In a large collaborative screening project, 370 men with idiopathic azoospermia or severe oligozoospermia wereanalysed for deletions of 76 DNA loci in Yq11. In 12 individuals, we observed de novo microdeletions involvingseveral DNA loci, while an additional patient had an inherited deletion. They were mapped to three differentsubregions in Yq11. One subregion coincides to the AZF region defined recently in distal Yq11. The second andthird subregion were mapped proximal to it, in proximal and middle Yq11, respectively. The different deletionsobserved were not overlapping but the extension of the deleted Y DNA in each subregion was similar in eachpatient analysed. In testis tissue sections, disruption of spermatogenesis was shown to be at the same phasewhen the microdeletion occurred in the same Yq11 subregion but at a different phase when the microdeletionoccurred in a different Yq11 subregion. Therefore, we propose the presence of not one but three spermatogenesisloci in Yq11 and that each locus is active during a different phase of male germ cell development. As the mostsevere phenotype after deletion of each locus is azoospermia, we designated them as: AZFa, AZFb and AZFc.Their probable phase of function in human spermatogenesis and candidate genes involved will be discussed. INTRODUCTIONGenes for male germ cell development are present on the Ychromosome in different species groups (1–3). In men, theposition of a spermatogenesis locus was mapped in theeuchromatic part of the long Y arm (Yq11). It was called‘azoospermia factor’ (AZF), as the first six men observed withterminal deletions in Yq were azoospermic (4). Mature spermcells were not detectable in their seminal fluid. In all cases, the Ydeletions included the large heterochromatin block of the long Yarm (Yq12) and an undefined amount of the adjacent euchromatin(Yq11). Subsequently, the presence of AZF in Yq11 wasconfirmed by numerous studies at both cytogenetic (5) andmolecular level (6–8). However, the genetic complexity of AZFcould not be revealed by these analyses.This first became possible by the detection of sterile patientswith small interstitial deletions (i.e. microdeletions) in Yq11. Ina study with 13 sterile men suffering from idiopathic azoospermiatwo different microdeletions in Yq11 were observed (9). Theywere mapped to two non overlapping positions in Yq11 interval6 (10). However, further studies of Yq11 microdeletionsassociated to the phenotype of male sterility, only confirmed theposition of an AZF locus in distal Yq11 (11,12). The mostextensive study was performed by Reijo et al. (13) on 89 sterile

Human Y Chromosome Azoospermia Factors (AZF) Mapped to Different Subregions in Yq11

We have detected deletions of portions of the Y chromosome long arm in 12 of 89 men with azoospermia (no sperm in semen). No Y deletions were detected in their male relatives or in 90 other fertile males. The 12 deletions overlap, defining a region likely to contain one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). Deletion of the AZF region is associated with highly variable testicular defects, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids. We find no evidence of YRRM genes, recently proposed as AZF candidates, in the AZF region. The region contains a single–copy gene, DAZ (Deleted in AZoospermia), which is transcribed in the adult testis and appears to encode an RNA binding protein. The possibility that DAZ is AZF should now be explored.

Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA–binding protein gene

Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

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Oxidative stress, DNA damage and the Y chromosome.

The world's population is increasing at an alarming rate and is projected to reach nine billion by 2050. Despite this, 15% of couples world-wide remain childless because of infertility. Few genetic causes of infertility have been identified in humans; nevertheless, genetic aetiologies are thought to underlie many cases of idiopathic infertility. Mouse models with reproductive defects as a major phenotype are being rapidly created and discovered and now total over 200. These models are helping to define mechanisms of reproductive function, as well as identify potential new contraceptive targets and genes involved in the pathophysiology of reproductive disorders. With this new information, men and women will continue to be confronted with difficult decisions on whether or not to use state-of-the-art technology and hormonal treatments to propagate their germline, despite the risks of transmitting mutant genes to their offspring.

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Genetic dissection of mammalian fertility pathways.

Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome

A deletion of the Y chromosome at the distal portion of band q11 was found in 6 men with normal male habitus but with azoospermia. Five of them were found during a survey of 1170 subfertile males while the sixth was karyotyped because of slight bone abnormalities. These findings, together with a review of the literature, suggest that on the distal portion of the nonfluorescent segment of the long arm of the Y, factors are located controlling spermatogenesis.

L. Tiepolo

Papers

Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm.