY/47,XXY suggest that only normal germ cells can complete meiosis (Kjessler 1966; Luciani et al. 1970; Laurent et al. 1973). However, to date, the sperm chromosome constitution of these patients has not been studied because of the frequent deficiency in spermatoge- nesis. Here, we report the first sperm chromosome analy- sis of an XY/XXY mosaic male using the technique of in vitro penetration of zona-free hamster eggs.

Materials and methods

The patient, a phenotypically normal 39-year-old man, was ascer- tained during routine karyotyping of voluntary sperm donors for IAD. He has two normal children. Cytogenetic analysis of his lym-

phocytes has revealed a mosaic karyotype 46,XY/47,XXY in the ratio 60:40. Two ejaculates were collected during a period of three weeks. Seminal analysis performed on both samples demonstrated normal parameters with a sperm density of 100 x 106/ml and 60% motility. Semen was frozen and stored in liquid nitrogen. Comple- ments were obtained after overnight capacitation of thawed sperm in test-yolk buffer at 4~ using the zona-free hamster oocytes penetration system. Details of sperm processing, egg collection, culture, fixation and chromosome analysis have been described previously by Brandriff et al. (1985).

Results

The frequencies of normal and abnormal sperm comple- ments and the type of sperm chromosome abnormalities are shown in Table 1. Out of 543 analysed chromosome metaphase spreads, 53 (9.8%) were abnormal, 37 (6.8%) were aneuploid, 15 (2.8%) had a structural abnormality and 1 (0.2%) complement presented both types.

Table 2 provides details of the sperm chromosomal ab- normalities. Of 37 aneuploid complements, five (0.9%) contained both X and Y gonosomes (Fig. 1), indicating a significant increase in sex chromosome hyperhaploidy (normal distribution), and two (0.4%) contained no gono- some. Autosomal chromosomal abnormalities were pre-

Table 1. Sperm chromosome complements from the 46,XY/ 47,XXY patient

Number of Frequency (%) complements

Normal 23,X 228 23,Y 262

Abnormal 53 Hyperhaploidy 5 Hypohaploidy 32

Structural abnormalities 15

Structural and numerical 1 abnormalities

42 48.25

9.76 0.92 5.9

2.75

0.18

Total 543 100 Correspondence to: B. Sble

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Table 2. Details and number of sperm complements with abnor- malities

Numerical Number Structural Number of spreads of spreads (32) (15)

Hypohaploid

22,X.-2 1 22,X,-8 2 22,X,-10 1 22.X,~ 1 22,X,-13 I 22,X,-14 1 22,X,-D 1 22,X,-16 2 22,X,-22 2 22.X,-G 1 21.X,-15,-17 1 21,X,-21.-22 1

22,-X or -Y 2

22,Y,-1 l 22,Y,-8 1 22,Y,-8,-11,+5 1 22,Y,-9 1 22,Y,-11 1 22,Y,-C 1 22,Y,-16 1 22,Y,-18 1 22,Y,-20 1 22,Y,-21 2 22,Y,-22 1 22,Y,-G 1 22,Y,-10,-20 1 22,Y,-17,-22 1

Hyperhaploid

24,XY 5

Numerical and structural

22,X,-6 +ace 1

23,X,csb(l)(p24) 1 23,X,cte(3,4) 1

(p23,q25)tr 23,X,csb(5)(ql 1) 1 23,X,csb(7)(q31 ) 1 23,X,csb(14)(ql 2) 1

23,Y,csb(1)(p21) 1 23,Y,csb(3)(pl 1) 1 23,Y,csb(3)(p13) 1 23,Y,csb(4)(q21) 1 23,Y,csb(6)(q25) 1 23,Y,csb(7)(q31 ) 1 23,Y,csb(8)(q21 ) 1 23,Y,csb(10)(q21) 1 23,Y,ctb(13)(q21) 1 23,Y,esb(15)(q14) 1

sent in 46 (8.5%) sperm complements: 26 (4.8%) sperm showed the absence of one chromosome, 4 (0.7%) sperm the absence of two chromosomes, 15 (2.8%) sperm showed a structural abnormality and 1 (0.2%) sperm showed both structural and numerical anomalies. There were 249 (46.5%) X-chromosome-bearing sperm and 287 (53.5%) Y-chromosome-bearing sperm (see Table 3); the sex ratio was thus not significantly different from the ex- pected 1 : 1 ratio (Chi square test, P > 0.05).

