Download On the origin and frequency of Y chromosome deletions responsible

Molecular Human Reproduction vol.3 no.7 pp. 549–554, 1997
OPINION
On the origin and frequency of Y chromosome deletions
responsible for severe male infertility
R.G.Edwards1,3 and Colin E.Bishop2
1Churchill
College, Cambridge, UK, and 2Department of Obstetrics and Gynecology and Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, Texas, USA
3To
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
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 10 000 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 41 257), 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
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 proliferation 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 G2–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).
549
R.G.Edwards and C.E.Bishop
Y deletions are transmitted in a dominant fashion and cause
aspermatogenesis or severe oligozoospermia in the offspring.
Many of them must arise de novo and be selected out of
the population within one or two generations. Under these
circumstances, the deletion frequency in the father’s spermatozoa must be close to 1 in 10 000 (i.e. the frequency at birth,
Reijo et al., 1995). Such a high frequency could be detected
by counting deletions in several thousand spermatozoa from
normozoospermic fathers of children with or without deletions.
This calculation gives the average proportion of deleted spermatozoa in a population of men, assuming that it is distributed
randomly and that random fertilization results in the same
proportion of deletions among offspring. The assumption about
random fertilization is probably correct. Evidence of any form
of selective advantage in mammals involving specific classes
of spermatozoa appears to be restricted to the Tt system in
mice, where spermatozoa carrying a particular t gene can
fertilize most of the ovulated eggs if ovulation occurs some
hours before mating (Braden and Weiler, 1964; Cebra-Thomas
et al., 1991). An average of several thousand spermatozoa
with deletions, among millions of normal spermatozoa, would
be present in ejaculates under these conditions. If specific
genetic or other factors were involved in deletion formation,
some men may produce higher proportions of spermatozooa
with de-novo deletions. Sufficient evidence indicates that
genetic and environmental factors may be effective in invoking
deletion formation under particular circumstances or in particular individuals.
The site of origin of deletions remains to be decided. A
father who is apparently normozoospermic and fully fertile
produces sons who are sterile. A meiotic or spermatid origin
seems to be the most likely cause, although deletions could
arise in fertilized eggs or embryos, to prevent the formation
of spermatogonia in the fetus and subsequently impair spermatogenesis in the adult. Evidence permitting a decision to
be made on the relative significance of a testicular or an
embryological origin of deletions is very limited. In one study,
two fathers of afflicted children did not carry the deletion in
leukocyte chromosomes, although their gametes were not
tested (Reijo et al., 1996). Spermatozoa and leukocytes taken
from each of their offspring carried DAZ, so the Y deletion
had obviously arisen de novo at some stage during the
formation of their germinal cells. A similar situation arose in
a second study where two infertile men who did not carry a
deletion in blood cells produced sons after intracytoplasmic
sperm injection (ICSI) with microdeletions located between
AZFb and AZFc (Kent-First et al., 1996b). Some men with nonobstructive azoospermia carrying AZF/DAZ deletions produce
sufficient spermatozoa capable of achieving conception and
normal embryonic development by means of the intracytoplasmic injection of a single spermatozoon into an oocyte
(Mulhall et al., 1997).
Testicular origin of deletions
A testicular origin of deletions could involve virtually any
spermatogenic or spermiogenic stage. Primary spermatocytes
in meiotic prophase are a most likely source of deletions
550
during chromosome alignment, pairing and crossing-over.
Spermatids are also a potential site, where major genetic
modifications include methylation, histone-protamine transition
and nuclear condensation. Adverse effects of such de-novo
deletions on the continued differentiation of germinal cells
could be covered in developing germinal cells by long-lived
spermatogenic mRNA synthesized before spermiogenesis
began.
