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Molecular analysis of the Y chromosome AZFc region in Japanese infertile males with spermatogenic defects
Journal of Reproductive Immunology 53 (2002) 37 – 44 www.elsevier.com/locate/jreprimm
Molecular analysis of the Y chromosome AZFc region in Japanese infertile males with spermatogenic defects Hideaki Sawai a,*, Shinji Komori a,b, Koji Koyama a,b a
Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, 1 – 1 Mukogawa-cho, Nishinomiya-shi, 663 – 8501, Japan b Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya-shi, 663 – 8501, Japan Accepted 16 April 2001
Abstract Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the human Y chromosome. Deletion in three Y chromosomal regions—AZFa, AZFb and AZFc— has been reported to disrupt spermatogenesis and cause infertility. Several candidate genes responsible for spermatogenesis have been identified in these regions and some of them are thought to be functional in human spermatogenesis. Here we report on clinical and molecular studies of Y chromosome micro-deletions in Japanese. In these studies the data from 157 infertile Japanese men with azoospermia and oligozoospermia was analyzed and divided into 5 categories based on spermatozoa count. Sixteen sets of primers were used for polymerase chain reaction (PCR) to amplify sequence tagged site markers. One common deletion in the AZFc region was identified in infertile men. On the other hand, no deletions around the AZFc region were identified in fertile men. Japanese infertile men in our study had a common deletion in the AZFc region of the Y chromosome. A genomic clone was obtained by PCR screening of the P1 phage artificial chromosome (PAC) library. This clone was analyzed by Southern blotting using a PCR amplified probe of sY240. Our analysis of the genomic sequence of the clone suggests that this locus may contain specific genes for spermatogenesis. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Y chromosome; Micro-deletion; Azoospermia factor (AZF); Deleted in azoospermia (DAZ)
* Corresponding author. Tel.: + 81-798-45-6481; fax: + 81-798-46-4163. E-mail address: [email protected] (H. Sawai). 0165-0378/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 0 1 ) 0 0 0 9 0 - 0
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H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
1. Introduction Human Y chromosome deletions that are possibly related to male infertility were first reported in the human chromosome by Tiepolo and Zuffardi (1976). Deletions of the distal part of the long chromosome arm (Yq) were detected in six infertile males. Chromosome banding indicated that these deletions were located in Yq11. Furthermore, these findings indicated that the genetic factor indispensable for spermatogenesis is also located in this locus (Yq11) of the Y chromosome. This region was defined as the azoospermic factor (AZF). However, further analysis of this region was difficult because a detailed map of the Y chromosome was not available at that time. Vergnaud et al. (1986) subdivided this region into two intervals, 5 and 6. Ma et al. (1992) then reported a precise map (14 intervals) in Yq11 using Y specific DNA probes. Finally, Vollrath et al. (1992) reported the first PCR based STS, sequence-tagged sites, interval map of the Y chromosome. In this study a deletion map of the human Y chromosome was constructed by testing 96 individuals with partial Y chromosomes for the presence or absence of many DNA loci. Most of the 132 Y chromosomal loci mapped were STS, detected by means of PCR. These studies resolved the euchromatic region (short arm, centromere, and proximal long arm) of the Y chromosome into 43 ordered intervals. The human Y chromosome was then mapped physically by Foote et al. (1992) by assembling 196 recombinant DNA clones, each containing a segment of the chromosome, into a single overlapping array including more than 98 percent of the euchromatic portion of the Y chromosome. Kobayashi et al. (1994) reported common deletions between DYS7C and DYS239 indicating the presence of at least one additional gene, deletion of which causes azoospermia. The AZF region in the 6C-6E interval of Yq11, the most distal region required for fertility, was then mapped by Reijo et al. (1995) and termed the ‘deleted in azoospermia’ (DAZ) region. DAZ is implicated in infertility and is the most common molecularly-defined cause of infertility in humans. Deletions in the DAZ region occur in 13% of azoospermic men and 6% of severely oligozoospermic men (Kostiner et al., 1998). Vogt et al. (1996) reported three different AZF regions in Yq11, AZFa, AZFb and AZFc. Their findings indicate that two different spermatogenesis genes are present in the corresponding genomic Y regions (AZFa and AZFb). They consider the AZFc region to correspond to the AZF region as defined by Reijo et al. (1995). We investigated whether azoospermia and severe oligozoospermia is caused by AZFc deletions in Japanese and, if so, which region is important for spermatogenesis.