Discussion

Compiled data on sperm cytogenetics of normal males or individuals with constitutional chromosome aberrations (Benet et al. 1991; Estop et al. 1991, 1992; Martin 1991,

1992; Martin et al. 1991; Goldman et al. 1992; Spriggs et al. 1992) have shown that the frequeny of gonosomal non- disjunctions, during male meiosis I, is low with only 0.06% 24,XY sets out of 14162. Our analysis of 543 sperm complements in a 46,XY/47,XXY male shows a signifi- cantly increased incidence of hyperhaploid gonosomal 24,XY sets (0.9%), with a lack of the expected correspond- ing gonosomal hypohaploidies, and a normal rate of auto- somal non-disjunctions. These 24,XY sperm complements may theoretically originate either from first meiotic gono- somal non-disjunctions in the normal 46,XY germ cell line, or from regular meiosis in the 47,XXY germ cell line.

In individuals with such mosaicisms, it has been as- sumed that only normal 46,XY germ cells are able to progress through spermatogenesis (Steinberger et al. 1965; Luciani and Stahl 1978; Vidal et al. 1984). Thus, all the observed 543 sperm complements should result only from the meiosis of 46,XY germ cells. The frequency of gono- somal non-disjunctions could have been increased by the presence of the abnormal 47,XXY germinal cell line, which may result in the disturbance of XY pairing in nor- mal germ cells, with a subsequent production of 24,XY sperm complements. Indeed, Hogg and McLaren (1985) have reported a lack of the sex vesicle in mice ectopic spermatocytes. However, according to this hypothesis, such a disturbance would also be expected for autosomes. Moreover, sex chromosome non-disjunctions in normal 46,XY spermatocytes should result in the same proportion of gonosomal hypohaploid 22,0 complements, but only two such karyotypes were observed. This suggests that the observed 24,XY spermatozoa probably do not result f rom sex chromosome non-disjunctions in the normal 46,XY germinal cell line.

On the other hand, 47,XXY germ cells can initiate meiosis (Skakkebaek et al. 1969; Vidal et al. 1984). Our results support this observation and indicate that these cells may be able to achieve meiosis thereby producing 24,XY spermatozoa. The ability of 47,XXY germ cells to complete meiosis may partially be related to the propor- tion of normal cell line populations surrounding them. In- deed, in Klinefelter mosaic cases, the presence of normal cells decreases the severity of the clinical manifestations (Sarkar and Marimuthu 1983).

No 24,XX sperm complements were observed, suggest- ing a preferential pairing of homologous sex chromo- somes in XXY spermatocytes. This has previously been suggested by Skakkebaek et al. (1969) and later by Vidal et al. (1984) in meiotic studies of two males with 46,XY/ 47,XXY mosaicisms. Both studies have described, in 13 spermatocytes out of a total of 35 analysed, a 24 chromo- somal constitution with no sex vesicle and 22 autosomal bivalents. A sex vesicle with a free X chromosome was not observed in any of the remaining spermatocytes.

Preferential pairing of homologous sex chromosomes has also been reported in 47,XYY males (Hult6n and Pearson 1971; Speed et al. 1991). However, in most 47,XYY cases only 46,XY spermatocytes have been ob- served, supporting the hypothesis that the extra Y chro- mosome is lost before meiosis (Evans et al. 1970).

To date, only 75 sperm complements from a 47,XYY patient have been reported (Benet and Martin 1988). In

34

Fig, 1. R-banded human sperm comple- ment showing a 24,XY chromosome constitution

Table 3. Sex rationS: frequencies of X- and Y-bearing sperm

Number of complements Frequency

X-bearing sperm 249 46.5

Y-bearing sperm 287 53.5

Total 536 100

a Only complements with one sex chromosome were counted

th i s case , n o i n c r e a s e d sex c h r o m o s o m a l a b n o r m a l i t i e s w e r e o b s e r v e d . T h i s r e s u l t c o u l d b e e x p l a i n e d e i t h e r b y t he los t o f the e x t r a Y as p r e v i o u s l y m e n t i o n e d , o r b y the f a i l u r e to d e t e c t s u c h r a r e a b n o r m a l i t i e s f r o m s m a l l s a m - p les o f s p e r m k a r y o t y p e s .

Acknowledgements. We thank Martine LainE for taking care of the hamsters and Mary Callanan for assistance with the preparation of the manuscript. We acknowledge the support of the ComitE de Ra- dioprotection d'ElectricitE de France (EDF), Institut National de la SantE et de la Recherche MEdicale (INSERM: grant 894013) and Fondation pour la Recherche MEdicale (FRM).

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