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Research Article |
Institute for Biogenesis Research,4 John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822
Division of Developmental Genetics,5 MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
ABSTRACT
In mouse and man, Y chromosome deletions are frequently associated with spermatogenic defects. XYTdym1qdelSry males have an extensive Yq deletion that almost completely abolishes the expression of two gene families, Ssty and Sly, located within the male-specific region of the mouse Y long arm. These males exhibit severe sperm defects and sterility. XYRIIIqdel males have a smaller interstitial Yq deletion, removing approximately two thirds of Ssty/Sly gene copies, and display an increased incidence of mild sperm head anomalies with impairment of fertility and an intriguing distortion in the sex ratio of offspring in favor of females. Here we used intracytoplasmic sperm injection (ICSI) to investigate the functional capacity of sperm from these Yq deletion males. Any selection related to the ability of sperm to fertilize in vitro is removed by ICSI, and we obtained two generations of live offspring from the infertile males. Genotyping of ICSI-derived offspring revealed that the YTdym1qdel deletion does not interfere with production of Y chromosome-bearing gametes, as judged from the frequency of Y chromosome transmission to the offspring. ICSI results for XYRIIIqdel males also indicate that there is no deficiency of Y sperm production in this genotype, although the data show an excess of females following in vitro fertilization and natural mating. Our findings suggest that 1) Yq deletions in mice do not bias the primary sex ratio and 2) YRIIIqdel spermatozoa have poorer fertilizing ability than their X-bearing counterparts. Thus, a normal complement of the Ssty and/or Sly gene families on mouse Yq appears necessary for normal sperm function. Summary: ICSI was successfully used to reproduce infertile mice with Yq deletions, and the analysis of sperm function in obtained offspring demonstrated that gene families located within the deletion interval are necessary for normal sperm function.
assisted reproductive technology, gamete biology, in vitro fertilization, sperm, spermatogenesis
INTRODUCTION
Recently it was shown that only two genes on the mouse Y chromosome are needed for spermatogenesis to complete meiosis, including both reduction divisions: the testis-determining factor Sry, and the translation initiation factor subunit Eif2s3y that is required for spermatogonial proliferation [1] (unpublished results). The remaining genes on the mouse Y are candidates for having a role in sperm differentiation (spermiogenesis) or function [2]. The male-specific region of the mouse Y chromosome (MSY) contains several genes and gene families. On the short arm (MSYp), there are seven known single-copy genes, one duplicated gene Zfy, and a multicopy gene Rbmy. On the long arm (MSYq) there are mostly repetitive sequences [3–8]. Among these repetitive sequences there is the complex Ssty gene family, encoding a protein of unknown function [2, 9–12], and multiple copies of a gene Sly, which has the potential to encode a putative chromatin-associated protein [13].
Studies on mice with Yq deficiencies imply that the genetic information on MSYq plays an important role in spermiogenesis that affects sperm quality and function. Males with a large interstitial deletion removing approximately two thirds of the MSYq (XYRIIIqdel) exhibit an increased incidence of mild sperm head defects (Fig. 1A), some impairment of sperm function, and a distortion of the offspring sex ratio in favor of females [11, 14–17]. Males with an even larger deletion removing approximately nine tenths of the MSYq (XYTdym1qdelSry) have most spermatozoa with gross head shape anomalies (Fig. 1B) and are infertile [2]. The fact that the severity of the sperm anomalies of males with MSYq deletions increases proportionally with the extent of deletion is consistent with the genetic information crucial for sperm differentiation and fertility being encoded by these multicopy MSYq genes [18].
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Intracytoplasmic sperm injection (ICSI), an injection of the spermatozoon directly into the cytoplasm of the oocyte, was first introduced in 1976 [19] and allows infertility caused by various sperm dysfunctions to be circumvented. Since 1992, when ICSI was successfully applied in humans [20], it has become the predominant technique in the treatment of severe male-factor infertility. Mouse ICSI was established in 1995 [21] and was shown successful in producing normal fertile mice [22, 23]. Recently it was demonstrated that mice with infertility caused by recessive autosomal mutations are able to produce live offspring when their spermatozoa or round spermatids are injected into the oocytes of normal females [24, 25] but no attempts have been made so far to overcome dominant mouse infertility. In addition to the obvious clinical and agricultural applications of ICSI, this method is also an important tool in basic research. Because any selection related to the ability of sperm to fertilize in vitro is removed by ICSI, the progeny obtained with this technique provide unique material for exploring various biological phenomena.
Here, we used ICSI to examine the effects of Y chromosome deletions on sperm function in fertilization in a mouse model. We provided the first evidence that offspring can be produced by ICSI from mice with dominant infertility resulting from Y chromosome deletions. We obtained two generations of XYRIIIqdel and XYTdym1qdelSry males and evaluated sperm from those males with respect to their genotype and fecundity in vitro. This study adds to our understanding of the role of Y chromosome deletions in gamete formation and distortion of the sex ratio.
MATERIALS AND METHODS
Chemicals
Mineral oil was purchased from Squibb and Sons, and eCG and hCG from Calbiochem. All other chemicals were obtained from Sigma Chemical Co. unless otherwise stated.
Animals
B6D2F1 (C57BL/6J x DBA/2) and CD-1 mice were obtained at 6 wk of age from the National Cancer Institute. B6D2F1 females were used as oocyte donors and CD-1 females as surrogate mothers. First-generation mice with Y chromosome deletions (and controls) were produced in house from cryopreserved sperm samples obtained from the MRC National Institute for Medical Research, UK (NIMR). Cryopreserved sperm samples were from mice on an outbred MF1 (NIMR colony) background. The samples were from the following males: 1) XYTdym1qdelSry. These males have a Y chromosome with an 11-kb deletion removing the testis determinant Sry [26], which is complemented by an autosomally-located Sry transgene [27]. They also have an extensive deletion removing approximately nine tenths of the male-specific region of the Y long arm (MSYq) [2]. These mice will be subsequently called 9/10MSYq–. 2) XYRIIIqdel. These males have a Y chromosome originating from the RIII inbred strain of mice and carry a deletion removing approximately two thirds of MSYq. The short arm of this Y chromosome is intact, providing an active Sry locus. These mice will be subsequently called 2/3MSYq–. 3) XYTdym1Sry and 4) XYRIII. These males are controls for the 9/10MSYq– and 2/3MSYq– males, and carry the Y chromosomes from which the deletion variants arose. The mice were fed ad libitum with a standard diet and were maintained in a temperature- and light-controlled room (22°C, 14L:10D), in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and guidelines presented in the National Research Council's Guide for Care and Use of Laboratory Animals published by the Institute for Laboratory Animal Research of the National Academy of Science, Bethesda, MD, 1996.
Media
Medium T6 [28] was used for in vitro fertilization (IVF) and Hepes-buffered CZB medium (Hepes-CZB; [21]) for gamete handling and ICSI. Medium CZB [29] was used for embryo culture. CZB and T6 were maintained in an atmosphere of 5% CO2 in air, and Hepes-CZB in air.
Sperm Collection
Epididymal spermatozoa were obtained from males 8–16 wk of age. The caudae epididymides were removed from each animal and placed in a 0.4-ml drop of T6 medium (capacitation drop) under oil. The epididymal contents were expressed from each cauda epididymis with needles and the tissue discarded. Spermatozoa were allowed to disperse for 2–3 min at room temperature. The sample of sperm suspension for ICSI was taken immediately after sperm dispersion. For IVF, a sample of sperm suspension was taken after 1.5 h of capacitation.
