A New Deletion of the Mouse Y Chromosome Long Arm Associated With the Loss of Ssty Expression, Abnormal Sperm Development and Sterility

Corresponding author: Paul S. Burgoyne, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom., pburgoy{at}nimr.mrc.ac.uk (E-mail)

Communicating editor: N. ARNHEIM

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The mouse Y chromosome carries 10 distinct genes or gene families that have open reading frames suggestive of retained functionality; it has been assumed that many of these function in spermatogenesis. However, we have recently shown that only two Y genes, the testis determinant Sry and the translation initiation factor Eif2s3y, are essential for spermatogenesis to proceed to the round spermatid stage. Thus, any further substantive mouse Y-gene functions in spermatogenesis are likely to be during sperm differentiation. The complex Ssty gene family present on the mouse Y long arm (Yq) has been implicated in sperm development, with partial Yq deletions that reduce Ssty expression resulting in impaired fertilization efficiency. Here we report the identification of a more extensive Yq deletion that abolishes Ssty expression and results in severe sperm defects and sterility. This result establishes that genetic information (Ssty?) essential for normal sperm differentiation and function is present on mouse Yq.

THE male-specific region of the Y chromosome (MSY) is an inherently hostile environment for genes (GRAVES 1995 ) and it is assumed that genes still retained on the mammalian MSY must afford some form of male benefit, for example, by functioning in spermatogenesis (LAHN and PAGE 1997 ; BURGOYNE 1998 ; SKALETSKY et al. 2003 ). However, we have recently shown that in the mouse only two Y genes—Sry to trigger testis development and Eif2s3y that has an essential role during spermatogonial proliferation—are needed for spermatogenesis to complete meiosis, including both reduction divisions (MAZEYRAT et al. 2001 ; P. S. BURGOYNE, unpublished observations). Thus, if other genes on the mouse Y chromosome have substantive roles in the normal spermatogenetic process, it is likely to be during the haploid phase of sperm differentiation (spermiogenesis). Studies of mice with Yq deficiencies have implicated genetic information on MSYq [the Y long arm excluding the pseudoautosomal region (PAR)] as having an important role in spermiogenesis that impacts on sperm quality and function. Males with large interstitial Yq deletions exhibit an increased incidence of mild sperm-head anomalies with some impairment of sperm function and an intriguing distortion of the offspring sex ratio in favor of females (MORIWAKI et al. 1988 ; SUH et al. 1989 ; STYRNA et al. 1991A , STYRNA et al. 1991B , STYRNA et al. 2002 ; XIAN et al. 1992 ; CONWAY et al. 1994 ). XSxr^aY*^X males, in which the only MSY contribution is from the Yp-derived Sxr^a factor and that thus lack MSYq, have grossly abnormal sperm and are sterile (BURGOYNE et al. 1992 ).

The mouse MSYq appears to be composed largely of repeats (NISHIOKA and LAMOTHE 1986 ; EICHER et al. 1989 ; NISHIOKA et al. 1992 , NISHIOKA et al. 1993A , NISHIOKA et al. 1993B ; FENNELLY et al. 1996 ; NAVIN et al. 1996 ; BERGSTROM et al. 1997 ). Among these repeats is a complex gene family, Ssty, which is transcribed exclusively in the testis during the spermatid stages (BISHOP et al. 1983 ; PRADO et al. 1992 ; CONWAY et al. 1994 ). This gene family probably originated from a retroposed transcript of the autosomal gene Spin (OH et al. 1997 , OH et al. 1998 ; TOURE et al. 2004 ) and at least some Ssty transcripts retain an open reading frame that encodes a putative SPIN-like protein. Very recently we have identified an Ssty protein product and have shown that this derives from only a minor subset of the transcribed Ssty genes (TOURE et al. 2004 ). It has been proposed that Ssty deficiency is responsible for the mild and severe sperm defects in mice with large interstitial Yq deletions and XSxr^aY*^X males, respectively (BURGOYNE et al. 1992 ; CONWAY et al. 1994 ).

The problem with attributing the severe sperm abnormalities and sterility of XSxr^aY*^X males exclusively to the lack of MSYq is that these males are now also known to lack the majority of copies of Rbmy, a gene family on Yp close to the centromere that has also been implicated in maintaining sperm quality (ELLIOTT et al. 1996 ; MAHADEVAIAH et al. 1998 ). Here we report the identification of a new mouse Yq deletion that removes all MSYq copies of Ssty, while leaving Yp (including all copies of Rbmy) intact. This deletion is also associated with severe sperm-head defects and sterility.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mice:
All the mice used in this study were produced on a random-bred albino MF1 strain (National Institute for Medical Research stock) background. The sex chromosome complement of the variant genotypes is illustrated in Fig 1. The new deletion was originally detected in male progeny of a cross of Sry-negative XXY^Tdym1 females (LOVELL-BADGE and ROBERTSON 1990 ; GUBBAY et al. 1992 ; MAHADEVAIAH et al. 1993 ) with males carrying an autosomally located Sry transgene. The proband was a putative XXY^Tdym1Sry male, but proved to be either weakly positive or negative for a PCR that detects the Ssty1 family, a subfamily of Ssty that is present in multiple copies throughout most of MSYq. This suggested that the Y^Tdym1 of the proband carried an extensive Yq deletion; we call this Y variant Y^Tdym1qdel. The XXY^Tdym1qdelSry male was sterile, as expected for an "XXY" male. However, the mother proved to carry the same chromosome and was used to establish a stock with this new Y variant.

