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In this section, genes that map to the azoosperm ia factor region o f the Y chrom osom e will be considered.

RBM Y RBM Y is a m ulticopy gene family, which is thought to be involved in spermatogenesis. R B M Y l, the first of this fam ily to be identified (Ma et a l , 1993) was isolated after the identification o f a CpG island in a cosm id clone m apped to Y q l 1. The RBM Y fam ily consists of approxim ately 30 genes and pseudogenes spread over both arms of the Y chrom osom e, with functional copies being localised within the AZFb region (Prosser et aL, 1996; Elliot et aL, 1997). RBM Y members code for proteins with a 90 aa RN A recognition m otif (RRM) and copies of a tandem ly repeated amino acid sequence of unknown function called the SRGY box (M a et al, 1993; Prosser et aL, 1996). Studies have indicated the presence of at least six RBM Y subfamilies termed R B M Y l to RBMY6. O f these,

RBM Y 1 is the largest and is the only fam ily to contain active members

(Prosser et aL, 1996; Chai et aL, 1997). Six highly hom ologous R B M Y l genes have been identified (RBM Y 1A to RBM Y IF), all of which differ by only one to seven bp, and each of which contains four SRGY repeats (Chai

The function of RBM Y 1 genes has not been elucidated. However, R B M Y l genes share around 67% hom ology with hnRN PG an autosom al gene located on chrom osom e 6. Both hnRN PG and RBM Y genes are members of the hnRN P (heterogeneous ribonucleoproteins) protein family, which share a com m on RNA recognition m otif (RRM) and are involved in pre-m RNA m etabolism and splicing (Soulard et a l , 1993; W eighardt et a l ,

1996). In contrast with RBM Y, hnRN PG protein contains no SRGY repeat units (Elliot et a l , 1996). RBM Y is expressed exclusively in m ale germ cells of foetal, prepubertal and adult testis. In adult m ale germ cells RBM Y is expressed after the cells have entered meiosis, which indicates that RBM Y is not absolutely essential for developm ent up to the meiotic stages of sperm atogenesis (EWioi et a l , 1997).

RBM Y 1 is evolutionarily conserved and hom ologues have been identified in many vertebrates, including marsupials (Ma et a l , 1993; Cooke

et a l , 1996; Delbridge et al., 1997). In mouse, an Rbm hom ologue with 66% similarity to hum an RBM Y 1 has been m apped on the short arm of the Y chrom osom e, betw een Sry and the centromere. Rbm seems to exist as a multicopy gene family, expressed only in the foetal and adult mouse germline, but unlike the hum an hom ologue, it contains only one SRGY m otif (Elliot et a l , 1996; Laval et a l , 1995).

Recently, an X-linked hom ologue of RBM Y designated RBM X has been found both in humans and m arsupials. It encodes the widely expressed protein hnRN PG and is thought to have arisen by retroposition of the

RBM Y gene. Like other genes with counterparts on both the X and the Y chrom osom e (Zfx/Zfy, Sox3/Sry), RBM X is widely expressed in m any tissues, in contrast to the testis-specific expression of R B M Y l. In addition, FISH has detected other sequences hom ologous to RBM Y on chrom osom es

1, 4, 9 and 11 (Delbridge et a l , 1999).

D A Z The DAZ (deleted in azoospermia) gene, was isolated after detailed deletion m apping o f the Y chrom osom es of infertile males,

follow ed by exon trapping to search for transcripts within a set of cosmids covering the com m on deleted region (Rejio et al., 1995). DAZ is located in the AZFc region on the long arm of the Y chrom osom e (Vogt et a l , 1996) and encodes an RN A-binding protein with a single RRM (RNA Recognition M otif) and seven to ten tandem ly arranged “D AZ specific”repeat units of 24 amino acids that appear to be polym orphic within the population (Reijo et aL, 1995; Y en et al., 1997). Saxena et al (1996) realised that these repeat units are the same as found at the D Y S l locus, a highly polym orphic fam ily of Yq specific sequences. Expression of DAZ is restricted to testis and exclusively to germ cells at early stages of spermatogenesis. It has been proposed that D A Z is involved in the maintenance of germ stem cell num ber and regulation of the first stages of sperm atogenesis (Rejio et al., 1995; M enke et al., 1997).

Like R B M Y l, DAZ is also a m em ber of a multigene family. In addition to the m ultiple copies present on the Y chrom osom e, there is an autosom al hom ologue, termed D A Z L l (DAZ-Like) (Yen et a l , 1996; Saxena et al., 1996; Rejio et a l , 1996; Cooke et a l , 1996) which maps to chrom osom e 3p24 in man and 17 in mouse. D A Z L l is a single copy gene expressed in testis and oocytes, that encodes a 3.3Kb transcript with only one “DAZ repeat” (Yen et al., 1996; Seboun et al., 1997; Nishi et al., 1999).