Oocyte Collection
B6D2F1 females, 8–12 wk old, were induced to superovulate with injections of 5 IU eCG and 5 IU hCG given 48 h apart. Oviducts were removed 14–15 h after the injection of hCG and placed in PBS in a petri dish. For IVF, oviducts were transferred beneath mineral oil in a plastic dish (Falcon, cat. no. 351007) close to the fertilization drop (T6 medium plus spermatozoa). The cumulus-oocyte complex was released from the ampullary region of each oviduct by rupturing the oviduct with the aid of a 25-gauge needle. The oviduct was discarded and the cumulus-oocyte complex moved into the fertilization drop. For ICSI, the cumulus-oocyte complexes were released from the oviducts into 0.1% of bovine testicular hyaluronidase (300 USP units/mg) in Hepes-CZB medium to disperse cumulus cells. The cumulus-free oocytes were washed with Hepes-CZB medium and used immediately for ICSI.
In Vitro Fertilization
The method for sperm capacitation and IVF using T6 medium has been described elsewhere [28]. Briefly, 200-µl drops of T6 medium (fertilization drops) were overlaid with mineral oil in a plastic culture dish (60-mm diameter) and equilibrated overnight at 37°C in a humidified atmosphere of 5% CO2 in air. The volume of sperm suspension added to the fertilization drop was dependent on the concentration of spermatozoa after dispersion in the capacitation drop. Generally, 10 µl of sperm suspension from the capacitation drop was added to each fertilization drop to give a final sperm concentration of approximately 2 x 106/ml. The contents of four oviducts were released into each fertilization drop. After gamete coincubation for 4 h, the oocytes were washed several times with Hepes-CZB medium followed by at least one wash with CZB medium. Only morphologically normal oocytes were selected for culture.
Intracytoplasmic Sperm Injection
ICSI was carried out as described recently by Szczygiel and Yanagimachi [30]. Briefly, a small drop of the incubated sperm suspension was mixed thoroughly with an equal volume of Hepes-CZB containing 12% (w/v) polyvinyl pyrrolidone (Mr 360 kDa) immediately before ICSI. ICSI was performed using Eppendorf Micromanipulators (Micromanipulator TransferMan; Eppendorf) with a Piezo-electric actuator (PMM Controller, model PMAS-CT150; Prima Tech). A single motile spermatozoon was drawn, tail first, into the injection pipette and moved back and forth until the head-midpiece junction (the neck) was at the opening of the injection pipette. The head was separated from the midpiece by applying one or more piezo pulses. After the midpiece and tail were discarded, the head was redrawn into the pipette and injected immediately into an oocyte. ICSI was done in Hepes-CZB within 1–2 h after oocyte collection. Whenever possible, motile spermatozoa were used, because earlier observations indicated that the incidence of abnormal sperm karyotypes increased when spermatozoa were not selected for motility. Motile spermatozoa were available in all groups with the exception of a few samples of cryopreserved sperm from 9/10MSYq– males. Sperm-injected oocytes were transferred into CZB medium and cultured at 37°C. The oocytes were examined 
6 h after ICSI for survival and activation.
Embryo Culture and Transfer
After IVF and ICSI, the oocytes were placed in 50-µl drops of CZB medium pre-equilibrated overnight with humidified 5% CO2 in air. The culture drops were contained in plastic culture dishes (Falcon) and overlaid with mineral oil. The survival of ICSI oocytes was scored 1–2 h after the commencement of culture. The number of 2-cell embryos (fertilized) was recorded after 24 h in culture. Embryos reaching the 2-cell stage were transferred to the oviducts (5–10 per oviduct) of CD-1 females mated during the previous night with vasectomized CD-1 males. Surrogate mothers were allowed to deliver and raise their offspring. The progeny were genotyped at weaning (21 days of age) and subsequently used for breeding or sperm analyses, or as sperm donors for IVF and the next series of ICSI.
