Meiotic and synaptonemal complex studies in a 14/2 1 translocation carrier

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1 INTERNATIONAL JOURNAL OFANDROLOGY 5 (1982) Department of Cell Biology', Faculty of Science, Department of Biologya, Faculty of Medicine, Fundacidn Puigvert3 and Instituto & Bwlogia FundumentuP, Uniumsidad Autdnoma de Barcelona, Barcelona, Spain Meiotic and synaptonemal complex studies in a 14/2 1 translocation carrier BY F. VidalV, C. Templado %4, J. Navarr~~,~, S. Manna3 and J. Ego~cue'9~ Meiotic studies in a sterile carrier of a 14/21 translocation showed a meiotic arrest, with degeneration of primary spermatocytes. Silver staining of pachytene cells revealed the presence of a trivalent. Its synaptonemal complex was quite similar to that described in previous light and electron microscopy studies in mammalian species. The observation of a trivalent with pairing in trans-configuration and the presence of desynaptic synaptonemal complexes in early pachytene could explain the relatively high incidence of non-disjunction in human D/G translocations and the spermatogenic disintegration sometimes seen in these cases. Key worh: synaptonemal complex - meiotic studies - centric fusion. The most common human translocation in parents of children with Down's syndrome involves chromosomes 14 and 21. Its incidence in newborns is about 0.01% (Hook et al. 1977). Approximately 1.5% of Down's syndrome cases are due to the abnormal segregation of the trivalent at meiosis. However, although the number of meiotic studies in translocation camers has been considerable (Kjessler 1966; Hecht et al. 1970; Chandley et al. 1972; Luciani et al. 1972; Laurent et al. 1973; Hultkn 1973; Koulischer & Schoysman 1974) no data are available on the pairing geometry of the multivalents, and no synaptonemal complex studies have ever been carried out in these patients. This lack of Received on April 29th,

2 information is important because, according to recent animal data (Moses et al. 1979; Elder 8c Pathak 1980) the risk of non-disjunction may vary considerably depending on the pairing characteristics of the chromosomes at meiosis. In this paper we report the meiotic and synaptonemal complex studies carried out in a sterile male with a 14/2 1 centric fusion. Materials and Methods The patient was a sterile, 27-year-old Caucasian male attending the infertility clinic of the Fundacibn Puigvert. The spermiogram showed oligoasthenoteratozospermy. G-banded mitotic metaphases (Gallimore 8c Richardson 1973) showed a 14/21 centric fusion. A small testicular biopsy was obtained under local anaesthesia. Part of it was collected in hypotonic solution (KC M), and regular meiotic preparations were made using the technique of Evans et al. (1964). Another portion of the biopsy was collected in F-10 medium with 20% foetal calf serum and processed for synaptonemal complex studies using a modification of the techniques of Fletcher (1979) and Pathak et al. (1980). The cells were spread on a 0.2 M sucrose surface and picked up by touching it with a clean glass slide. After allowing the cells to settle on the slide they were fixed in formaldehyde (adjusted to ph 9.0, using 1.0 N sodium hydroxide). After 5-10 min the slide was washed for a few seconds in 0.4% photoflo (Kodak) and allowed to dry at room temperature. Staining was carried out in a 50% AgN03 solution in deionized water which also contained 0.03% formalin. Slides were incubated in a moist chamber for 2 h at 65 C. Results The meiotic preparations contained a large number of prophase I figures, but only 3 diakineses could be found, and all of them appeared incomplete and with a clear degeneration of the bivalents. Synaptonemal complex (SC) studies by light microscopy revealed the presence of a trivalent in all well spread figures (Fig. 1). In 99 profase figures, pairing of the trivalent was in the cis configuration (Figs. 1 and 2a, b, c); only one cell showed pairing in trans (Fig. 2d). Pairing of the centromeric ends of the acrocentric elements of the trivalents was incomplete, as described in some stages of prophase I by Moses et al. (1979) and by Elder 8c Pathak (1980) (see Fig. 3). Some of the early pachytene figures contained desynaptic complexes. 22

3 Fig. 1. SC, in a prophase I cell. The trivalent is easily identifiable (box). Pairing of the acrocentric elements is incomplete (X 1500). Fig. 2. a), b) and c): silver stained trivalents with pairing in cis; arrows point to telomeres. d): trivalent with pairing in trans; although the image can correspond to twisting of incompletely paired trivalent (see Discussion). 23

4 - NORMAL Chromosomea -- TRANSLOCATION CARRIER - Cis Trans -m Fig. 3. carrier of a 14/21 translocation. Schematic representation of incomplete meiotic pairing (SCs) in normal individual and a Discussion The possibility to analyse in some detail trivalent or multivalent associations by light microscopy of silver stained pachytene cells (Elder & Pathak 1980) may be valuable for the study of human subfertility, because in subfertile patients the amount of testicular material that can be used for cytogenetic studies is restricted (part of the biopsy is for histology). The number of divisions in suitable stages may also be low, and the information provided by classical meiotic preparations is limited, specially in cases such as our patient who, as sometimes occurs in translocation carriers (Chandley 1979), showed a partial meiotic arrest with degeneration of the primary spermatocytes. As shown by Moses et al. (1979), lemur hybrids heterozygous for as many as 5 centric fusions are fertile due to the fact that all trivalents pair in cis, thus probably predisposing for a comparable arrangement of the kinetochores in the metaphase plate, and hence a normal/balanced segregation of the chromosomes involved in the translocation. The same happens in the cotton rat Sigmodon fulviventer, where the heterozygous carriers of a 10/11 centric fusions always show pairing in cis (Elder & Pathak 1980). Although the trans configuration of acrocentric/metacentric elements may not be permissible in some cases, it is evident that in human carriers of centric fusions 24