Identifying the site of origin of deletions is reminiscent of
similar classical studies on varying rates of sensitivity to
mutagenesis in successive spermatogenic and spermiogenic
stages in mammals. Sensitivity was originally measured by
relating the degree of induced mutation to different spermatogenic stages in male mice exposed to X-rays or alkylating
agents. The frequency of induced mutations fluctuated as
successive cohorts of differentiating germinal cells appeared
in ejaculates (Leblond and Clermont, 1950; Roosen-Runge
and Geisel, 1950; Oakberg, 1956). A more direct assay involved
correlating the pattern of mutation frequency with the rate of
migration of [14C]-labelled germinal cells between the stages
of DNA synthesis in primary spermatocytes and the occurrence
of ejaculation. The frequencies of mutations induced by lowlevel irradiation varied again and was related to successive
spermatogenic stages (Oakberg, 1957; Sirlin and Edwards,
1957, 1958). Meiotic and early-post meiotic stages, and mature
testicular spermatozoa were highly sensitive, and new
mutations did not seem to impair the capacity of spermatozoa
to achieve fertilization. Deletion frequencies could be measured
today in a similar manner.
Deletions could be induced at virtually all spermatogenic
and spermiogenic stages. Zygotene could be highly sensitive,
when associations form between homologous and heterologous
chromosomes. Various forms of mutation including inversions,
deletions and unequal exchanges could be equated with preliminaries to full chiasma formation. Initial pairing could be
associated with trial-and-error mismatching and misalignment
in telomeres as GC-rich sequences promote an initial attachment or association (Chandley, 1987, 1988). Recombination
frequencies are much higher in telomeric regions of spermatocytes than in oocytes, so that spermatozoa carry more recombinants. Telomerase is active in human gonads, where it
presumably maintains telomere length (Wright et al., 1996).
Secondary or intrachromosomal pairing and translocations are
caused by exposure to external agents including X-irradiation
and alkylating agents. A high frequency of de-novo deletions
in spermatozoa causing infertility, termed ‘germ cell maturation
impairment’ and involving an accumulation of small chromosome rearrangements in combination with environmental
agents, could explain the paternal origin of structural autosomal
rearrangements (see Forejt et al., 1981; Chandley, 1988; Olson
and Magenis, 1988). Exchanges also occur between autosomes
and the sex vesicle, as in X/autosome translocation carriers,
and quadrivalents sometimes protude from the sex vesicle
(Lifschytz and Lindsley, 1972; A.C.Chandley, personal communication).
Normal rules of deletion and mutation formation need not
apply to the Y chromosome in view of its limited pairing sites
with the X chromosome. Heterochromatin and highly repeat
Y chromosome deletions and severe male infertility
sequences may also predispose sites on this chromosome to
induced genetic changes, as in sulphatase deficiency, spinal
muscular atrophy and some X chromosome deletions (Yen
et al., 1990; Reijo et al., 1995). Recombination nodules are
associated with synaptonemal complexes in spermatocytes,
without necessarily being related to the later formation of
chiasmata. These nodules are uncommon in heterochromatic
regions. Interval 6 may be particularly susceptible to deletion
formation in view of its proximity to heterochromatin. Highly
specific mechanisms involving a high density of repeated
sequences could cause lead loop formation, deletion of intervening DNA and deletions in Yq. The human Y chromosome
back-folds with itself, seen in meiotic preparations, perhaps
due to similar repeat sequences in different locations and
interchanges within the chromosome (Chandley, 1987, 1988
and personal communication).
A second pseudoautosomal region exists in the X/Y bivalent,
with genetic exchange occurring betwen Xq-Yq. Telomeres of
Xq and Yq associate during meiosis, to form a short synaptonemal complex in rare cases. Sequence homologies extending
over 400 kilobases of Xqter and Yqter in a region called PAR2
form the basis of genetic exchanges in this region (Freije
et al., 1992). This hypothesis was tested using two highly
informative microsatellite markers from YAC clones carrying
Xqter sequences, and by consulting a set of reference pedigrees
in the Centre d’Etude du Polymophisme in Paris. Four recombinations were identified among a total of 195 informative
meioses in which paternal X alleles were inherited by male
offspring and a paternal Y allele in one female offspring
(Affara et al., 1996). In another study, multiple transcription
initiation sites and binding motifs were found in the 59 flanking
regions of the gene IL9R, which has been identified at Xq28
and Yq12 in the pseudoautosomal region of long arm in the
vicinity of the telomere (Kermouni et al., 1995).