Genotyping Offspring of the Sry Transgenic Males
The autosomal Sry transgene is fully penetrant and segregates independently of the Sry-negative Y chromosome; any male offspring therefore carry the transgene. The offspring of the 9/10MSYq– males and their XYTdym1Sry controls potentially comprise XX and "XY" females together with XXSry and "XY"Sry males. To identify the Y-bearing males and females, genomic DNA was obtained from an ear punch at weaning and isolated by overnight incubation at 55°C in GNTK buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl pH 8.5, 0.45% Triton X-100, and 0.45% Tween 20) containing 0.02% Proteinase K, followed by 15 min incubation at 95°C, and PCR analysis was performed using primers ZFYP1, 5'-AAgATAAgCTTACATAATCACATggA-3', and ZFYP2, 5'-CCTATgAAATCCTTTgCTgCACATgT-3', to test for the presence of Zfy on the short arm of the Y chromosome (Yp). The PCR conditions were as follows: initial denaturation at 94°C for 3 min followed by 30 cycles of denaturation at 96°C for 10 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, with final elongation at 72°C for 5 min. The amplification products were analyzed on ethidium bromide-stained agarose gels.
Statistics
Chi-square, likelihood ratio, and Fisher exact probability tests were used for analyzing all responses. Lack of statistical significance was reported when all three tests gave P > 0.05. Presence of statistical significance was noted when at least one of the three tests showed P 
0.01 or P 0.05. The computations were done using KyPlot version 2.0 beta 13 software (developed by Koichi Yoshioka and available online: http://www.woundedmoon.org/win32/kyplot.html).
RESULTS
Production of First and Second Generation of ICSI Offspring
Cryopreserved sperm samples from 9/10MSYq– and 2/3MSYq– males and their appropriate controls XYTdym1Sry and XYRIII were shipped from NIMR, London. The thawed spermatozoa were injected into oocytes from B6D2F1 females by ICSI (Table 1). When spermatozoa from 9/10MSYq– males were injected, a high proportion of oocytes (71%) reached the 2-cell stage, but this proportion was significantly less than that of control (84%, P < 0.01). No differences were observed in the proportion of 2-cell embryos obtained with spermatozoa from 2/3MSYq– males and their controls (81% and 82%, P > 0.05). Embryos at the 2-cell stage were transferred into the oviducts of pseudopregnant females. Live offspring were obtained with high efficiency (37%–46%) and there were no statistically significant differences between groups.
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The males with MSYq deletions (and controls) derived by ICSI (ICSI1 males) were used for mating, for in vitro fecundity testing, and as sperm donors for the next series of ICSI. When spermatozoa from ICSI1 males were injected into the oocytes from B6D2F1 females, a high proportion of embryos (82%–85%) was obtained, with no differences among groups (Table 2). Live offspring (second-generation ICSI offspring, i.e., ICSI2) were produced with high efficiency, ranging from 38% to 54% live-born from embryos transferred, with no differences between groups.
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Fecundity of Males with MSYq Deletions
To test for sperm function, an in vitro fecundity assay was performed for the frozen-thawed original samples and the fresh spermatozoa obtained from ICSI1 males. When frozen-thawed spermatozoa were used for in vitro fertilization, the fertilization rate (the proportion of 2-cell embryos from oocytes inseminated) was low both in deletion and in control groups (9/10MSYq–: 7%, 11/158; 2/3MSYq–: 3%, 5/190; XYTdym1Sry: 2%, 2/113; and XYRIII: 6%, 7/121). This was presumably because of low sperm survival rate after cryopreservation.
When fresh spermatozoa from ICSI1 9/10MSYq– males were used for IVF only three 2-cell embryos (3/385) were obtained in six IVF attempts (Table 3). Spermatozoa from control ICSI1 XYTdym1Sry males fertilized 64% of oocytes (P < 0.001). Spermatozoa from ICSI1 2/3MSYq– males fertilized almost 50% of oocytes inseminated, but this proportion was significantly lower than when spermatozoa from control ICSI1 XYRIII males were used in IVF (81%, P < 0.001).
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Gamete Genotype Frequency in Sperm from 9/10MSYq– Males
Offspring produced by ICSI with sperm from 9/10MSYq– males (and XYTdym1Sry controls) were genotyped by PCR. Four types of progeny, originating from four genotypes of spermatozoa, are predicted from these males: 1) XYTdym1qdelSry males; 2) XX females; 3) XXSry males; and 4) XYTdym1qdel females. The presence of the YTdym1qdel (and YTdym1) chromosome in the progeny was confirmed by detecting Zfy on Yp.
