On the origin and frequency of Y chromosome deletions responsible for severe male infertility

Transcription

1 Molecular Human Reproduction vol.3 no.7 pp , 1997 OPINION On the origin and frequency of Y chromosome deletions responsible for severe male infertility R.G.Edwards 1,3 and Colin E.Bishop 2 1 Churchill College, Cambridge, UK, and 2 Department of Obstetrics and Gynecology and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA 3 To whom correspondence should be addressed The origin of deletions associated with non-obstructive severe oligozoospermia in men are discussed. Deletions could arise during various stages of meiosis, at later stages in spermatids, or post-fertilization. Certain embryonic stages may be highly sensitive. The possibilities of an inherited propensity to these and other deletions, and of mosaicism in embryos are assessed. Key words: deletions/meiosis/mosaicism/oligozoospermia/post-fertilization deletions Introduction in others who inherited conditions variously described as maturation arrest and extreme severe oligozoospermia. The underlying genetic situation may be more complex than this, since more than one locus may be involved (Kobayashi et al., 1994). Three distinct interstitial deletions causing azoospermia or severe oligozoospermia could occur in Yq. They have been named AZFa, b and c (Vogt et al., 1996), and they occur in three distinct non-overlapping subregions of Yq11. AZFc coincides with DAZ while the others are located more proximally (Vogt et al., 1996). The three deletions affect distinct and separate phases of spermatogenesis and sperm development. AZFa and b are seemingly active before prolifera- tion or at meiosis respectively, and c is associated with a heterogeneous phenotype. The role of DAZ in causing azoospermia in men has been questioned, e.g. by Shan et al. (1996), on the basis that an autosomal gene expressed in the testis may impair spermatogenesis. It should be emphasized that the Y deletions which include DAZ are all very large (~1 1.5 mb). It remains a good possibility that other genes will be found in this region which also play a role, perhaps in association with DAZ, in male infertility. To date, no small interstitial deletions or mutations have been found within the DAZ gene (Vereb et al., 1997), so formal proof of its role is in fact lacking. An autosomal homologue of DAZ (DAZLA, DAZH or SPGYLA) located on human chromosome 3 (Saxena et al., 1996; Yen et al., 1996; Seboun et al., 1997), and shows high homology to the Drosophila spermatogenesis gene boule. Its role in mammalian spermatogenesisis is currently under investigation. It encodes an RNA binding protein and may be involved in the G 2 M transition. Some investigators even question whether AZF actually operates in germinal cells, since it may instead impair Sertoli cell functions in sustaining spermatogenesis. Evidence from polymerase chain reaction (PCR) and in-situ hybridization nevertheless indicates that it is transcribed only in germ cells in the testis (Menke et al., 1997; Niedeberger et al., 1997). Cytologically-detected deletions on the distal part of the Yq were first identified, and their possible role in causing azoospermia in infertile men first suggested, by Tiepolo and Zuffardi (1976). Later work revealed that microdeletions on the Y chromosome were also associated with infertility (Ma et al., 1992, 1993; Chandley and Cooke, 1994), and led to the suggestion that a gene, azoospermia factor (AZF), was involved in the regulation of spermatogonial development. Ma et al. (1992) proposed that a region containing genes they named as YRRM1 and YRRM2 was deleted in aspermatogenesis. This was later questioned since sequences in these regions were found to be identical and present in most of a group of azoospermic men (Reijo et al., 1995). Instead, a series of deletions in interval 6 of the human Y chromosome were identified as a potential cause of infertility in 12 out of a group of 89 men with azoospermia or very severe oligozoospermia. In all, ~13% of azoospermic men had de-novo deletions of interstitial or terminal portions of Yq which overlap the AZF region, indicating that ~1 in men is afflicted with the deletion (Reijo et al., 1995). This evidence implies that one or more genes needed for normal spermatogenesis must be present in this region of the Y chromosome. These deletions involved the loss of a novel transcription unit called DAZ (deleted in azoospermia), which is usually present in the AZF region in men of normal fertility (Reijo et al., 1995). The DAZ gene regulates a protein of 366 amino acids (molecular weight ), which appears to bind to RNA or single-stranded DNA. Deletions of varying length were identified in different men (Reijo et al., 1995), although exact relationships between the nature of the deletions and male infertility, and between the various deleted sequences involved in the regulation of spermatogenesis, have not been resolved. The resulting loss of spermatogonial stem cells was complete in many azoospermic men and almost complete European Society for Human Reproduction and Embryology 549

