Genetic contribution to male infertility.

Worldwide, of couples trying for a child, 2-7% fail to conceive. Extensive screening programmes of men attending infertility clinics show that chromosomal and gene disorders make a significant contribution to spermatogenic impairment. It appears that an orderly genome is essential for normal germ cell development, since numerical and structural chromosome abnormalities are found in association with germ cell breakdown. The most recent research indicates that genes on the Y chromosome and autosomes are involved in spermatogenic control.

Chromosome anomalies and Y chromosome microdeletions as causal factors in male infertility.

Among the 10% or so of men who are diagnosed as oligo- or azoospermic in the absence of any physical obstruction, research is now showing that between 8 and 15% carry a microdeletion in the long arm of the Y chromosome which, by loss of specific DNA segments, leads to loss of vital genes for sperm production. Chromosomal anomalies account for approximately 2% of all men who attend infertility clinics, rising to 15% among those with azoospermia. There are serious implications for couples seeking help by intracytoplasmic sperm injection (ICSI), since chromosomal or gene defects which might normally be lost or eliminated by natural means could be transmitted in offspring. The need for genetic testing of ICSI donors and their offspring is raised, and a requirement for counselling is recommended.

Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development.

RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.

Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm.

The association of abnormal spermatogenesis in men with Y chromosome deletions suggests that genes important for spermatogenesis have been removed from these individuals. Recently, genes encoding two putative RNA-binding proteins (RBM and DAZ/SPGY) have been mapped to two different regions of the human Y chromosome. Both of these genes encode proteins that contain a single RNA recognition motif and a (different) internally repeating sequence. Y-linked RBM homologues are found in all mammalian species. We have raised an antiserum to RBM and used it to show that RBM is a nuclear protein expressed in fetal, prepubertal, and adult male germ cells. The distribution of RBM protein in the adult correlates with the pattern of transcriptional activity in spermatogenesis, suggesting that RBM is involved in the nuclear metabolism of newly synthesized RNA. RBM sequences are found on both arms of the Y chromosome making genotype-phenotype correlations difficult for this gene family. To address the location of the functional genes and the consequences of their deletion, we examined a panel of men with Y chromosome deletions and known testicular pathologies using this antiserum. This approach enabled us to map a region of the Y chromosome essential for RBM expression. In the absence of detectable RBM expression we see stages of germ cell development up to early meiosis, but not past this point into the haploid phase of spermatogenesis.

A human candidate spermatogenesis gene, RBM1, is conserved and amplified on the marsupial Y chromosome.

Three genes, RBM1, DAZ and TSPY, map to a small region of the long arm of the human Y chromosome which is deleted in azoospermic men. RBM1, but not DAZ or TSPY, has a Y-linked homologue in marsupials which is transcribed in the testis. This suggests that RBM1 has been retained on the Y chromosome because of a critical male-specific function. Marsupial RBM1 is closely related to human RBM1, but, like the related autosomal gene hnRNPG, lacks the amplification of an exon. This suggests that RBM1 evolved from hnRNPG at least 130 million years ago and has undergone internal amplification in primates, as well as independent amplification in several therian [corrected] lineages.

Fertility investigations in the F1 hybrid and backcross progeny of cattle (Bos taurus) and yak (B. grunniens) in Mongolia.

Investigations conducted in Mongolia into the sterility of the male khainag, an F1 hybrid animal resulting from crossing cattle (Bos taurus, 2n = 60) with yaks (B. grunniens, 2n = 60), are reported. Reduced numbers of spermatogonia appear to characterise the testicular tubules of the khainag, and despite the identical cytological appearance of the two parental karyotypes, synaptic anomalies are seen at meiotic prophase in primary spermatocytes. The female khainag is fertile and can be backcrossed to cattle or yak bulls to produce a B1 backcross animal, the ortoom. Further backcrossing of ortoom females to cattle or yaks will yield a B2 backcross animal, the usanguzee. The impression is gained of better meiotic pairing in the backcross animals than in the khainag. The "Haldane Rule" is followed perfectly by the cattle x yak hybrid; namely, sterility is confined to the male.

Polymerase chain reaction screening for Y chromosome microdeletions: a first step towards the diagnosis of genetically-determined spermatogenic failure in men.