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
39
2. Materials and methods
2.1. Subjects To screen Y chromosome deletions by PCR, genomic DNA was prepared from peripheral blood samples from 157 Japanese males who presented to infertility clinics and whose sperm count was azoospermic or severe oligozoospermic (sperm count less than 1×106/ml). Patients were classified into 5 categories as shown in Table 1.
2.2. PCR We examined each male for the presence of Y chromosome STS as shown in Table 2. These sequences and amplification conditions were described previously (Vollrath et al., 1992; Reijo et al., 1995). Amplified products were electrophoresed on 3% agarose gel and stained by ethidium bromide and photographed.
2.3. Southern blotting PCR products were subjected to Southern blotting when amplification was not observed by ethidium bromide staining. Amplified products were transferred to a nylon membrane, and then Southern blotting was performed using standard procedures with PCR products from healthy individuals as probes.
Table 1 Classification of male infertility Group
Sperm count
Number of patients
A OA OI OII OIII
Azoospermia (unknown etiology) Azoospermia (obstruction) B1×105/ml 1×105/ml5spermatozoaB1×106/ml 1×106/ml5
24 20 33 30 50
Group A, azoospermia; Group OA, azoospermia with obstruction; Group OI, spermatozoa B1×105/ml; Group OII, 1×105/ml5spermatozoaB1×106/ml, Group OIII; 1×106/ml5 spermatozoa.
40
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
Table 2 Primer sequence for PCR amplification STS marker
Forward primer
Reverse primer
Product length
sY138
CACATGAAGCACTG GAACTG AGTTAGTAAGCCCC AGTTATCCTCC TCAAATAGCAGCAA TTTAATAT TTACTTCCTTAAGTC AAAGCGG GGGTTTTGCCTGCAT ACGTAATTA GGGTGTTACCAGAA GGCAAA CAGTGATACACTCG GACTTGTGTA GTTACAGGATTCGG CGTGAT CCCCATCGGTAAAC CAAATCA GAATGTGTATTCAA GGACTTCTCG GGTGAGTCAAATTA GTCAATGTCC CTCTGGGACAAGTG TTCCTTG GGTCTTTAAAAGGT GAGTCAAATT CTCAGAAGTCCTCC TAATAGTTCC TACATGTTATGTGC TATGCC TACGGGTCTCGAAT GGAATA
AGGGCCTGAGTCTCC AGG TTTGGAAAAGGACAC CTTATTAGCCA GCACCTGAAGAGCTG CTTG CTGAGACAGCAAGAC CAATCC CCTAAAAGCAATTCTA AACCTCCAG GAACCGTATCTACCAA AGCAGC GTTATTTGAAAAGCTA CACGGG CTCGTCATGTGCAGCC AC CCCATTGAAGTTTCAA GGTGTCA TACTTCCTTCGGGGCC TCT CCTTACCACAGGACAG AGGG CATTGGCATGAATGTG TATTCA AGACAGAGGGAACTT CAAGACC ACAGTGGTTTGTAGCG GGTA CACATTATATAATATG TATGTTGTC TCATTGCATTCCTTTCC ATT
170
sY233 sY240 sY245 sY277 sY254 sY283 sY255 sY236 sY267 sY272 sY269 sY273 sY158 sY159 sY160
115 247 101 275 107 375 126 94 102 93 94 95 231 550 236
2.4. PCR screening of human genome To obtain a clone that included the common deleted region, the P1 artificial chromosome (PAC) library of the human genome was screened using PCR. Actual screening was performed by Incyte Genomics, Inc. (Palo Alto, CA).