View larger version (27K): In this window In a new window Download PPT slide   Figure 1. Diagrams of the sex chromosome complement of variant mice, highlighting the MSY deficiencies. (A) The known gene content of the mouse MSY. Yp (shown expanded) carries seven single-copy genes, one duplicated gene (Zfy), and multiple copies of Rbmy. MSYq carries multiple copies of the Ssty gene family. (B) The variant YTdym1 has a complete Y-gene complement except that an 11-kb deletion has removed the testis determinant Sry. XYTdym1 mice are female, but when the Sry deficiency is complemented by an Sry transgene, the mice are normal fertile males. (C) The new variant YTdym1qdel, in addition to the deletion of Sry, has a large deletion removing most of MSYq. (D) The variant YRIIIqdel is an RIII strain Y chromosome with a deletion removing about two-thirds of Yq. (E) XSxraY*X males are male because of the presence of the YRIII-derived sex-reversal factor Sxra attached distal to the X PAR. Sxra comprises most of Yp except for a marked reduction in copies of Rbmy. The Y*X chromosome in effect is an X chromosome with a deletion from just proximal to Amel (close to the X PAR boundary) to within the DXH XF34 sequence cluster adjacent to the centromere. It includes no MSY, but provides a second PAR, which is essential to avoid meiotic arrest. TEL, telomere; CEN, centromere; Yp, Y short arm; MSYq, male-specific region of the Y long arm; PAR, pseudoautosomal region of Yq.

To assess the effects of the new Yq deletion on spermatogenesis, it was necessary to produce XY^Tdym1qdelSry males for comparison with XY^Tdym1Sry males (MAHADEVAIAH et al. 1998 ), which are the appropriate controls. XY^Tdym1qdel females were therefore mated to XY^d1Sry males (MAHADEVAIAH et al. 1998 ) to provide the same Sry transgene that is present in the XY^Tdym1Sry males. The production of the XY^Tdym1qdelSry males proved very difficult since the XY^Tdym1qdel females bred very poorly (cf. XY^Tdym1 females; MAHADEVAIAH et al. 1993 ). The XY^Tdym1qdelSry males were identified by PCR (see below) as being positive for the Tdym1 deletion and negative for Ssty1 and were initially distinguished from XXY^Tdym1Sry males by testis palpation on the assumption that they would have substantially larger testes (100 mg vs. 20–25 mg). The initial diagnosis was subsequently checked cytogenetically in air-dried bone marrow metaphases stained with pH 6.8-buffered Giemsa.

XSxr^aY*^X males (BURGOYNE et al. 1992 ) used in comparisons with the new Yq deletion males were produced by mating XY*^X females (EICHER et al. 1991 ; BURGOYNE et al. 1998 ) to XYSxr^a males (CATTANACH 1987 ). XY^RIIIqdel males used to provide sperm and testis samples were derived from a stock originating from the mice described by CONWAY et al. 1994 .

PCR assays:
In defining the new Yq deletion, PCR assays were used that detect markers in Yp, Yq, and the PAR. These markers, together with the primers used, are listed in Table 1.

Southern blot analysis:
The probes used for Southern blot analysis were the Ssty1 cDNA clone pYMT2/B (BISHOP and HATAT 1987 ), an Ssty1 intron probe (TOURE et al. 2004 ), the Ssty2 cDNA clone pC11 (PRADO et al. 1992 ), and an Rbmy cDNA (MAHADEVAIAH et al. 1998 ). Genomic DNA was extracted from tail biopsies and 15 µg was digested with EcoRI, electrophoresed through a 0.8% agarose gel, and transferred to a Hybond-N membrane (Amersham, Buckinghamshire, UK). After fixation, the membrane was hybridized overnight at 60° with ³²P-labeled probes in hybridization buffer (6x SSC, 5x Denhart's, 0.5% SDS, 100 µg/ml salmon sperm DNA). After two 60° washes (30 min 0.5x SSC, 0.1% SDS; 30 min 0.1x SSC, 0.1% SDS) the membrane was exposed to X-ray film or a phosphorimager screen overnight. An additional blot that was loaded with only 4 µg of EcoRI-digested control male DNA to allow longer exposures to X-ray film was prepared. This was hybridized to Ssty probes at very high stringency (hybridization at 65° and two 60-min washes at 65° with 0.1x SSC, 0.1% SDS) and was exposed to X-ray film for 2 weeks.

Northern blot analysis:
The probes used for Northern analysis were the Ssty1/2 cDNAs used for Southern analysis and an actin probe that recognizes - and ß-actin transcripts (MINTY et al. 1981 ). Total RNA (20 µg) was electrophoresed in a 1.4% formaldehyde/agarose gel and transferred to Hybond-N membrane (Amersham) using 20x SSC buffer. The membrane was fixed for 2 hr at 80° and hybridized overnight at 60° with ³²P-labeled probes in hybridization buffer (6x SSC, 5x Denhart's, 0.1% SDS, 50 mM sodium phosphate, 100 µg/ml salmon sperm DNA). After two 60° washes (30 min 0.5x SSC, 0.1% SDS; 30 min 0.1x SSC, 0.1% SDS) the membrane was exposed to X-ray film for 5 hr and subsequently to a phosphorimager screen to allow quantitation of hybridization using ImageQuant software.

Western blot analysis:
Testicular protein lysates were obtained by homogenization in liquid nitrogen and resuspension in Laemmli buffer at 10% w/v. Lysates were then denatured for 10 min at 95° and 5–10 µl were electrophoresed through an SDS/polyacrylamide minigel. Transfer to Hybond-C membrane was performed at 110 mA for 2 hr and the membrane was then processed for immunodetection. The membrane was blocked (PBS, 0.1% Tween, 5% milk powder) for 1 hr and incubated with first antibody diluted in the blocking solution for 2 hr at room temperature: rabbit anti-SSTY1 antibody, 1:500 (TOURE et al. 2004 ); rabbit anti-RBMY antibody, 1:2000 (TURNER et al. 2002 ); and mouse anti-ACTIN, 1:100 (sc-8432, Santa Cruz). After three washes (PBS, 0.1% Tween), the membrane was incubated with the secondary antibody for 45 min at room temperature (anti-rabbit or anti-mouse antibody coupled with HRP from Dako, Carpintaria, CA). Following three washes (PBS, 0.1% Tween), the signal was revealed by chemiluminescence (SuperSignal, Pierce, Rockford, IL) and recorded on X-ray film. Quantitation was carried out using National Institutes of Health Image 1.62 software.

Analysis of the XY^Tdym1qdelSry males:
The males were mated to two normal females for at least 3 weeks and in three cases the added females were checked for copulatory plugs as evidence of mating. When the males were killed, testes were weighed and sperm samples from the initial segment of the capita epididymides were used for sperm counts as previously described (MAHADEVAIAH et al. 1998 ) and/or for sperm smears (see below). One testis was fixed in Bouin and wax embedded and sections were stained with hematoxylin and eosin. XY^Tdym1Sry males were used to provide control material.

Comparisons of sperm-head morphology:
Silver-stained sperm smears were produced using sperm from the initial segment of the capita epididymides (MAHADEVAIAH et al. 1998 ) of four XY^Tdym1qdelSry males together with five XY^Tdym1Sry controls. The slides were coded and randomized along with slides from two XY^RIIIqdel males and one XSxr^aY*^X male for comparative purposes, and 100 sperm from each of two to four slides from each male were classified as described by MAHADEVAIAH et al. 1998 . Slides from two of the XY^Tdym1qdelSry males were subsequently recoded and scored along with the slides from two XSxr^aY*^X males, this time including a new category of "extreme 1a" (see RESULTS and Fig 6C).

View larger version (59K): In this window In a new window Download PPT slide   Figure 2. PCR analysis of the new deletion (here denoted YTdym1qdel). Positive and negative controls are indicated by "+" and "-" throughout. (A) Identification of the proband. Samples 1–4 are from brothers derived from the XXYTdym1 x XYSry cross. Samples 1 and 2 are from XXSry males and 4 is from an XYSry male. Sample 3 is positive for the Tdym1 deletion in Yp that has removed Sry, implying that YTdym1 is present; it should therefore also be positive for Ssty1 located on Yq. However, only a faint Ssty1 band is seen and a similar faint band is seen in the XX (-) negative control. This implies that most or all Ssty1 copies on Yq have been deleted. (B) Evidence that the deletion is an interstitial deletion restricted to Yq. (B1) Analysis of Yp markers. The proband 3 and his brother 4 (XYSry) are positive for all Yp markers tested. (B2) Analysis of Ssty1 using more stringent conditions and of Ssty2. Samples 5 and 6 are from XSxraY*X males that lack MSYq, and samples 7 and 8 are from XYTdym1qdelSry males. Neither Ssty1 nor Ssty2 is detectable in the deletion males under stringent conditions. Thus the PCR results suggest that all Ssty copies have been deleted in YTdym1qdel. (B3) Analysis of Sts located in the distal PAR. We have previously shown that the 129 strain has a variant Sts locus that is not detected by our standard PCR. The samples are 129 XY (-), MF1 XY (+), X129O (9), X129YTdym1qdel (10), and X129XMF1 (11). The deletion mouse is Sts positive and since the X is from the 129 strain, the PCR product must have been amplified from an Sts copy present on YTdym1qdel.

View larger version (61K): In this window In a new window Download PPT slide   Figure 3. Southern blot analysis of YTdym1qdel. (A–D) A blot of EcoRI-digested XX, XYTdym1qdelSry, XSxraY*X, XYRIIIqdel, and XYRIII DNA was consecutively hybridized with Ssty1 cDNA, Ssty1 intron, Ssty2 cDNA, and Rbmy intron probes, which all detect fragments present in multiple copies in normal males. The XYTdym1qdelSry male appears to be negative with all three Ssty probes, but is equivalent to the control male with respect to Rbmy hybridization. As expected, the XSxraY*X DNA samples show markedly reduced Rbmy hybridization, because the Yp-derived Sxra has only about one-eighth of the normal Yp complement of Rbmy copies. (E and F) Southern blot of EcoRI-digested XX, XYTdym1qdelSry, XSxraY*X, and XYRIII DNA, for which only 4 µg (instead of 15 µg) of XYRIII DNA was loaded. After exposure for 2 weeks, only two very faint Ssty1/2-hybridizing fragments are detected in the deletion males; these DNA fragments are not Y specific since they are also seen in XX female DNA.

View larger version (85K): In this window In a new window Download PPT slide   Figure 4. The YTdym1qdel chromosome in mitotic metaphase. (A) The YTdym1qdel chromosome is minute compared to chromosome 19. (B) The YTdym1 from which the deleted chromosome arose is a little larger than chromosome 19.

View larger version (132K): In this window In a new window Download PPT slide   Figure 5. Impaired sperm shedding in XYTdym1qdelSry males. (A–C) Control XYTdym1Sry testis tubule sections. (A) Early stage VIII tubule with step 8 round spermatids together with a layer of mature sperm (arrow). (B) Late stage VIII tubule from which the mature sperm have been shed. (C) Late stage IX tubule with elongated stage 9 spermatids. (D) XYTdym1qdelSry late stage VIII tubule with retention of a layer of mature sperm (arrow). (E) XYTdym1qdelSry late stage IX testis tubule showing continued retention of pockets of mature sperm (arrows). Inset is a higher-power view of step 9 spermatids and a pocket of mature sperm that have not been shed.

View larger version (45K): In this window In a new window Download PPT slide   Figure 6. Sperm abnormalities in XYTdym1qdelSry males. (A) Diagram of our standard categories used in classifying abnormal sperm. (B–E) Examples of silver-stained sperm from control XYTdym1Sry males and the three genotypes with Yq deficiencies. In the XYTdym1qdelSry males, many of the sperm fell into a category (extreme 1a) that was intermediate between abnormalities 1a and 3. The sperm abnormality classes 1a, extreme 1a, 3, and 4 can be viewed as showing progressively more severe acrosome abnormalities. (F) The percentage of normal, slightly abnormal, and grossly abnormal sperm, together with the proportions of the more prevalent sperm abnormalities, in XYTdym1qdelSry males compared with those in XYTdym1Sry controls and the previously characterized genotypes XYRIIIqdel (predominantly slightly abnormal sperm) and XSxraY*X (all grossly abnormal sperm). The sperm from XYTdym1qdelSry males are much more severely affected than those from XYRIIIqdel males, but do not show the 100% gross abnormality characteristic of XSxraY*X males. Numbers of animals and of sperm per genotype are: XYTdym1Sry 5, 2000; XYTdym1qdelSry 4, 1500; XYRIIIqdel 2, 400; and XSxraY*X 1, 400.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Origin of the Yq deletion:
The proband was a putative XXY^Tdym1Sry male (see MATERIALS AND METHODS), but, although shown by PCR analysis to be carrying the Tdym1 deletion that has removed Sry from Yp, the male appeared to lack Ssty1, a multicopy gene distributed over most of MSYq (Fig 2A). This male had received this putative variant Y chromosome from his mother; she nevertheless had five sisters carrying the original Ssty-positive Y^Tdym1 chromosome. We concluded that a Yq deletion event removing most or all copies of Ssty1 had likely occurred during meiosis in the proband's XXY^Tdym1 grandmother.

Characterization of the deletion:
Once the initial PCR diagnosis on the proband was found to be replicated in the mother and a brother, more Y PCR assays were carried out to assess whether the deletion was restricted to MSYq (Fig 2B). First, PCR assays that detect a GATA/GACA (Y207) repeat near the Yp telomere and the Rbmy gene cluster on Yp near the centromere were carried out. Both these PCRs were positive, suggesting that most, if not all, of Yp was present, except for the preexisting 11-kb Tdym1 deletion that had removed Sry. Three other Yp-located genes were also shown to be present (Fig 2B1). We subsequently checked more mice with the new deletion for both Ssty1 and Ssty2, using XSxr^aY*^X DNA as a control in which most of Yp is present but MSYq is absent. These two genotypes were indistinguishable, suggesting that all MSYq-located copies of Ssty have been deleted (Fig 2B2). To test for the PAR-located Sts gene, we introduced the strain 129 X chromosome, which has a variant Sts that is not detected by the standard Sts PCR assay (BURGOYNE et al. 1998 ), thus enabling the Sts status of the new Y variant to be assessed. This showed that Sts is present (Fig 2B3). These results strongly suggest that the deletion is an interstitial Yq deletion removing all Ssty copies but leaving at least the distal PAR intact.

We subsequently checked the extent of the Ssty deficiency by Southern analysis; DNA samples from Yq-deficient XSxr^aY*^X and XY^RIIIqdel males were included for comparison (Fig 3). Using an Ssty1 cDNA probe and an Ssty1 intron probe that is present in a subset of Ssty1 copies (TOURE et al. 2004 ), we could detect no hybridization in the male with the new deletion; furthermore, with an Ssty2 cDNA probe, no hybridizing bands remained (Fig 3, A–C). This suggested that all MSYq-located Ssty copies are absent, as in XSxr^aY*^X males. Consistent with Yp being intact, a probe detecting the multicopy Rbmy gene family located on Yp hybridized with normal intensity (Fig 3D). As expected, Rbmy hybridization was markedly reduced in XSxr^aY*^X males because the Yp-derived Sxr^a factor has only approximately one-eighth of the normal Yp complement of Rbmy copies (MAHADEVAIAH et al. 1998 ). We produced a further blot loaded with much less control DNA to allow longer exposures of the filter with the Ssty probes (Fig 3E and Fig F). Two very faint bands were seen with the Ssty probes with a 2-week exposure, but these bands were also present in XSxr^aY*^X males that lack MSYq and in XX females. We presume these bands are due to hybridization to X or autosomally located Ssty1-related copies, since we have previously cloned and sequenced the PCR product intermittently obtained in XX females with Ssty1 primers (see Fig 2A) and found two distinct Ssty-related sequences (P. S. BURGOYNE and S. H. LAVAL, unpublished results).

In summary, the deletion is an interstitial deletion within Yq that has occurred in a Y^Tdym1 chromosome and that has removed all MSYq-located copies of Ssty. The extent of the Yq deletion is evident in bone marrow metaphases in which this chromosome is minute (Fig 4). The chromosome is here denoted "Y^Tdym1qdel."

XY^Tdym1qdelSry males have gross sperm defects and are sterile:
The XXY^Tdym1qdelSry proband lacked spermatogenic cells as expected for any male with two X chromosomes. We therefore attempted to produce some XY^Tdym1qdelSry transgenic males (see MATERIALS AND METHODS) to enable any effects of the deletion on spermatogenesis to be assessed. After a period of a few months, a single putative XY^Tdym1qdelSry male was produced with testes that palpation suggested were of normal size. However, when the male was mated to two females, they failed to become pregnant. The male was killed and the genotype confirmed by bone marrow metaphase analysis. Testis size (122 and 103 mg) fell within the range (94–122 mg) that we have previously observed for XY^Tdym1Sry males (MAHADEVAIAH et al. 1998 ); these latter males are of normal fertility. We have since test mated eight more XY^Tdym1qdelSry males and for three of these males, mating was confirmed by the presence of copulatory plugs. Testis weights were again comparable to controls but all proved to be sterile (Table 2).