Overall, approximately 11% of men attending infertility clinics suffer unexplained oligo- or azoospermia. Cytogenetic observations of loss of the distal portion of the Y chromosome long arm (Yq) were found to be associated with disrupted spermatogenesis. The existence of a gene locus involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was thus postulated. It is suggested that microdeletions, or mutations, at the AZF locus could result in impaired spermatogenesis in chromosomally normal men. In order to test this hypothesis we have carried out Y chromosome genetic screening of 100 oligo- or azoospermic 46XY patients. We have also assessed phenotype/genotype relationships in those patients whose infertility has an underlying genetic aetiology. Patients were screened by polymerase chain reaction (PCR) with a set of Y chromosome-specific sequence tagged sites (STS) for submicroscopic deletions of their Y chromosome. Our results show that as many as 8% of cases of unexplained male infertility may have an underlying genetic aetiology related to microdeletions in two specific regions of the Y chromosome. Positive results from such a screen will be important when deciding the suitability of a patient for assisted conception schemes such as intracytoplasmic sperm injection.

An RBM homologue maps to the mouse Y chromosome and is expressed in germ cells.

We have isolated a murine homologue of the human Y-linked RBM genes (previously termed YRRM), a gene family implicated in spermatogenesis and which encodes proteins containing an RNA recognition motif. A number of very similar copies of this gene (called Rbm) are present in the mouse. These mouse homologues are also Y-encoded, mapping on the short arm of the chromosome, proximal to Sry. Expression is confined to the testis, specifically the germ line on the basis of lack of expression in the germ-line negative testes of adult sex-reversed mice. The timing of Rbm transcription is regulated, with fetal message levels reaching a peak at 15 d.p.c. Transcripts are clearly detectable by 4 days after birth and reach their highest level at 14 d.p.p. which is the time at which the Y chromosome condenses during meiotic prophase. These results suggest that Rbm is functionally involved in germline RNA metabolism.

High-resolution fluorescence in situ hybridization of RBM- and TSPY-related cosmids on released Y chromatin in humans and pygmy chimpanzees.

Applying two-colour fluorescence in situ hybridization (FISH) we simultaneously hybridized RBM- and TSPY-related cosmids to Y chromosomes in prophase and to released Y chromatin in interphase nuclei of man and pygmy chimpanzee. Whereas, even on prophasic Y chromosomes, no resolution of the overlapping RBM and TSPY signal clusters could be achieved, the RBM and TSPY signals are completely separated from each other in our maximum released Y chromatin stretches in interphase nuclei. These results unequivocally lend support to the view that the RBM and TSPY families have an interspersed organization on the Y chromosomes of man and higher apes. Thus, the distribution of RBM and TSPY signals might well go back to a common organization of these genes next to each other on an ancient Y chromosome.

Different distributions of homologous chromosomes in adult human Sertoli cells and in lymphocytes signify nuclear differentiation.

Using whole chromosome painting probes for human chromosomes 3,7,8,13,17 and 21 and X and the probe pHY2.1 for the Y chromosome coupled with fluorescent in situ hybridization (FISH) analysis, the distribution of chromosomes is reported in nuclei of Sertoli cells of the adult testis and in stimulated blood lymphocytes. The distribution of chromosomes in the two cell types is significantly different. A strong tendency for each pair of homologues to pair is inferred from the observation of only a single detectable signal in the majority of Sertoli cell nuclei. The sex chromosomes, by contrast, give two clearly separated signals. Interphase nuclei in dividing blood lymphocytes, analysed as controls, also show mainly two separated signals for all non-acrocentric autosomal pairs, but acrocentric pairs no. 13 and 21 show some tendency to associate, probably reflecting satellite association.

Y chromosome microdeletions and male subfertility.

Men with azoospermia and severe oligozoospermia have been investigated by molecular probing of the long arm of the Y chromosome. We find microdeletions affecting various parts of the long arm of the Y chromosome in approximately 10% of men with non-obstructive azoospermia and severe oligozoospermia but not in a fertile comparison population. This work needs further confirmation in different countries and different racial groups but it would appear that microdeletions (and presumably genetic defects) are commonly associated with defects of spermatogenesis. These findings have implications for the management of severe male subfertility with in vitro fertilization/intracytoplasmic sperm injection.

A reassessment of Y chromosomal behaviour in germ cells and Sertoli cells of the mouse as revealed by in situ hybridisation.