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
41
3. Results We detected Y chromosome deletions in 12 males in the AZFc region (Fig. 1). All of these patients were severely oligozoospermic or azoospermic. No deletions were observed in any of the healthy controls. Eleven males had the same in the sY240 region. Three of these individuals (OI-389, OII-390, OII-207) had deletions only in the sY240 region. The other seven patients had additional deletions around the sY240 region. Individuals OII-40 and OII-132 had continuous deletions around the sY240 region. On the other hand, individuals A-401, OA-469, A-401, OA-469, OI-191, OI-342 and OI-430 had intermittent deletions. OII-30 had no deletions in the sY240 region, but deletions were observed in the sY267 region. These results are summarized in Fig. 1. To confirm deletions, Southern blotting was performed when deletions were detected by PCR. Although the PCR products were not easily discernable, Southern blotting rendered amplification clearly visible (Fig. 2). A genomic clone of approximately 120 kb was obtained by screening the P1 phage artificial chromosome (PAC) library with PCR. The clone was then analyzed by Southern blotting using a PCR amplified probe of sY240. Genomic sequence analysis suggested that this locus may contain specific genes for spermatogenesis. A 6.56 kb fragment was observed after digestion of the clone with Bam HI and Not I. Eco RI and Not I digestion produced
Fig. 1. Micro-deletions of Y chromosome detected in male infertility. Black bars indicate the amplified region (no deletions). Group A, azoospermia; Group OA, azoospermia with obstruction; Group OI, spermatozoa B1 ×105/ml; Group OII, 1 × 105/ml5 spermatozoaB 1× 106/ml; Group OIII, 1 ×106/ml5spermatozoa.
42
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
Fig. 2. PCR amplification and Southern blotting. (a) PCR products show faint bands in patients I-55, I-167 and I-197. M: FX174/HaeIII (b) Southern blotting to confirm PCR amplification. No deletions were observed in these three patients.
a 9.42 kb fragment. The lengths of these fragments are analogous to those produced by similar digestion of the Homo sapiens BAC clone RP11214M24 (Gene Bank AC025735).
4. Discussion Our findings indicate that severe oligozoospermia and azoospermia are caused in some patients by deletions on the Y chromosome. In our study 12 patients had deletions in the AZFc region. These microscopic deletions were confined to the AZFc region, with none observed in the DAZ region (Kato et al., 2001 in press). Our results show that some hot spots exist in Yq11. In this region several gene families with testis-specific expression have been mapped. RBMY and DAZ are well-known genes and they encode an RNA-binding motif (Reijo et al., 1995; Vogt et al., 1996). In addition to these genes, several other genes have been reported in this region. It may be possible that the common deleted region observed in our study may involve important genes for spermatogenesis. Although these deletions have been reported to be associated with male infertility, no gene specific mutations that lead to azoospermia have been reported (Vereb et al., 1997). If some genes are important for spermatogenesis, both gene mutations and deletions in the AZF region should have been
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
43
observed. One possible explanation is a repetitive sequence of the genes. Some candidate genes (DAZ et al.) are known to be repetitive on the Y chromosome. Infertility may be caused by the loss of repetitive DAZ gene clusters. Deletions of one or some DAZ genes may be silent or cause mild reduction of sperm counts. Some recent reviews are helpful for precise understanding of the molecular and clinical significance of Y chromosome deletions (Kostiner et al., 1998; Vogt, 1998) One of the serious points under discussion is whether Y chromosome analysis and genetic counseling should be offered to infertile couples (Kostiner et al., 1998). Transmission of infertility to offspring is probable if Y chromosome deletions are detected. At the present time there is insufficient information to predict the degree of spermatogenic impairment. Our recent study has shown that if the microdeletion of Y chromosome is detected in peripheral blood lymphocyte of an infertile male, the same deletion exists in each Y chromosome of the spermatozoa (Komori et al., 2001). When Y chromosome testing is performed and deletions are observed, the significance and limitations should be properly explained to the patient. Although the deletion may be passed on to the male offspring, the severity of spermatogenic impairment is difficult to predict.