With Y chromosome deletions, the likely causes of sterility relate to impairment of sperm production and/or sperm quality. From the testis histology, sperm production appeared qualitatively normal. However, it was noted that there was a problem with sperm shedding, late spermatids (step 16) being retained along with step 9 spermatids in stage IX tubules and beyond, instead of being shed during stage VIII (Fig 5). Sperm counts for the initial segment of the caput epididymis for the first four males suggested a substantial reduction in sperm output (0.2–0.5 million vs. 2.2–3.7 million for controls). It was then realized that an unusually high number of detached abnormally shaped sperm heads were present in the sperm suspension (see examples in Fig 6C); these were being missed in the counting procedure. For four subsequent males for which care was taken to include the detached misshapen sperm heads, the sperm counts of 0.5–1.1 million were within the fertile range, although significantly reduced (P < 0.001) compared to controls (Table 2).

To assess the extent of the sperm abnormalities, silver-stained sperm smears were analyzed from four of the XY^Tdym1qdelSry males together with four XY^Tdym1Sry controls. For comparative purposes, sperm smears were also included from two XY^RIIIqdel males (predominantly mild sperm-head defects; CONWAY et al. 1994 ) and from one XSxr^aY*^X male (100% grossly abnormal sperm; BURGOYNE et al. 1992 ). The results (Fig 6C and Fig F) show that all the sperm in XY^Tdym1qdelSry males are abnormal, and the majority severely so. They are thus much more severely affected than the sperm of XY^RIIIqdel males (Fig 6D and Fig F). Nevertheless, all four males had sperm in the "slightly abnormal" category (range 8–24%) and thus were less severely affected than the XSxr^aY*^X male analyzed, which had 100% grossly abnormal sperm (Fig 6E and Fig F), as did previous males of this genotype. In the course of scoring some of the slides (all of which proved to be from XY^Tdym1qdelSry males), difficulty was experienced in categorizing a prevalent sperm anomaly, which appeared to be an extreme form of the mild 1a anomaly characteristic of XY^RIIIqdel males. Most of these extreme 1a sperm (see Fig 6C, e1a) were initially assigned to class 3, since they could not be classified as a mild anomaly. Rescoring coded slides from two of the XY^Tdym1qdelSry males along with two XSxr^aY*^X males, while including a new category of extreme 1a, confirmed that this is a prevalent sperm anomaly in XY^Tdym1qdelSry males and reinforced the conclusion that they are less severely affected than XSxr^aY*^X males (Table 3).

Ssty and Rbmy expression in relation to abnormal sperm development:
The more severe sperm anomalies in XY^Tdym1qdelSry compared to XY^RIIIqdel males parallels the increase in Ssty gene deficiency. However, with multi-copy Y genes it is particularly important to establish whether the gene deficiencies are reflected in equivalent reductions at the level of RNA and/or protein. We therefore compared Ssty expression in these males with that of control males by Northern and Western analysis. As previously reported (CONWAY et al. 1994 ; TOURE et al. 2004 ), XY^RIIIqdel males have a clear (54% by phosphorimager analysis) reduction in Ssty2 transcripts, but Ssty1 transcript levels are less affected (32% reduction; Fig 7A). Paradoxically, despite the modest reduction in Ssty1 transcripts, SSTY1 protein appeared to be present at higher levels than in the XY^RIII control (Fig 7B). This apparent increase was confirmed in a further comparison of two XY^RIIIqdel and two XY^RIII males; quantitation indicated a greater than twofold increase in SSTY1 expression (Fig 7C). Consistent with the Southern analysis, XY^Tdym1qdelSry males had only very faint hybridization on Northerns with Ssty1 and Ssty2 probes (98% reduction in both cases); there was no detection of SSTY1 protein by Western analysis (Fig 7A and Fig B). We also assessed Ssty expression in XSxr^aY*^X males and found it to be indistinguishable from that in XY^Tdym1qdelSry males (Fig 7A and Fig B).

View larger version (72K): In this window In a new window Download PPT slide   Figure 7. A comparison of Ssty and Rbmy expression in XYRIIIqdel, XSxraY*X, and XYTdym1qdelSry males. (A) Two Northern blots of testis RNA probed with Ssty1 and Ssty2 cDNA probes with actin serving as a loading control. In XYRIIIqdel males, after allowing for loading, there is a 32% reduction in Ssty1 transcripts relative to XYRIII controls (Ssty1/actin ratios from phosphorimager analysis: XYRIIIqdel 56, 51; XYRIII 37, 35) and a 54% reduction in Ssty2 transcripts (Ssty2/actin ratios: XYRIIIqdel 74, 68; XYRIII 33, 33). In XSxraY*X and XYTdym1qdelSry males, there was only a trace of hybridization with Ssty1 or Ssty2. (B and C) Western analysis with an antibody specific for an Ssty1-encoded protein (SSTY1). There is an increase (more than twofold by quantitation) of SSTY1 protein in XYRIIIqdel males, but no SSTY1 protein is detected in XSxraY*X and XYTdym1qdelSry males. (D) Western analysis using an antibody that detects RBMY protein. XYRIIIqdel and XYTdym1qdelSry males have normal levels of RBMY, which is consistent with their having a normal number of copies of Rbmy. By contrast, XSxraY*X males, which have an 80% reduction in Rbmy copies, have a markedly reduced amount of RBMY protein.

Rbmy expression was checked by Western analysis. XY^Tdym1qdelSry and XY^RIIIqdel males had normal levels of RBMY protein. XSxr^aY*^X males, which have only a few copies of the multi-copy Rbmy gene family remaining in the Yp-derived Sxr^a factor, have a marked (95%) reduction in RBMY protein expression (Fig 7D).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have identified an extensive new interstitial mouse Yq deletion that results in male sterility, thus establishing that the mouse Y long arm carries genetic information essential for fertility. The only MSY genes known to be present on Yq are multiple copies of the complex Ssty gene family. The Southern analysis using Ssty probes at high stringency suggested that all Y-located copies of Ssty have been deleted. It has been previously reported that there are some Yp-located Ssty copies, but these are diverged noncoding copies that would probably not hybridize at high stringency (BOETTGER-TONG et al. 1998 ). In terms of Ssty expression, the new deletion males are indistinguishable from XSxr^aY*^X males that lack all of MSYq (BURGOYNE et al. 1992 ), there being only trace levels of hybridization on Northerns and no expression of the only known Ssty-encoded protein, SSTY1.