2 R.G.Edwards and C.E.Bishop Y deletions are transmitted in a dominant fashion and cause during chromosome alignment, pairing and crossing-over. aspermatogenesis or severe oligozoospermia in the offspring. Spermatids are also a potential site, where major genetic Many of them must arise de novo and be selected out of modifications include methylation, histone-protamine transition the population within one or two generations. Under these and nuclear condensation. Adverse effects of such de-novo circumstances, the deletion frequency in the father s spermato- deletions on the continued differentiation of germinal cells zoa must be close to 1 in (i.e. the frequency at birth, could be covered in developing germinal cells by long-lived Reijo et al., 1995). Such a high frequency could be detected spermatogenic mrna synthesized before spermiogenesis by counting deletions in several thousand spermatozoa from began. normozoospermic fathers of children with or without deletions. Identifying the site of origin of deletions is reminiscent of This calculation gives the average proportion of deleted spermatozoa similar classical studies on varying rates of sensitivity to in a population of men, assuming that it is distributed mutagenesis in successive spermatogenic and spermiogenic randomly and that random fertilization results in the same stages in mammals. Sensitivity was originally measured by proportion of deletions among offspring. The assumption about relating the degree of induced mutation to different spermatogenic random fertilization is probably correct. Evidence of any form stages in male mice exposed to X-rays or alkylating of selective advantage in mammals involving specific classes agents. The frequency of induced mutations fluctuated as of spermatozoa appears to be restricted to the Tt system in successive cohorts of differentiating germinal cells appeared mice, where spermatozoa carrying a particular t gene can in ejaculates (Leblond and Clermont, 1950; Roosen-Runge fertilize most of the ovulated eggs if ovulation occurs some and Geisel, 1950; Oakberg, 1956). A more direct assay involved hours before mating (Braden and Weiler, 1964; Cebra-Thomas correlating the pattern of mutation frequency with the rate of et al., 1991). An average of several thousand spermatozoa migration of [ 14 C]-labelled germinal cells between the stages with deletions, among millions of normal spermatozoa, would of DNA synthesis in primary spermatocytes and the occurrence be present in ejaculates under these conditions. If specific of ejaculation. The frequencies of mutations induced by low- genetic or other factors were involved in deletion formation, level irradiation varied again and was related to successive some men may produce higher proportions of spermatozooa spermatogenic stages (Oakberg, 1957; Sirlin and Edwards, with de-novo deletions. Sufficient evidence indicates that 1957, 1958). Meiotic and early-post meiotic stages, and mature genetic and environmental factors may be effective in invoking testicular spermatozoa were highly sensitive, and new deletion formation under particular circumstances or in particu- mutations did not seem to impair the capacity of spermatozoa lar individuals. to achieve fertilization. Deletion frequencies could be measured The site of origin of deletions remains to be decided. A today in a similar manner. father who is apparently normozoospermic and fully fertile Deletions could be induced at virtually all spermatogenic produces sons who are sterile. A meiotic or spermatid origin and spermiogenic stages. Zygotene could be highly sensitive, seems to be the most likely cause, although deletions could when associations form between homologous and heterologous arise in fertilized eggs or embryos, to prevent the formation chromosomes. Various forms of mutation including inversions, of spermatogonia in the fetus and subsequently impair spermatogenesis deletions and unequal exchanges could be equated with prelim- in the adult. Evidence permitting a decision to inaries to full chiasma formation. Initial pairing could be be made on the relative significance of a testicular or an associated with trial-and-error mismatching and misalignment embryological origin of deletions is very limited. In one study, in telomeres as GC-rich sequences promote an initial attachment two fathers of afflicted children did not carry the deletion in or association (Chandley, 1987, 1988). Recombination leukocyte chromosomes, although their gametes were not frequencies are much higher in telomeric regions of spermatocytes tested (Reijo et al., 1996). Spermatozoa and leukocytes taken than in oocytes, so that spermatozoa carry more recomtested from each of their offspring carried DAZ, so the Y deletion binants. Telomerase is active in human gonads, where it had obviously arisen de novo at some stage during the presumably maintains telomere length (Wright et al., 1996). formation of their germinal cells. A similar situation arose in Secondary or intrachromosomal pairing and translocations are a second study where two infertile men who did not carry a caused by exposure to external agents including X-irradiation deletion in blood cells produced sons after intracytoplasmic and alkylating agents. A high frequency of de-novo deletions sperm injection (ICSI) with microdeletions located between in spermatozoa causing