References Foote, S., Vollrath, D., Hilton, A., Page, D.C., 1992. The human Y chromosome: overlapping DNA clones spanning the euchromatic region. Science 258, 60 –66. Kato H., Komori S., Nakata Y., Sakata K., Kanazawa R., Handa M., Kobayashi S., Koyama K., Isojima S. Screening for deletions of the D1623 of the Y chromosome in Japanese azoospermic and oligozoospermic men, J. Hum. Genet. (in press). Kobayashi, K., Mizuno, K., Hida, A., Komaki, R., Tomita, K., Matsushita, I., Namiki, M., Iwamoto, T., Tamura, S., Minowada, S., Nakahori, Y., Nakagome, Y., 1994. PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis. Hum. Mol. Genet. 3, 1965 –1967. Komori, S., Nakata, Y., Sakata, K., Kato, H., Koyama, K., 2001. Analysis for microdeletions of Y chromosome in a single spermatozoon from a man with severe oligozoospermia. J. Hum. Genet. 46, 76 –79. Kostiner, D.R., Turek, P.J., Reijo, R.A., 1998. Male infertility: analysis of the markers and genes on the human Y chromosome. Hum. Reprod. 13, 3032 –3038. Ma, K., Sharkey, A., Kirsch, S., Vogt, P., Keil, R., Hargreave, T.B., McBeath, S., Chandley, A.C., 1992. Towards the molecular localization of the AZF locus: mapping of microdeletions in azoospermic men with 14 subintervals of interval 6 of the human Y chromosome. Hum. Mol. Genet. 1, 29 –33. Reijo, R., Lee, T.-Y., Salo, P., Alagappan, R., Brown, L.G., Rosenberg, M., Rozen, S., Jaffe, T., Straus, D., Hovatta, O., de la Chapelle, A., Silber, S., Page, D., 1995. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genet. 10, 383 – 393.
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Tiepolo, L., Zuffardi, O., 1976. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum. Genet. 34, 119– 124. Vereb, M., Agulnik, A.I., Houston, J.T., Lipschultz, L.I., Lamb, D.J., Bishop, C.E., 1997. Absence of DAZ gene mutations in cases of non-obstructed azoospermia. Mol. Hum. Reprod. 3, 55 –59. Vergnaud, G., Page, D.C., Simmler, M.C., Brown, L., Rouyer, F., Noel, B., Botstein, D., de la Chapelle, A., Weissenbach, J.A., 1986. A deletion map of the human Y chromosome based on DNA hybridization. Am. J. Hum. Genet. 38, 109 –124. Vogt, P.H., Edelmann, A., Kirsch, S., Henegariu, O., Hirschmann, P., Kiesewetter, F., Ko¨ hn, F.M., Schill, W.B., Farah, S., Ramos, C., Hartmann, M., Hartschuh, W., Meschede, D., Behre, H.M., Castel, A., Nieschlag, E., Weidner, W., Gro¨ ne, H.-J., Jung, A., Engel, W., Haidl, G., 1996. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet. 5, 933 –943. Vogt, P.H., 1998. Human chromosome deletions in Yq11, AZF candidate genes and male infertility: history and update. Mol. Hum. Reprod. 4, 739 –744. Vollrath, D., Foote, S., Hilton, A., Brown, L.G., Beer-Romero, P., Bogan, J.S., Page, D.C., 1992. The human Y chromosome: a 43-interval map based on naturally occurring deletions. Science 258, 52 –59.