The pedigree data indicate that the deletion originated in an XXY^Tdym1 female. We have previously identified Y^Tdym1 chromosomes with Yq deletions among the progeny of XXY^Tdym1 females and have postulated that the frequent self-synapsis of the univalent Y^Tdym1 in these females to form a "hairpin" structure may facilitate intrachromosomal events that result in deletion (MAHADEVAIAH et al. 1993 ). These in fact may be recombination events between runs of Yq repeats that are present in opposite orientations, as in the large palindromic repeats on the human Y (SKALETSKY et al. 2003 ).

In assessing the cause of the sterility of the XY^Tdym1qdelSry males, it was established that they successfully mate with females. Sperm numbers assessed from the initial segment of the caput epididymis were substantially reduced compared to controls, perhaps because shedding of sperm from the epithelium was impaired. Nevertheless, the sperm counts were all higher than those expected to lead to sterility (RODRIGUEZ and BURGOYNE 2000 , RODRIGUEZ and BURGOYNE 2001 ). The sterility is therefore likely to be predominantly due to the defects of sperm differentiation reflected in the distortion of head shape. This is supported by some preliminary in vitro fertilization data: 0/62 eggs incubated with sperm from an XY^Tdym1qdelSry male progressed to the two-cell stage compared with 40/94 and 42/83 for the controls.

The observed consequences of this new Yq deletion support the view (BURGOYNE et al. 1992 ) that the long arm of the mouse Y chromosome carries factors of crucial importance for sperm differentiation and fertility, with the severity of the phenotype being related to the extent of the deletion. Currently, members of the Ssty gene family are the only genes known to be present in MSYq. Since Ssty is expressed in spermatids (CONWAY et al. 1994 ) and is present in multiple copies widely distributed on MSYq, it has the right credentials to explain the relationship between the size of Yq deletions and the severity of the sperm defects. However, interpretation of this putative relationship is not straightforward for two reasons. First, some uncertainties remain as to the protein-coding potential of Ssty transcripts. There is circumstantial evidence that the SSTY1 protein derives from only a subset of Ssty1 genes that contain a 5'-intron that promotes subsequent transcription (TOURE et al. 2004 ). No Ssty2-encoded protein has been identified, nor have we been able to identify any intron-containing members of this subfamily. Second, because partial Yq deletions lead to sex-ratio distortion in favor of females, we have long considered the possibility that the multi-copy Ssty family may function to negate the effects of an X-linked meiotic driver that favors the transmission of the X chromosome (CONWAY et al. 1994 ). This role for Ssty could be mediated at the RNA level, as has been shown in the case for the Stellate/Suppressor of Stellate system in Drosophila (ARAVIN et al. 2001 ). The effects of Yq deletions may therefore depend not only on changes in the levels of Ssty-encoded protein(s), but also on changes in the profile of Ssty transcripts. It is noteworthy in this regard that SSTY1 expression increases in XY^RIIIqdel males, despite the reduction in Ssty1 transcripts; this could be due to an altered balance between protein-coding and noncoding Ssty transcripts, the latter acting to inhibit translation of SSTY1.

The XY^Tdym1qdelSry males described here proved to be indistinguishable from XSxr^aY*^X males with respect to Ssty expression and both genotypes are sterile, yet the analysis of sperm-head morphology showed that the sperm-head defects in XSxr^aY*^X males are more severe. One possible explanation for this increased severity is that XSxr^aY*^X males have a marked reduction in the multicopy Rbmy gene family (MAHADEVAIAH et al. 1998 ), which we show here results in a 95% reduction in RBMY expression. RBMY deficiency has already been implicated as a cause of impaired spermiogenesis in other mice with a deletion removing most of the Rbmy gene cluster from Yp (MAHADEVAIAH et al. 1998 ).

The case for Ssty deficiency being responsible for the abnormal sperm development in XY^Tdym1qdelSry males and the additional Rbmy deficiency for the more severe sperm defects in XSxr^aY*^X males would be strengthened if it could be established that no other genes map to the respective Yq and Yp regions. However, obtaining reliable and complete sequence information for large domains with extensive repeats is a daunting task (KURODA-KAWAGUCHI et al. 2001 ). We are now using microarray analysis to identify transcriptional differences between mice with Yq deficiencies. In conjunction with the sequence information for the mouse genome, this should eventually allow the chromosomal assignment of all downregulated transcripts. However, proof that these gene deficiencies are responsible can probably derive only from transgene rescue approaches. We have already produced a transgenic line with an expressing Rbmy transgene, but for Ssty we need to generate new transgenic lines that, unlike previous lines (TOURE et al. 2004 ), not only transcribe but also translate Ssty.

	ACKNOWLEDGMENTS

We thank Michael Mitchell for sharing unpublished PCR primer sequence information, David Elliott for the RBMY antibody, Ian Harrigan and Wendy Hatton for testis sections, and the Biological Services staff for tailing mice for PCR genotyping. A.T. was supported by a 1-year Institut National de la Santé et Recherche Medicale overseas training fellowship followed by a 2-year European Community "Marie Curie" individual fellowship.