Mutation rates in minisatellites are regulated genetically.
Minisatellites are not randomly distributed, and are common
near telomeres, where synapsis is initiated (Jeffreys et al.,
1994). High frequencies of conversion products include deletions and insertions, sometimes even of a single nucleotide.
Such mutations may represent search errors for homology
during recognition and synapsis of homologues initiated during
meiosis (Carpenter, 1987). Astonishingly high and complex
mutation rates in some human minisatellites, and independent
of the length of the allele, arise in many single spermatozoa,
e.g. a value of almost 1% per gamete for MS32. These
deletions are seemingly germ-line specific and their ubiquitous
presence in every human spermatozoon may confer an individual genetic identity on each of these gametes. Complex
changes in nucleotide sequences in MS32 in the human male
germline arise at one end of the tandem repeat array (Monckton
et al., 1994). Regulatory genes flanking such arrays control
their mutation rates, and similar initiating factors could control
the initiation of meiotic recombination (Jeffreys et al., 1994).
This type of hypervariability is reduced in some men by a
change associated with G→C transversion upstream of the
minisatellite and by the presence of the C variant in their
spermatozoa. Such mechanisms may also regulate synaptonemal pairing, associations between double strand breaks, tandem
repeat arrays and gap repair as in yeast hypersensitive sites,
regulated by genes such as ARG4 (Schultes and Szostak, 1991;
Massey and Nicholas, 1993). Sister chromatid exchange due
to a high frequency of short or long tandem repeats may
explain the transmission of an expanded deletion from a fertile
father to an infertile son (Kobayashi et al., 1994).
Genetic factors regulating chromosomal associations and
chiasma formation during meiosis may thus predispose some
men to disturbances in the onset and normality of meiotic
pairing. In some individuals, these factors could also lead to
microdeletions and minisatellite instability and to complex
chromosome associations with clinical consequences such as
a predisposition to cancer or other disorders in the offspring.
This situation was achieved experimentally in mice, when
homologous human genes of the MSH2 [mutS (Escherichia
coli) homologue 2] or PMS2 (post-meiotic segregation
increased) were inactivated by gene targetting. They then
displayed minisatellite instability and an early onset of tumours
(Reitmar et al., 1995). Mice defective in the DNA mismatch
repair gene PMS2 display male infertility associated with a
disruption in the synaptic pairing of homologous chromosomes,
the formation of many univalents and a total absence of
spermatids (Baker et al., 1995). Minisatellite instability is
characteristic of mice deficient for another mismatch repair
gene Mlh1, in which both sexes are infertile (Baker et al., 1996).
Genetic regulatory systems may also control other human
deletion syndromes. A total of 13 novel microdeletions of
varying size within and near to the gene POU35 are associated
with X-linked deafness type 3 (DFN3) (de Kok et al., 1996).
Sites were located for eight of them, six being proximal and
two located within the gene. The authors suggested that
proximally-located POU35 loci are sites of transcription regulating factors, and considered the possibility of their being
mutation initiating factors. The situation with DFN3 could be
a model for the DiGeorge syndrome where banding, fluorescence in-situ hybridization (FISH) and Southern blotting identified a microdeletion at 22q11 in 1 in 9700 births (Tézenas du
Montcel et al., 1996). It might also be relevant to DAZ and other
Y deletions causing azoospermia or severe oligozoospermia.
Certain genotypes confer a sensitivity to mutagenic agents, as
in carriers of the autosomal recessive for Rothmund–Thomson
syndrome who have a predisposition to malignancy. DNA
repair can be impaired in their isolated cell lines and trisomy
8 or i(8q) clones were identified in two out of three patients.
Among the parents of two siblings with the syndrome, the
father had a normal karyotype, and the mother was 45.X/
46.XX[2/26] with rare cells of 46X,-X,115 and
46,XX,del(9)q11 (Lindor et al., 1996). Mental retardation in
men can be invoked by Xq-Yq interchanges (Lahn et al., 1994).