All four types of progeny were obtained, showing that spermatozoa of all four genotypes of sperm were present in the epididymides of 9/10MSYq– males. The frequencies of sperm genotypes are shown in Table 4. The transmission of the YTdym1qdel chromosome was 40% in ICSI1 offspring and 46% in ICSI2 offspring and not significantly different from that of YTdym1 in control males, in which the transmission was 53% and 44% in ICSI1 and ICSI2, respectively. Furthermore, there were no significant differences in the transmission of the YTdym1 chromosome by the control XYTdym1Sry males following ICSI, IVF, or mating. However, there was a significant increase in Sry transgene frequency in pooled ICSI progeny derived from 9/10MSYq– males compared to ICSI progeny derived from control males (65% vs. 42%, P < 0.01).
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Sex Ratio in Offspring of 2/3MSYq– Males Obtained by Three Methods: ICSI, IVF, and Mating
In laboratory breeding conditions, 2/3MSYq– are of apparently normal fertility, despite their lower fecundity in IVF assays. However, the litters of these males exhibit a distortion in the sex ratio in favor of females [11]. In the present study, when 2/3MSYq– males were used for mating and IVF, a similar sex ratio distortion in favor of females was observed (Table 5). However, when spermatozoa from these males were used for ICSI, the sex ratio distortion toward females was no longer present in the offspring. A statistically significant difference (P < 0.05) was noted when comparing the proportion of females in the litters produced by ICSI1 (or in pooled results of ICSI1 and ICSI2) and by mating. There were no statistically significant differences in the sex ratios between litters produced by ICSI and mating in control males (Table 5).
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DISCUSSION
In this study we evaluated the effects of MSYq deletions on sperm function using ICSI. Y chromosome deletions are responsible for reproductive failure in a significant number of infertile men. These men exhibit various spermatogenic defects, with impairment in sperm number and morphology as prevailing abnormalities. However, with the advance of ICSI, they are now able to conceive. Although ICSI is commonly used to treat infertility in men carrying Y chromosome deletions, no studies have been done in an adequate mouse model. Here, for the first time, we present the results of ICSI with spermatozoa from two types of mice with the deletions of the Y chromosome long arm: 2/3MSYq– and 9/10MSYq– males.
It was previously reported that 2/3MSYq– males, although able to reproduce, exhibit significant impairment in fertilization [31–33]. Thus, they can be considered subfertile. When spermatozoa from 2/3MSYq– males were injected into the oocytes by ICSI, fertilization efficiency was high. Two generations of 2/3MSYq– males were produced, and the proportion of live offspring obtained with ICSI was similar to that obtained in controls. ICSI efficiency to achieve fertilization and live offspring was even more spectacular when it was used with spermatozoa from males with extensive 9/10MSYq– deletion. 9/10MSYq– males were originally reported infertile [2]. To verify this, all 9/10MSYq– males obtained in the course of this study were mated with normal B6D2F1 females for at least 2 mo, and the females were changed each 2 wk. Out of 16 males tested, only one induced pregnancy in one of the females (two offspring were born, with genotypes XX and XXSry). In repeated attempts of IVF, almost no fertilization was achieved. However, 9/10MSYq– males were able to sire offspring when their spermatozoa were injected into the oocytes. More than one third of oocytes injected resulted in live pups.
The original production of XYTdym1qdelSry males was very difficult [2]. Obtaining the first XYTdym1qdelSry male by breeding from an XXYTdym1 sister of the XXYTdym1qdelSry proband took several months. Subsequently 9/10MSYq– males were obtained through breeding XYTdym1qdel females, which are of very poor fertility, and the process was tedious and required a large breeding program. With ICSI, the first nine males (first-generation ICSI 9/10MSYq– males) were produced within a period of 3 mo. Thus, we demonstrate that ICSI not only allows overcoming infertility of a valuable mouse model, but is also highly efficient in multiplying mice that would otherwise be very difficult to obtain.