infertility, termed germ cell maturation AZFb and AZFc (Kent-First et al., 1996b). Some men with non- impairment and involving an accumulation of small chromosome obstructive azoospermia carrying AZF/DAZ deletions produce rearrangements in combination with environmental sufficient spermatozoa capable of achieving conception and agents, could explain the paternal origin of structural autosomal normal embryonic development by means of the intracytoplasmic rearrangements (see Forejt et al., 1981; Chandley, 1988; Olson injection of a single spermatozoon into an oocyte and Magenis, 1988). Exchanges also occur between autosomes (Mulhall et al., 1997). and the sex vesicle, as in X/autosome translocation carriers, and quadrivalents sometimes protude from the sex vesicle Testicular origin of deletions (Lifschytz and Lindsley, 1972; A.C.Chandley, personal communication). A testicular origin of deletions could involve virtually any Normal rules of deletion and mutation formation need not spermatogenic or spermiogenic stage. Primary spermatocytes apply to the Y chromosome in view of its limited pairing sites in meiotic prophase are a most likely source of deletions with the X chromosome. Heterochromatin and highly repeat 550

3 Y chromosome deletions and severe male infertility sequences may also predispose sites on this chromosome to repeat arrays and gap repair as in yeast hypersensitive sites, induced genetic changes, as in sulphatase deficiency, spinal regulated by genes such as ARG4 (Schultes and Szostak, 1991; muscular atrophy and some X chromosome deletions (Yen Massey and Nicholas, 1993). Sister chromatid exchange due et al., 1990; Reijo et al., 1995). Recombination nodules are to a high frequency of short or long tandem repeats may associated with synaptonemal complexes in spermatocytes, explain the transmission of an expanded deletion from a fertile without necessarily being related to the later formation of father to an infertile son (Kobayashi et al., 1994). chiasmata. These nodules are uncommon in heterochromatic Genetic factors regulating chromosomal associations and regions. Interval 6 may be particularly susceptible to deletion chiasma formation during meiosis may thus predispose some formation in view of its proximity to heterochromatin. Highly men to disturbances in the onset and normality of meiotic specific mechanisms involving a high density of repeated pairing. In some individuals, these factors could also lead to sequences could cause lead loop formation, deletion of inter- microdeletions and minisatellite instability and to complex vening DNA and deletions in Yq. The human Y chromosome chromosome associations with clinical consequences such as back-folds with itself, seen in meiotic preparations, perhaps a predisposition to cancer or other disorders in the offspring. due to similar repeat sequences in different locations and This situation was achieved experimentally in mice, when interchanges within the chromosome (Chandley, 1987, 1988 homologous human genes of the MSH2 [muts (Escherichia and personal communication). coli) homologue 2] or PMS2 (post-meiotic segregation A second pseudoautosomal region exists in the X/Y bivalent, increased) were inactivated by gene targetting. They then with genetic exchange occurring betwen Xq-Yq. Telomeres of displayed minisatellite instability and an early onset of tumours Xq and Yq associate during meiosis, to form a short synaptone- (Reitmar et al., 1995). Mice defective in the DNA mismatch mal complex in rare cases. Sequence homologies extending repair gene PMS2 display male infertility associated with a over 400 kilobases of Xqter and Yqter in a region called PAR2 disruption in the synaptic pairing of homologous chromosomes, form the basis of genetic exchanges in this region (Freije the formation of many univalents and a total absence of et al., 1992). This hypothesis was tested using two highly spermatids (Baker et al., 1995). Minisatellite instability is informative microsatellite markers from YAC clones carrying characteristic of mice deficient for another mismatch repair Xqter sequences, and by consulting a set of reference pedigrees gene Mlh1, in which both sexes are infertile (Baker et al., 1996). in the Centre d Etude du Polymophisme in Paris. Four recombinations Genetic regulatory systems may also control other human were identified among a total of 195 informative deletion syndromes. A total of 13 novel microdeletions of meioses in which paternal X alleles were inherited by male varying size within and near to the gene POU35 are associated offspring and a paternal Y allele in one female offspring with X-linked deafness type 3 (DFN3) (de Kok et al., 1996). (Affara et al., 1996). In another study, multiple transcription Sites were located for eight of them, six being proximal and initiation sites and binding motifs were found in the 5 flanking two located within the gene. The authors suggested that regions of the gene IL9R, which has been identified at Xq28 proximally-located POU35 loci are sites of transcription regu- and Yq12 in the pseudoautosomal region of long arm in the lating factors, and considered the possibility of their being vicinity of the telomere (Kermouni et al., 1995). mutation initiating