Certain individuals genetically predisposed to deletion
formation may produce sufficient spermatozoa with de-novo
deletions to compete successfully at fertilization with nondeleted spermatozoa. They could be identified by assessing
deletion frequencies among single spermatozoa of men who
have produced sons with deletions. A general causative agent,
e.g. an environmental factor, might induce mutations at several
sites simultaneously, to induce two or more deletion syndromes
in a single genome.
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R.G.Edwards and C.E.Bishop
Pronuclear and embryonic origins of deletions
The risks of deletion formation elsewhere than in the testis,
especially in early embryos, would at first sight seem to
be slim. There are no counterparts to the intimate pairing
associations and recombinations occurring during meiosis.
Nevertheless, distinct genetic phenomena affecting chromatin
structure, gene expression and differentiation could sensitize
pronuclei or early blastomeres to genetic changes. Mitotic
disorders in cleaving human embryos seem to be so frequent
as to cause immense numbers of chromosomal mosaics.
Deletions and other genetic disorders arising in male and
female pronuclei could well be mistakenly attributed to a
gonadal origin in the father or mother. Pronuclear anomalies
could display sexual differences. Distinct genetic phenomena
involve DNA condensation and hyperacetylation during protamine/histone conversion in male pronuclei. Transcription
occurs in both pronuclei, but at levels five times greater in the
male pronucleus. Simultaneously, a near-total inhibition of
transcription in the female pronucleus, and possibly some
imprinting, may be imposed by a modified chromatin structure
which virtually extinguishes promoter activity (Tesarik and
Kopecny, 1989, 1990; Nothias et al., 1995; Aoki et al., 1997).
The late pronuclear stage is highly sensitive to teratogenic
changes induced by alkylating agents (Rutledge et al., 1992).
One copy of the paternal Y is present before the S phase in
the early male pronucleus, and a single deletion event would
produce offspring uniform for deletions. Embryological and
hereditary consequences would be similar to those arising
meiotically. A deletion in pronuclei involving one chromatid
of the Y chromosome in the G2 phase could produce mosaicism
in 2-cell embryos, although virtually all analyses on blood
samples of afflicted adults have failed to detect any mosaicism.
Vogt (1995) suggests that the loss of spermatogonia is agerelated, so that a severe oligozoospermic man with deletions
will become azoospermic as he ages. Nevertheless, chromosomal breakage in a pronucleus was offered as an explanation
of rod/ring mosaicism in chromosome 2 of a child with mild
mental retardation (Wyandt et al., 1982).
Embryonic activation in mouse embryos involves an initial
‘minor’ activation in 1-cell embryos independent of the first
DNA replication, and a second ‘major’ activation’ in S and
G2 phases in 2-cell stages (Flach et al., 1982). The latter
depends on DNA replication in 1-cell eggs, which activates
one or more transcription initiating factors (Aoki et al., 1997),
and is characterized by the synthesis of many polypeptides
and a reduction in genomic methylation (Monk et al., 1987).
DNA replication in 2-cell embryos inactivates some factors
regulating transcription and translation (Nothias et al., 1995;
Aoki et al., 1997). Uniform or mosaic deletions could arise
during somatic pairing or chromosome breakage in one blastomere of 2-cell embryos or in later cleavage stages. The
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;
Munné et al., 1997). Spindle anomalies and the presence
of multinucleated blastomeres may be associated with this
enormous frequency of chromosomal mosaicism (Delhanty
552
and Handyside, 1996; R.G.Edwards and H.K.Beard, manuscript
submitted).
Diverse forms of genetic activity in morulae and blastocysts
could also expose their constituent tissues to genetic changes.
Such genetic processes include heterochromatin formation
during X-inactivation, imprinting, and fragile X amplification.