5 there is a decrease in fertility (Chandley 1979) and unbalanced gametes are produced, as exemplified in the birth of children with Down s syndrome. The decrease in fertility may be due to a meiotic breakdown of unknown etiology (desynapsis?) sometimes observed in these individuals, and/or to miscarriage of unbalanced zygotes, while the production of unbalaned gametes could be due to pairing in trans, where the indifferent orientation of the fusion product would predispose to non-disjuntion. Since, as shown by Moses et al. (1979), pairing of the trivalents takes place from qter to pter of the acrodentrics and from the tips of the arms to the centromere of the fusion product, and the synaptonemal complexes show considerable twisting, the only thing that can be concluded from light or electron microscopy observations is that in lemur hybrids and cotton rats there is convergent co-orientation of the centromeres of the acrocentrics which, of course, also pre-dispose to normall balanced segregation of the trivalent; it is necessary to observe completely paired trivalents, in order to conclude that there is convergent co-orientation of the acrocentrics. In cases with only incomplete pairing, a twist of the trivalent may give the impression that pairing is in trans, an objection that can reasonably be made to our Fig. 2d. From a clinical point of view, D/G translocation male carriers are usually given an empirical recurrence risk for Down s syndrome of 2-3%. In our opinion, and taking into account that meiotic (Templado et al. 1980) and synaptonemal complex studies (Vidal et al. in press) can now be carried out in ejaculated cells, thus obviating the need for a testicular biopsy, genetic counseling of male translocation carriers may possibly in the future be improved and enhanced by such meiotic studies. Acknowledgments This investigation received financial support from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization. References Chandley A C, Christies S, Fletcher J, Frackiewicz A &Jacobs P A (1972): Translocation heterozygosity and associated subfertility in man. Cytogenetics 1 1 : 516. Chandley A C (1979): The chromosomal bases of human infertility. Br Med Bull 35: 181. Elder F F B & Pathak S (1980): Light microscopy observations on the behaviour of silver-stained trivalents in pachytene cells of Sigmodon fulviventer (Rodentia, Muridae) heterozygous for centric fusions. Cytogenet Cell Genet 27: 31. Evans E P, Breckon G & Ford C E (1964): An air-drying method for meiotic preparations from mammalian testes. Cytogenetics 3: 289. Fletcher J M (1979): Light microscope analysis of meiotic prophase chromosomes by silver staining. Chromosoma 72 :

6 Gallimore P M & Richardson C R (1973): An improved banding technique exemplified in the karyotype analysis of two strains of rat. Chromosoma 41 : 259. Hecht F, Delay M, Seely J R & Stoddard G R (1970): Meiotic evidence in Down s syndrome for 2 1/21 chromosome translocation or isochromosome. J Pediat 76: 298. Hook E B & Hamerton J L (1977): The frecuency of chromosome abnormalities detected in consecutive newborn studies. Differences between studies. Results by sex and by seventy of phenotypic involvement. In: Hook E B & Porter I H (eds). Population Cytogenetics. Studies in Humans. Academic Press, New York. Hultin M (1973): Cytogenetic aspects of human male meiosis. In: Harris H & Hirschhorn K (eds). Advances in Human Genetics, Vol4. Plenum Press, New York. Kjessler B (1966): Karyotype, meiosis and spermatogenesis in a sample of men attending an infertility clinic. Karger, Basel. Koulischer L & Schoysman R (1974): Chromosomes and human infertility. I. Mitotic and meiotic chromosome studies in 202 consecutive male patients. Clin Genet 5: 116. Laurent C, Papathanassiou Z, Haour P & Cognat M (1973): Facteurs gknetiques dans la stidit6 masculine. Lyon MCd 229: 883. Luciani J M, Mat& A & Stahl A (1972): La meiose dans les sterilitcs masculines. In: Thibault C (ed). FPcondite et st6rilite du mile. Acquisitions rccentes. Masson, Paris. Moses M J, Karatsis P A & Hamilton A E (1979): Synaptonemal complex analysis of heteromorphic trivalents in Lemur hybrids. Chromosoma 70: 141. Pathak S & Elder F B (1 980): Silver stained accessory structures on human sex chromosomes. Hum Genet 54: 171. Templado C, Marina S, Coll M D & Egozcue J (1980): Meiotic studies in human semen. Report of 180 cases. Hum Genet 53: 335. Vidal F, Ponsa M, Navarro J, Guitart M, Coll M D & Egozcue J: Estudi de complexes sinaptonemics en microscopia electronica. Bull SOC Cat biol (in press). Author s address: F. Vidal, Instituto de Biologia Fundamental, Universidad Autonoma de Barcelona, Avda. San Antonio Ma Claret, 171, Barcelona-25, Spain. 26