Molecular analysis of the Y chromosome AZFc region in Japanese infertile males with spermatogenic defects Hideaki Sawai a,*, Shinji Komori a,b, Koji Koyama a,b a
Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, 1 – 1 Mukogawa-cho, Nishinomiya-shi, 663 – 8501, Japan b Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya-shi, 663 – 8501, Japan Accepted 16 April 2001
Abstract Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the human Y chromosome. Deletion in three Y chromosomal regions—AZFa, AZFb and AZFc— has been reported to disrupt spermatogenesis and cause infertility. Several candidate genes responsible for spermatogenesis have been identified in these regions and some of them are thought to be functional in human spermatogenesis. Here we report on clinical and molecular studies of Y chromosome micro-deletions in Japanese. In these studies the data from 157 infertile Japanese men with azoospermia and oligozoospermia was analyzed and divided into 5 categories based on spermatozoa count. Sixteen sets of primers were used for polymerase chain reaction (PCR) to amplify sequence tagged site markers. One common deletion in the AZFc region was identified in infertile men. On the other hand, no deletions around the AZFc region were identified in fertile men. Japanese infertile men in our study had a common deletion in the AZFc region of the Y chromosome. A genomic clone was obtained by PCR screening of the P1 phage artificial chromosome (PAC) library. This clone was analyzed by Southern blotting using a PCR amplified probe of sY240. Our analysis of the genomic sequence of the clone suggests that this locus may contain specific genes for spermatogenesis. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Y chromosome; Micro-deletion; Azoospermia factor (AZF); Deleted in azoospermia (DAZ)
* Corresponding author. Tel.: + 81-798-45-6481; fax: + 81-798-46-4163. E-mail address: [email protected] (H. Sawai). 0165-0378/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 0 1 ) 0 0 0 9 0 - 0
38
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
1. Introduction Human Y chromosome deletions that are possibly related to male infertility were first reported in the human chromosome by Tiepolo and Zuffardi (1976). Deletions of the distal part of the long chromosome arm (Yq) were detected in six infertile males. Chromosome banding indicated that these deletions were located in Yq11. Furthermore, these findings indicated that the genetic factor indispensable for spermatogenesis is also located in this locus (Yq11) of the Y chromosome. This region was defined as the azoospermic factor (AZF). However, further analysis of this region was difficult because a detailed map of the Y chromosome was not available at that time. Vergnaud et al. (1986) subdivided this region into two intervals, 5 and 6. Ma et al. (1992) then reported a precise map (14 intervals) in Yq11 using Y specific DNA probes. Finally, Vollrath et al. (1992) reported the first PCR based STS, sequence-tagged sites, interval map of the Y chromosome. In this study a deletion map of the human Y chromosome was constructed by testing 96 individuals with partial Y chromosomes for the presence or absence of many DNA loci. Most of the 132 Y chromosomal loci mapped were STS, detected by means of PCR. These studies resolved the euchromatic region (short arm, centromere, and proximal long arm) of the Y chromosome into 43 ordered intervals. The human Y chromosome was then mapped physically by Foote et al. (1992) by assembling 196 recombinant DNA clones, each containing a segment of the chromosome, into a single overlapping array including more than 98 percent of the euchromatic portion of the Y chromosome. Kobayashi et al. (1994) reported common deletions between DYS7C and DYS239 indicating the presence of at least one additional gene, deletion of which causes azoospermia. The AZF region in the 6C-6E interval of Yq11, the most distal region required for fertility, was then mapped by Reijo et al. (1995) and termed the ‘deleted in azoospermia’ (DAZ) region. DAZ is implicated in infertility and is the most common molecularly-defined cause of infertility in humans. Deletions in the DAZ region occur in 13% of azoospermic men and 6% of severely oligozoospermic men (Kostiner et al., 1998). Vogt et al. (1996) reported three different AZF regions in Yq11, AZFa, AZFb and AZFc. Their findings indicate that two different spermatogenesis genes are present in the corresponding genomic Y regions (AZFa and AZFb). They consider the AZFc region to correspond to the AZF region as defined by Reijo et al. (1995). We investigated whether azoospermia and severe oligozoospermia is caused by AZFc deletions in Japanese and, if so, which region is important for spermatogenesis.