The recombinase-activating gene Rag-1 is expressed in mouse
morulae and blastocysts; with Rag-2, it is associated with with
genetic rearrangements in T cell receptor and immunoglobulin
(Ig) genes in immature T or B lymphocytes (Hayakawa et al.,
1996). These authors suggest that its role in blastocysts may
be related to a loss of totipotency or X-inactivation. At least
three cell lineages have apparently differentiated in fullyexpanded mouse blastocysts (R.G.Edwards and H.K.Beard,
manuscript submitted). Two of them, inner cell mass and
trophectoderm, are familiar. The third could be the mammalian
germ line, identified by the expression of the gene oct-4 under
the regulation of a distal enhancer (Palmieri et al., 1994; Yeom
et al., 1996). This gene may be one of the primary regulators
of the germ cell lineage in mamalian embryos.
This very early separation of germ line from soma and
placenta, when embryos only contain a dozen or so stem cells,
could explain why some early-onset genetic disorders are
differentially expressed in one or more of these tissues.
Mutations, deletions, amplified fragile X sequences and Xinactivation originating in a single stem cell could have extreme
implications in one cell line without affecting the others.
Mosaicism could arise independently in inner cell mass or in
germinal cells. A mutation occurring as a mitotic event
in early embryogenesis, before the separation of ectoderm,
mesoderm and endoderm, was offered as the cause of
mosaicism for a de-novo deletion within the dystrophin gene
in both somatic and germinal cells of a mother; her daughter
carried a uniform deletion (Bunyan et al., 1994). These authors
described other similar cases of mosaicism. Mosaicism arose
in siblings with Rothmund–Thomson syndrome, as described
earlier (Lindor et al., 1996). Kent-First et al. (1996a,b) raised
the possibility of Y deletion mosaicism arising in the germcell lineage in two children where the deletion was absent in
the fathers’ blood. This early separation of germline and soma
would also explain why germline cells can be imprinted while
somatic tissues are not, and how many repeat sequences can
form in somatic cells, but not in germinal cells, of human
embryos with fragile X. The CGG triplet is amplified in
somatic cells of male fetuses with fragile X, but not in their
germinal cells, so that in the adult the spermatozoa do not
express the full mutation (Reyniers et al., 1993). The gene
Rad51 may regulate gamete formation, X-inactivation and the
expansion of triplet repeats in fragile X; two human loci,
DFFRX and DFFRY mapping to Xp11.4 and Yq11.2, are
transcribed in embryonic and adult tissues where DFFRX
escapes X-inactivation (Hayakawa et al., 1996).
Conclusions
Deletion analyses on single spermatozoa of the fathers and
their offspring to assess the frequency of deletions should help
to clarify the origins of DAZ and other Y deletions. Most
Y chromosome deletions and severe male infertility
deletions presumably arise in zygotene, but other meiotic or
spermiogenic stages, or various stages of early embryogenesis,
may also be predisposed to deletion formation. More knowledge is needed on the frequency of de-novo deletions in male
germinal cells to decide if they arise randomly or among a
restricted number of genetically-sensitive individuals.
Microdeletions occur in very high frequency in the male
germline and in human spermatozoa, but apparently not in the
female germline. The evolutionary significance of such an
immense rate of genetic change in one germline must be
analysed. Perhaps only the male germline can tolerate such a
high rate of variability, since such enormous numbers of
spermatozoa are normally produced per ejaculate. These numbers would permit a high rate of selection against deleterious
changes. Such an evolutionary development may be the basis of
the immense numbers of misshapen and immotile spermatozoa
characterizing human spermatozoa. The occurrence and nature
of deletions in single human spermatozoa should be related to
the individual morphology and motility to identify any causative links (Vogt, 1995).
Various clinical conditions have been correlated with deletion formation. An increasing awareness of these genetic
systems in the human male stresses the need for care and
follow-up during the application of new methods to the
alleviation of male infertility. Careful counselling of patients
is mandatory, especially on the risk of offspring inheriting
several deletions simultaneously. Fortunately, genetic testing
and counselling is being introduced into many clinics as in
one where microdeletions in AZFc were identified in seven
out of 11 oligozoospermic men and in none of, 19 azoospermic
men or in controls (Kremer et al., 1997).
Acknowledgements
We thank Mary Jo Kent-First, Ann Chandley and Pat Jacobs for their
constructive criticisms of this manuscript.
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Received on October 14, 1996; accepted on May 12, 1997