Manuscript received July 28, 2003; Accepted for publication October 27, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ARAVIN, A. A., N. M. NAUMOVA, A. V. TULIN, V. V. VAGIN, and Y. M. ROZOVSKY et al., 2001  Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr. Biol. 11:1017-1027.[CrossRef][Medline]

BERGSTROM, D. E., H. YAN, M. M. SONTI, S. NARAYANSWAMI, and J. K. BAYLERAN et al., 1997  An expanded collection of mouse Y chromosome RDA clones. Mamm. Genome 8:510-512.[CrossRef][Medline]

BISHOP, C. E. and D. HATAT, 1987  Molecular cloning and sequence analysis of a mouse Y chromosome RNA transcript expressed in the testis. Nucleic Acids Res. 15:2959-2969.[Abstract/Free Full Text]

BISHOP, C. E., G. GUELLAEN, D. GELDWERTH, R. VOSS, and M. FELLOUS et al., 1983  Single-copy DNA sequences specific for the human Y chromosome. Nature 303:831-832.[CrossRef][Medline]

BOETTGER-TONG, H. L., A. I. AGULNIK, T. I. TY, and C. E. BISHOP, 1998  Transposition of RhoA to the murine Y chromosome. Genomics 49:180-187.[CrossRef][Medline]

BURGOYNE, P. S., 1998  The mammalian Y chromosome: a new perspective. BioEssays 20:363-366.[CrossRef][Medline]

BURGOYNE, P. S., S. K. MAHADEVAIAH, M. J. SUTCLIFFE, and S. J. PALMER, 1992  Fertility in mice requires X-Y pairing and a Y-chromosomal "spermiogenesis" gene mapping to the long arm. Cell 71:391-398.[CrossRef][Medline]

BURGOYNE, P. S., S. K. MAHADEVAIAH, J. PERRY, S. J. PALMER, and A. ASHWORTH, 1998  The Y* rearrangement in mice: new insights into a perplexing PAR. Cytogenet. Cell Genet. 80:37-40.[CrossRef][Medline]

CATTANACH, B. M., 1987  Sex-reversed mice and sex determination. Ann. NY Acad. Sci. 513:27-29.[CrossRef][Medline]

CONWAY, S. J., S. K. MAHADEVAIAH, S. M. DARLING, B. CAPEL, and Á. M. RATTIGAN et al., 1994  Y353/B: a candidate multiple-copy spermiogenesis gene on the mouse Y chromosome. Mamm. Genome 5:203-210.[CrossRef][Medline]

EICHER, E. M., K. W. HUTCHISON, S. J. PHILIPS, P. K. TUCKER, and B. LEE, 1989  A repeated segment on the mouse Y chromosome is composed of retroviral-related Y-enriched and Y-specific sequences. Genetics 122:181-192.[Abstract/Free Full Text]

EICHER, E. M., D. W. HALE, P. A. HUNT, B. K. LEE, and P. K. TUCKER et al., 1991  The mouse Y* chromosome involves a complex rearrangement, including interstitial positioning of the pseudoautosomal region. Cytogenet. Cell Genet. 57:221-230.[Medline]

ELLIOTT, D. J., K. MA, S. M. KERR, R. THAKRAR, and R. SPEED et al., 1996  An RBM homologue maps to the mouse Y chromosome and is expressed in germ cells. Hum. Mol. Genet. 5:869-874.[Abstract/Free Full Text]

FENNELLY, J., K. HARPER, S. LAVAL, E. WRIGHT, and M. PLUMB, 1996  Co-amplification to tail-to-tail copies of MuRVY and IAPE retroviral genomes on the Mus musculus Y chromosome. Mamm. Genome 7:31-36.[CrossRef][Medline]

GRAVES, J. A. M., 1995  The origin and function of the mammalian Y chromosome and Y-borne genes: an evolving understanding. BioEssays 17:311-321.[CrossRef][Medline]

GUBBAY, J., N. VIVIAN, A. ECONOMOU, D. JACKSON, and P. GOODFELLOW et al., 1992  Inverted repeat structure of the Sry locus in mice. Proc. Natl. Acad. Sci. USA 89:7953-7957.[Abstract/Free Full Text]

KING, T. R., G. J. CHRISTIANSON, M. J. MITCHELL, C. E. BISHOP, and D. SCOTT et al., 1994  Deletion mapping by immmunoselection against the H-Y histocompatibility antigen further resolves the Sxr^a region of the mouse Y chromosome and reveals complexity of the Hya locus. Genomics 24:159-168.[CrossRef][Medline]

KURODA-KAWAGUCHI, T., H. SKALETSKY, L. G. BROWN, P. J. MINX, and H. S. CORDUM et al., 2001  The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat. Genet. 29:279-286.[CrossRef][Medline]

LAHN, B. T. and D. C. PAGE, 1997  Functional coherence of the human Y chromosome. Science 278:675-680.[Abstract/Free Full Text]

LAVAL, S. H., V. REED, H. J. BLAIR, and Y. BOYD, 1997  The structure of DXF34, a human X-linked sequence family with homology to a transcribed mouse Y-linked repeat. Mamm. Genome 8:689-691.[CrossRef][Medline]

LOVELL-BADGE, R. and E. ROBERTSON, 1990  XY female mice resulting from a heritable mutation in the primary testis-determining gene, Tdy.. Development 109:635-646.[Abstract]

MAHADEVAIAH, S. K., R. LOVELL-BADGE, and P. S. BURGOYNE, 1993  Tdy-negative XY, XXY and XYY female mice: breeding data and synaptonemal complex analysis. J. Reprod. Fertil. 97:151-160.[Abstract/Free Full Text]

MAHADEVAIAH, S. K., T. ODORISIO, D. J. ELLIOTT, A. RATTIGAN, and M. SZOT et al., 1998  Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y deletion interval associated with a high incidence of sperm abnormalities. Hum. Mol. Genet. 7:715-727.[Abstract/Free Full Text]

MAZEYRAT, S., N. SAUT, V. GRIGORIEV, S. K. M. MAHADEVAIAH, and O. A. OJARIKRE et al., 2001  A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis. Nat. Genet. 29:49-53.[CrossRef][Medline]

MINTY, A. J., M. CARAVATTI, B. ROBERT, A. COHEN, and P. DAUBAS et al., 1981  Mouse actin mRNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA. J. Biol. Chem. 256:1008-1014.[Free Full Text]

MORIWAKI, K., D.-S. SUH, and J. STYRNA, 1988  Genetic factors effecting sperm morphology in the mouse. Mouse News Lett. 82:138.