D A Z L l has a 130bp segment at the 3’ end of the coding region that is absent in the Y-linked DAZ copies (Yen et a l , 1996; Cooke et a l , 1996). Recently, Slee et al (1999), has tested the capacity of hum an D A Z gene, to com plem ent the sterile phenotype of the Dazl knockout m ale m ouse (DazL^'),

w hich is characterised by severe germ cell depletion and meiotic failure. A lthough introduction of the hum an transgene failed to com pletely restore

fertility, histological exam ination revealed a partial and variable rescue of the m utant phenotype.

Autosomal DAZ homologues have been identified in marsupials, old and new world monkeys and various other m am m als (Seboun et aL,

1997; Delbridge et aL, 1997). Y-linked DAZ copies have been identified only in old world monkeys, apes and humans, which suggests that the Y chrom osom e has only recently acquired a copy of the DAZ gene probably within the last 30-50 m illion years ago (Seboun et aL, 1997; V ogt et aL,

1997; A gulnik et aL, 1998). A second hom ologue of DAZ, boule, has been described in Drosophila. Loss of function of boule results in azoosperm ia due to blockage of meiotic divisions and limited sperm atid differentiation (Eberhart et aL, 1996).

Agulnik et al (1998) have shown that the intron and exon sequences o f the Y-linked DAZ copies are acquiring base pair changes at an equal rate. This implies that the exons are subject to a neutral genetic drift and an absence of any functional selective pressures on these genes. However some, but not all, studies, have shown deletions within the DAZ cluster in 5-

15% of infertile and subfertile patients (Rejio et aL, 1995; V ereb et aL,

1997; Ferlin et aL, 1999).

T S P Y In addition to RBM Y and DAZ, there is another m ulticopy gene fam ily on the hum an Y chromosome thought to be a candidate AZF gene, the Testis-specific protein Y-encoded gene (TSPY) (Arnem ann et aL,

1987). The gene fam ily consists of around 20 to 40 copies some o f which are functional and some pseudogenes. The sequence of m embers of this fam ily are highly conserved; functional genes share hom ology as high as 98- 99% with each other and up to 90% with their pseudogenes. M embers of this gene fam ily are widespread along both arms of the Y chrom osom e.

where they are arranged in clusters. On Yp, there are two clusters, TSPYA and TSPYB, within interval 3; two further clusters on Y q are located in intervals 4 and 5 (intervals seen in Fig 1.5) (Arnemann et aL, 1991; V ogt et aL, 1997). TSPY transcription units are around 2.8Kb and are organised as com ponents of the 20Kb DYZ5 repeat units (Manz et aL, 1993).

The TSPY genes, with m inor differences to each other, are com posed of six exons, five introns, and a prom oter region of undefined length and sequence (Schnieders et aL, 1996). TSPY products have been found as splice variants, but whether these are generated by alternative splicing of the same transcript or derive from different transcripts is still unclear. The 1.3Kb TSPY transcript encodes a 33KD protein, hom ologous to the proto-oncogene SET (myeloid leukem ia associated) and the

nucleosom e assem bly protein 1 (NAP 1) (Schnieders et aL, 1996).

Expression of TSPY is restricted to testis in both embryos and adults prim arily to the cytoplasm of a subset of spermatogonial cells and around the basal lam ina of the seminiferous tubules (Arnemann et aL, 1987; Zhang

et aL, 1992; Schnieders et aL, 1996). W ith these data in mind, Schnieders et al (1996), suggested that TSPY might regulate the normal proliferation of sperm atogonia, and their entry into meiotic differentiation. TSPY is

expressed aberrantly in tumors with germ cell origins and in epithelial cells of prostate cancers and may play a role in the genesis of these tumors. The case for TSPY as an im portant gene in gonadoblastom a is review ed by Lau (1999). It is relevant that the “gonadoblastom a” critical region has been located to the short arm of the Y chromosome, where TSPY copies are located (Tsuchiya et aL, 1995).

TSPY hom ologous sequences are conserved in great apes (Schem pp

et aL, 1995), and m ammals (Vogel et aL, 1997). In mouse, Tspy has been identified as a Y-linked, single copy gene. However, it produces only low

(M azeyrat and M itchell, 1998). In rat, there are two TSPY genes located to Yp, one is functional and expressed in testis and the other is truncated and possibly non-functional (M azeyrat and M itchell, 1998; Dechend et aL,

1998). Hum an and cow represent the most phylogenetically distant species in which functional TSPY sequences were found. In the cow, the TSPY cluster is located on Yp and comprises 50-200 copies. It is not yet clear whether TSPY is present on the marsupial Y chrom osom e (Delbridge et aL,

1997).

Recently, searches of EST databases have identified a num ber of novel genes, with significant hom ology to the TSPY -SET -N A Pl family. These have been designated TSPY-Like (TSPYL) and occur in m ouse and man. One TSPY L gene is located to human chrom osom e 6 and m ouse chrom osom e 10 and is ubiquitously expressed (Vogel et aL, 1998). Both the hum an and mouse Tspyl hom ologues are intronless, indicating that TSPY L has arisen by an ancient retroposition event.