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
39
2. Materials and methods
2.1. Subjects To screen Y chromosome deletions by PCR, genomic DNA was prepared from peripheral blood samples from 157 Japanese males who presented to infertility clinics and whose sperm count was azoospermic or severe oligozoospermic (sperm count less than 1×106/ml). Patients were classified into 5 categories as shown in Table 1.
2.2. PCR We examined each male for the presence of Y chromosome STS as shown in Table 2. These sequences and amplification conditions were described previously (Vollrath et al., 1992; Reijo et al., 1995). Amplified products were electrophoresed on 3% agarose gel and stained by ethidium bromide and photographed.
2.3. Southern blotting PCR products were subjected to Southern blotting when amplification was not observed by ethidium bromide staining. Amplified products were transferred to a nylon membrane, and then Southern blotting was performed using standard procedures with PCR products from healthy individuals as probes.
Table 1 Classification of male infertility Group
Sperm count
Number of patients
A OA OI OII OIII
Azoospermia (unknown etiology) Azoospermia (obstruction) B1×105/ml 1×105/ml5spermatozoaB1×106/ml 1×106/ml5
24 20 33 30 50
Group A, azoospermia; Group OA, azoospermia with obstruction; Group OI, spermatozoa B1×105/ml; Group OII, 1×105/ml5spermatozoaB1×106/ml, Group OIII; 1×106/ml5 spermatozoa.
40
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
Table 2 Primer sequence for PCR amplification STS marker
Forward primer
Reverse primer
Product length
sY138
CACATGAAGCACTG GAACTG AGTTAGTAAGCCCC AGTTATCCTCC TCAAATAGCAGCAA TTTAATAT TTACTTCCTTAAGTC AAAGCGG GGGTTTTGCCTGCAT ACGTAATTA GGGTGTTACCAGAA GGCAAA CAGTGATACACTCG GACTTGTGTA GTTACAGGATTCGG CGTGAT CCCCATCGGTAAAC CAAATCA GAATGTGTATTCAA GGACTTCTCG GGTGAGTCAAATTA GTCAATGTCC CTCTGGGACAAGTG TTCCTTG GGTCTTTAAAAGGT GAGTCAAATT CTCAGAAGTCCTCC TAATAGTTCC TACATGTTATGTGC TATGCC TACGGGTCTCGAAT GGAATA
AGGGCCTGAGTCTCC AGG TTTGGAAAAGGACAC CTTATTAGCCA GCACCTGAAGAGCTG CTTG CTGAGACAGCAAGAC CAATCC CCTAAAAGCAATTCTA AACCTCCAG GAACCGTATCTACCAA AGCAGC GTTATTTGAAAAGCTA CACGGG CTCGTCATGTGCAGCC AC CCCATTGAAGTTTCAA GGTGTCA TACTTCCTTCGGGGCC TCT CCTTACCACAGGACAG AGGG CATTGGCATGAATGTG TATTCA AGACAGAGGGAACTT CAAGACC ACAGTGGTTTGTAGCG GGTA CACATTATATAATATG TATGTTGTC TCATTGCATTCCTTTCC ATT
170
sY233 sY240 sY245 sY277 sY254 sY283 sY255 sY236 sY267 sY272 sY269 sY273 sY158 sY159 sY160
115 247 101 275 107 375 126 94 102 93 94 95 231 550 236
2.4. PCR screening of human genome To obtain a clone that included the common deleted region, the P1 artificial chromosome (PAC) library of the human genome was screened using PCR. Actual screening was performed by Incyte Genomics, Inc. (Palo Alto, CA).