NAVIN, A., R. PREKERIS, N. A. LISITSYN, M. M. SONTI, and D. A. GRIECO et al., 1996  Mouse Y-specific repeats isolated by whole chromosome representational difference analysis. Genomics 36:349-353.[CrossRef][Medline]

NISHIOKA, Y. and E. LAMOTHE, 1986  Isolation and characterization of a mouse Y chromosomal repetitive sequence. Genetics 113:417-432.[Abstract/Free Full Text]

NISHIOKA, Y., B. M. DOLAN, A. FIORELLINO, and V. F. PRADO, 1992  Nucelotide sequence analysis of a mouse Y chromosomal DNA fragment containing Bkm and LINE elements. Genetica 85:7-15.[CrossRef]

NISHIOKA, Y., B. M. DOLAN, V. F. PRADO, L. ZAHED, and H. TYSON, 1993a  Comparison of mouse Y-chromosomal repetitive sequences isolated from Mus musculus, M. spicilegus, and M. spretus.. Cytogenet. Cell Genet. 64:54-58.[Medline]

NISHIOKA, Y., B. M. DOLAN, and L. ZAHED, 1993b  Molecular characterization of a mouse Y chromosomal repetitive sequence amplified in distantly related species in the genus Mus.. Genome 36:588-593.[Medline]

OH, B., S.-Y. HWANG, D. SOLTER, and B. KNOWLES, 1997  Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo. Development 124:493-503.[Abstract]

OH, B., A. HAMPL, J. J. EPPIG, D. SOLTER, and B. B. KNOWLES, 1998  SPIN, a substrate in the MAP kinase pathway in mouse oocytes. Mol. Reprod. Dev. 50:240-249.[CrossRef][Medline]

PRADO, V. F., C.-H. LEE, L. ZAHED, M. VEKEMANS, and Y. NISHIOKA, 1992  Molecular characterization of a mouse Y chromosomal repetitive sequence that detects transcripts in the testis. Cytogenet. Cell Genet. 61:87-90.[Medline]

RODRIGUEZ, T. A. and P. S. BURGOYNE, 2000  Evidence that sex chromosome asynapsis, rather than excess Y gene dosage, is responsible for the meiotic impairment of XYY mice. Cytogenet. Cell Genet. 89:38-43.[CrossRef][Medline]

RODRIGUEZ, T. A. and P. S. BURGOYNE, 2001  Spermatogenic failure in male mice with four sex chromosomes. Chromosoma 110:124-129.[Medline]

SALIDO, E. C., X. M. LI, P. H. YEN, N. MARTIN, and T. K. MOHANDAS et al., 1996  Cloning and expression of the mouse pseudoautosomal steroid sulphatase gene (Sts). Nat. Genet. 13:83-86.[CrossRef][Medline]

SKALETSKY, H., T. KURODA-KAWAGUCHI, P. J. MINX, H. S. CORDUM, and L. HILLIER et al., 2003  The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423:825-837.[CrossRef][Medline]

STYRNA, J., H. T. IMAI, and K. MORIWAKI, 1991a  An increased level of sperm abnormalities in mice with a partial deletion of the Y chromosome. Genet. Res. 57:195-199.[Medline]

STYRNA, J., J. KLAG, and K. MORIWAKI, 1991b  Influence of partial deletion of the Y chromosome on mouse sperm phenotype. J. Reprod. Fertil. 92:187-195.[Abstract/Free Full Text]

STYRNA, J., B. BILINSKA, and H. KRZANOWSKAA, 2002  The effect of a partial Y chromosome deletion in B10.BR-Ydel mice on testis morphology, sperm quality and efficiency of fertilization. Reprod. Fertil. Dev. 14:101-108.[CrossRef][Medline]

SUH, D.-S., J. STYRNA, and K. MORIWAKI, 1989  Effect of Y chromosome and H-2 complex derived from Japanese wild mouse on sperm morphology. Genet. Res. 53:17-19.[Medline]

TOURÉ, A., V. GRIGORIEV, S. K. MAHADEVAIAH, Á. RATTIGAN, and O. A. OJARIKRE et al., 2004  A protein encoded by a member of the multicopy Ssty gene family located on the long arm of the mouse Y chromosome is expressed during sperm development. Genomics 83:140-147.[CrossRef][Medline]

TURNER, J. M. A., S. K. MAHADEVAIAH, R. BENAVENTE, H. H. OFFENBERG, and C. HEYTING et al., 2000  Analysis of male meiotic "sex-body" proteins during XY female meiosis provides new insights into their functions. Chromosoma 109:426-432.[Medline]

TURNER, J. M. A., S. K. MAHADEVAIAH, D. J. ELLIOTT, H. J. GARCHON, and J. R. PEHRSON et al., 2002  Meiotic sex chromosome inactivation in male mice with targeted disruptions of Xist. J. Cell Sci. 115:4097-4105.[Abstract/Free Full Text]

WRIGHT, W. E., D. A. SASSOON, and V. K. LIN, 1989  Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56:607-617.[CrossRef][Medline]

XIAN, M., S. AZUMA, K. NAITO, T. KUNIEDA, and K. MORIWAKI et al., 1992  Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa. Biol. Reprod. 47:549-553.[Abstract]

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