factors. The situation with DFN3 could be Mutation rates in minisatellites are regulated genetically. a model for the DiGeorge syndrome where banding, fluorescence Minisatellites are not randomly distributed, and are common in-situ hybridization (FISH) and Southern blotting identi- near telomeres, where synapsis is initiated (Jeffreys et al., fied a microdeletion at 22q11 in 1 in 9700 births (Tézenas du 1994). High frequencies of conversion products include deletions Montcel et al., 1996). It might also be relevant to DAZ and other and insertions, sometimes even of a single nucleotide. Y deletions causing azoospermia or severe oligozoospermia. Such mutations may represent search errors for homology Certain genotypes confer a sensitivity to mutagenic agents, as during recognition and synapsis of homologues initiated during in carriers of the autosomal recessive for Rothmund Thomson meiosis (Carpenter, 1987). Astonishingly high and complex syndrome who have a predisposition to malignancy. DNA mutation rates in some human minisatellites, and independent repair can be impaired in their isolated cell lines and trisomy of the length of the allele, arise in many single spermatozoa, 8 or i(8q) clones were identified in two out of three patients. e.g. a value of almost 1% per gamete for MS32. These Among the parents of two siblings with the syndrome, the deletions are seemingly germ-line specific and their ubiquitous father had a normal karyotype, and the mother was 45.X/ presence in every human spermatozoon may confer an indi- 46.XX[2/26] with rare cells of 46X,-X, 15 and vidual genetic identity on each of these gametes. Complex 46,XX,del(9)q11 (Lindor et al., 1996). Mental retardation in changes in nucleotide sequences in MS32 in the human male men can be invoked by Xq-Yq interchanges (Lahn et al., 1994). germline arise at one end of the tandem repeat array (Monckton Certain individuals genetically predisposed to deletion et al., 1994). Regulatory genes flanking such arrays control formation may produce sufficient spermatozoa with de-novo their mutation rates, and similar initiating factors could control deletions to compete successfully at fertilization with non- the initiation of meiotic recombination (Jeffreys et al., 1994). deleted spermatozoa. They could be identified by assessing This type of hypervariability is reduced in some men by a deletion frequencies among single spermatozoa of men who change associated with G C transversion upstream of the have produced sons with deletions. A general causative agent, minisatellite and by the presence of the C variant in their e.g. an environmental factor, might induce mutations at several spermatozoa. Such mechanisms may also regulate synaptone- sites simultaneously, to induce two or more deletion syndromes mal pairing, associations between double strand breaks, tandem in a single genome. 551

4 R.G.Edwards and C.E.Bishop Pronuclear and embryonic origins of deletions and Handyside, 1996; R.G.Edwards and H.K.Beard, manuscript The risks of deletion formation elsewhere than in the testis, submitted). especially in early embryos, would at first sight seem to Diverse forms of genetic activity in morulae and blastocysts be slim. There are no counterparts to the intimate pairing could also expose their constituent tissues to genetic changes. associations and recombinations occurring during meiosis. Such genetic processes include heterochromatin formation Nevertheless, distinct genetic phenomena affecting chromatin during X-inactivation, imprinting, and fragile X amplification. structure, gene expression and differentiation could sensitize The recombinase-activating gene Rag-1 is expressed in mouse pronuclei or early blastomeres to genetic changes. Mitotic morulae and blastocysts; with Rag-2, it is associated with with disorders in cleaving human embryos seem to be so frequent genetic rearrangements in T cell receptor and immunoglobulin as to cause immense numbers of chromosomal mosaics. (Ig) genes in immature T or B lymphocytes (Hayakawa et al., Deletions and other genetic disorders arising in male and 1996). These authors suggest that its role in blastocysts may female pronuclei could well be mistakenly attributed to a be related to a loss of totipotency or X-inactivation. At least gonadal origin in the father or mother. Pronuclear anomalies three cell lineages have apparently differentiated in fully- could display sexual differences. Distinct genetic phenomena expanded mouse blastocysts (R.G.Edwards and H.K.Beard, involve DNA condensation and hyperacetylation during protrophectoderm, are familiar. The third could be the mammalian manuscript submitted). Two of them, inner cell mass and tamine/histone conversion in male pronuclei. Transcription occurs in both pronuclei, but at levels five times greater in the germ line, identified by the expression of the gene oct-4 under male pronucleus. Simultaneously, a near-total inhibition of the regulation of a distal enhancer (Palmieri et al., 1994; Yeom transcription in the female pronucleus, and possibly some et al., 1996). This gene may be one of the primary regulators imprinting, may be imposed by a modified chromatin structure of the germ cell lineage in mamalian embryos. This very