In situ hybridisation experiments were carried out to reappraise the state of condensation of the Y chromosome in germ cells and Sertoli cells of the mouse. Previous work had suggested that all testicular cells showed a condensed Y chromosome prior to the adult stage. We now demonstrate that, although the Y chromosome is condensed in pre-pubertal Sertoli cells, it is greatly expanded in primordial germ cells (gonocytes). An expanded Y-signal is first seen in Sertoli cell nuclei at or around day 21 of postnatal development, coinciding with the first appearance of spermatids in the germinal epithelium.

Comparative mapping of YRRM- and TSPY-related cosmids in man and hominoid apes.

Using chromosomal in situ hybridization it has been demonstrated that specific members of the YRRM and the TSPY families are multicopy and Y chromosome specific in hominoids. After hybridization with the YRRM-related cosmid A5F and the TSPY-related cosmids cos36 and cY91, a reverse and complementary pattern of main and secondary signals is detected on the Y chromosomes of the human, the pygmy chimpanzee and the gorilla, while the location of signals coincides on the Y chromosomes of the chimpanzee, both orang-utan subspecies and the white hand gibbon. This complementary distribution of YRRM and TSPY sequences on the hominoid Y chromosomes possibly originates from a similar sequence motif that is shared by and evolutionarily conserved between certain members of both gene families and/or repeated elements flanking those genes. Otherwise this complementary distribution could go back to a common organization of these genes next to each other on an ancient Y chromosome which was disrupted by chromosomal rearrangements and amplification of one or other of the genes at each of the locations.

Condensation behaviour of the human X chromosome in male germ cells and Sertoli cells examined by fluorescence in situ hybridization.

The chromatin condensation behaviour of the human X chromosome has been studied by fluorescence in situ hybridization (FISH) analysis in germ cells and Sertoli cells of the adult testis, and comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this stage of meiosis, could be a prerequisite for XY pairing and crossing-over. By in situ hybridization analysis, the sex chromosomes of patients with 'Sertoli-cell-only' syndrome appear extremely contracted compared with the normally extended state of those in adult Sertoli cells of fertile men. By contrast, the state of expansion for chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to patterns of expression noted for sex-linked genes in the human testis.

Human male fertility--Y-linked genes and spermatogenesis.

Using a positional cloning approach, we have isolated a new gene family, the Y-located RNA Recognition Motif genes (YRRM), which constitutes a candidate for AZF, the 'azoospermia factor' located principally within band Yq11.23, and thought to be important in the control of human spermatogenesis. The YRRM gene family has at least 15 members, more than one of which are transcribed, some of which are pseudogenes. RNA in situ hybridization to adult human testis tissue indicates that gene expression of the YRRM family is confined to germ cells, notably spermatogonia and/or primary spermatocytes. Similar patterns of hybridization are seen for a second gene family, TSPY, clustered mainly on the short arm of the human Y chromosome. Both YRRM and TSPY show Y chromosome conservation in several mammalian species.

Chromatin condensation behaviour of the Y chromosome in the human testis. I. Evidence for decondensation of distal Yq in germ cells prior to puberty with a switch to Sertoli cells in adults.

The condensation behaviour of the human Y chromosome in germ cells and Sertoli cells of pre- and post-pubertal testes was followed by fluorescence in situ hybridisation using probes for three different regions of the Y chromosome. Patterns of expansion or contraction of signal are taken to reflect degrees of condensation of the related Y chromatin and hence its potential for genetic activity. For probe pHY2.1, which labels the distal non-fluorescent and fluorescent heterochromatin of the Y chromosome (Yq12), an expanded signal seen in gonocytes of the prepubertal testis is superseded by a condensed signal seen in adult germ cells at all but the zygotene stage of meiotic prophase when meiotic pairing takes place. In contrast, Sertoli cells show a condensed signal pre-pubertally but a greatly expanded signal in the adult testis. A totally condensed pHY2.1 signal is found in a chromosomally normal man with Sertoli-cell-only syndrome. It is hypothesised that control over at least some facets of spermatogenesis may not, in the adult, be autonomous to the germ cells, but rather may emanate from the Sertoli cells. Chromatin expansion at zygotene could, however, be important for pairing and crossing over in the XY bivalent, successful synapsis ensuring survival of spermatocytes into the post-meiotic stages.