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
41
3. Results We detected Y chromosome deletions in 12 males in the AZFc region (Fig. 1). All of these patients were severely oligozoospermic or azoospermic. No deletions were observed in any of the healthy controls. Eleven males had the same in the sY240 region. Three of these individuals (OI-389, OII-390, OII-207) had deletions only in the sY240 region. The other seven patients had additional deletions around the sY240 region. Individuals OII-40 and OII-132 had continuous deletions around the sY240 region. On the other hand, individuals A-401, OA-469, A-401, OA-469, OI-191, OI-342 and OI-430 had intermittent deletions. OII-30 had no deletions in the sY240 region, but deletions were observed in the sY267 region. These results are summarized in Fig. 1. To confirm deletions, Southern blotting was performed when deletions were detected by PCR. Although the PCR products were not easily discernable, Southern blotting rendered amplification clearly visible (Fig. 2). A genomic clone of approximately 120 kb was obtained by screening the P1 phage artificial chromosome (PAC) library with PCR. The clone was then analyzed by Southern blotting using a PCR amplified probe of sY240. Genomic sequence analysis suggested that this locus may contain specific genes for spermatogenesis. A 6.56 kb fragment was observed after digestion of the clone with Bam HI and Not I. Eco RI and Not I digestion produced
Fig. 1. Micro-deletions of Y chromosome detected in male infertility. Black bars indicate the amplified region (no deletions). Group A, azoospermia; Group OA, azoospermia with obstruction; Group OI, spermatozoa B1 ×105/ml; Group OII, 1 × 105/ml5 spermatozoaB 1× 106/ml; Group OIII, 1 ×106/ml5spermatozoa.
42
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
Fig. 2. PCR amplification and Southern blotting. (a) PCR products show faint bands in patients I-55, I-167 and I-197. M: FX174/HaeIII (b) Southern blotting to confirm PCR amplification. No deletions were observed in these three patients.
a 9.42 kb fragment. The lengths of these fragments are analogous to those produced by similar digestion of the Homo sapiens BAC clone RP11214M24 (Gene Bank AC025735).
4. Discussion Our findings indicate that severe oligozoospermia and azoospermia are caused in some patients by deletions on the Y chromosome. In our study 12 patients had deletions in the AZFc region. These microscopic deletions were confined to the AZFc region, with none observed in the DAZ region (Kato et al., 2001 in press). Our results show that some hot spots exist in Yq11. In this region several gene families with testis-specific expression have been mapped. RBMY and DAZ are well-known genes and they encode an RNA-binding motif (Reijo et al., 1995; Vogt et al., 1996). In addition to these genes, several other genes have been reported in this region. It may be possible that the common deleted region observed in our study may involve important genes for spermatogenesis. Although these deletions have been reported to be associated with male infertility, no gene specific mutations that lead to azoospermia have been reported (Vereb et al., 1997). If some genes are important for spermatogenesis, both gene mutations and deletions in the AZF region should have been
H. Sawai et al. / Journal of Reproducti6e Immunology 53 (2002) 37–44
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observed. One possible explanation is a repetitive sequence of the genes. Some candidate genes (DAZ et al.) are known to be repetitive on the Y chromosome. Infertility may be caused by the loss of repetitive DAZ gene clusters. Deletions of one or some DAZ genes may be silent or cause mild reduction of sperm counts. Some recent reviews are helpful for precise understanding of the molecular and clinical significance of Y chromosome deletions (Kostiner et al., 1998; Vogt, 1998) One of the serious points under discussion is whether Y chromosome analysis and genetic counseling should be offered to infertile couples (Kostiner et al., 1998). Transmission of infertility to offspring is probable if Y chromosome deletions are detected. At the present time there is insufficient information to predict the degree of spermatogenic impairment. Our recent study has shown that if the microdeletion of Y chromosome is detected in peripheral blood lymphocyte of an infertile male, the same deletion exists in each Y chromosome of the spermatozoa (Komori et al., 2001). When Y chromosome testing is performed and deletions are observed, the significance and limitations should be properly explained to the patient. Although the deletion may be passed on to the male offspring, the severity of spermatogenic impairment is difficult to predict.
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