early separation of germ line from soma and which virtually extinguishes promoter activity (Tesarik and placenta, when embryos only contain a dozen or so stem cells, Kopecny, 1989, 1990; Nothias et al., 1995; Aoki et al., 1997). could explain why some early-onset genetic disorders are The late pronuclear stage is highly sensitive to teratogenic differentially expressed in one or more of these tissues. changes induced by alkylating agents (Rutledge et al., 1992). Mutations, deletions, amplified fragile X sequences and X- One copy of the paternal Y is present before the S phase in inactivation originating in a single stem cell could have extreme the early male pronucleus, and a single deletion event would implications in one cell line without affecting the others. produce offspring uniform for deletions. Embryological and Mosaicism could arise independently in inner cell mass or in hereditary consequences would be similar to those arising germinal cells. A mutation occurring as a mitotic event meiotically. A deletion in pronuclei involving one chromatid in early embryogenesis, before the separation of ectoderm, of the Y chromosome in the G 2 phase could produce mosaicism mesoderm and endoderm, was offered as the cause of in 2-cell embryos, although virtually all analyses on blood mosaicism for a de-novo deletion within the dystrophin gene samples of afflicted adults have failed to detect any mosaicism. in both somatic and germinal cells of a mother; her daughter Vogt (1995) suggests that the loss of spermatogonia is agecarried a uniform deletion (Bunyan et al., 1994). These authors related, so that a severe oligozoospermic man with deletions described other similar cases of mosaicism. Mosaicism arose will become azoospermic as he ages. Nevertheless, chromoin siblings with Rothmund Thomson syndrome, as described somal breakage in a pronucleus was offered as an explanation earlier (Lindor et al., 1996). Kent-First et al. (1996a,b) raised of rod/ring mosaicism in chromosome 2 of a child with mild the possibility of Y deletion mosaicism arising in the germmental retardation (Wyandt et al., 1982). cell lineage in two children where the deletion was absent in Embryonic activation in mouse embryos involves an initial the fathers blood. This early separation of germline and soma minor activation in 1-cell embryos independent of the first would also explain why germline cells can be imprinted while DNA replication, and a second major activation in S and somatic tissues are not, and how many repeat sequences can G 2 phases in 2-cell stages (Flach et al., 1982). The latter form in somatic cells, but not in germinal cells, of human depends on DNA replication in 1-cell eggs, which activates embryos with fragile X. The CGG triplet is amplified in one or more transcription initiating factors (Aoki et al., 1997), somatic cells of male fetuses with fragile X, but not in their and is characterized by the synthesis of many polypeptides germinal cells, so that in the adult the spermatozoa do not and a reduction in genomic methylation (Monk et al., 1987). express the full mutation (Reyniers et al., 1993). The gene DNA replication in 2-cell embryos inactivates some factors Rad51 may regulate gamete formation, X-inactivation and the regulating transcription and translation (Nothias et al., 1995; expansion of triplet repeats in fragile X; two human loci, Aoki et al., 1997). Uniform or mosaic deletions could arise DFFRX and DFFRY mapping to Xp11.4 and Yq11.2, are during somatic pairing or chromosome breakage in one blasto- transcribed in embryonic and adult tissues where DFFRX mere of 2-cell embryos or in later cleavage stages. The escapes X-inactivation (Hayakawa et al., 1996). frequency of chromosomal mosaicism in human embryos cleaving in vitro is extremely high, and may even afflict as many as 75% of them (Delhanty and Handyside, 1996; Conclusions Munné et al., 1997). Spindle anomalies and the presence Deletion analyses on single spermatozoa of the fathers and of multinucleated blastomeres may be associated with this their offspring to assess the frequency of deletions should help enormous frequency of chromosomal mosaicism (Delhanty to clarify the origins of DAZ and other Y deletions. Most 552

5 Y chromosome deletions and severe male infertility deletions presumably arise in zygotene, but other meiotic or Chandley, A.C. and Cooke, H.J. (1994) Human male fertility Y-linked genes and spermatogenesis. Hum. Mol. Genet., 3, spermiogenic stages, or various stages of early embryogenesis, de Kok, Y.J.M., Vossenaar, E.R., Cremers, C.W.R.J. et al. (1996) Identification may also be predisposed to deletion formation. More know- of a hot spot for microdeletions in patients with X-linked deafness type 3 ledge is needed on the frequency of de-novo deletions in male (DFN3) 900 kb proximal to the DNF3 gene POU3F4. Hum. Mol. Genet., 5, germinal cells to decide if they arise randomly or among a Delhanty, J.D.A. and Handyside, A.H. (1996) The origin of genetic defects restricted number of genetically-sensitive individuals. in the human and their detection in the preimplantation embryo. Hum. Microdeletions occur in very high frequency in the male Reprod. 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