To test for the sperm function in males with Yq deletions, we performed an in vitro fecundity assay. The impairment in sperm function may be influenced by genetic background, the source of the Y chromosome, and the effects of MSYq deletion, as well as interactions between these factors. Mating of 2/3MSYq– males on a CBA-inbred background gave 100% successful fertilization, but the fertilization was delayed, as shown by evaluation of percentages of fertilized ova on Day 1 vs. Day 2 postcoitus [33]. When males on a B10.BR background were mated, more than half of them produced sterile copulations. The rest gave fertilization rates that were reasonable but lower than those found in control males (79% vs. 98%) [32]. These males also gave lower fertilization rates in IVF (22%) as compared to controls (79%) [31]. In mice carrying the YRIII chromosome, no difference in fertility was initially found between the Yq-deleted and control males from breeding records [11]. Later, however, Burgoyne [18] reported that males with a mixture of XY and XYqdel germ cells transmit almost no Yqdel chromosomes to the offspring, although Yqdel sperm are present in the epididymides. Thus, although the differences between various genetic backgrounds were evident, all previous reports suggested that sperm from 2/3MSYq– males were inefficient in fertilization. Our first-generation ICSI offspring were produced by injecting spermatozoa from males on an MF1 background into the oocytes from B6D2F1 females. The males obtained were thus 50% MF1, 25% C57BL/6, and 25% DBA/2. The proportion of oocytes fertilized by sperm from ICSI1 2/3MSYq– males was significantly lower than that fertilized by sperm from controls on the same background. Deterioration of sperm quality was observed when comparing 2/3MSYq– and 9/10MSYq– males. Spermatozoa from ICSI1 9/10MSYq– males were unable to fertilize oocytes in vitro ([2] and this study). This supports previous indications that deletion of the long arm of the mouse Y chromosome affects sperm capacity to fertilize oocytes, and that this effect increases in proportion to deletion size.
With Y chromosome deletions, the likely causes of infertility are impaired sperm production and/or sperm quality. It was found that in 9/10MSYq– males, sperm production appeared relatively normal, as judged by testis histology, and sperm counts were within fertile range but reduced when compared to controls [2]. One of the unresolved problems was whether 9/10MSYq– males produce spermatozoa of all four predicted genotypes. Taking into account that sperm counts were lower than those found in controls, one might consider that sperm carrying the defective Y chromosome were eliminated during maturation. Attempts to genotype spermatozoa directly, via FISH, did not provide satisfactory results (unpublished observations). Here, we used ICSI to test for the presence of YTdym1qdel spermatozoa as evidenced by their transmission to viable offspring. We obtained offspring of all four possible genotypes, and there were no significant differences in the frequency of progeny carrying a Y chromosome between 9/10MSYq– males and controls. This enabled us to conclude that the 9/10MSYq deletion does not result in a deficiency of Y-bearing relative to X-bearing sperm, and that the deletion does not preclude Y chromosome transmission.
We did observe an unexpected increase in the transmission of the autosomally located Sry transgene in 9/10MSYq– males as compared to controls. The motility of sperm from 9/10MSYq– males (especially the motility of frozen-thawed sperm) was significantly lower than in other groups. This suggests the intriguing possibility that sperm carrying the Sry transgene were less prone to motility loss than sperm without Sry. This can be tested in the future by comparing Sry transmission frequency for 9/10MSYq– males using sperm samples with and without prior selection for motile sperm.
The sex ratio bias toward females previously observed in offspring of 2/3MSYq– males [11, 14–17] was also noted in preimplantation-stage embryos [34]. Until now it was not known whether distortion in sex ratio toward females in offspring of 2/3MSYq– males was caused by a reduced frequency Y-bearing sperm in the ejaculate or their preferential impairment in fertilization. In ICSI, in which spermatozoa are injected directly into the oocyte, any selection related to ability of sperm to fertilize on its own is omitted, and the progeny obtained reflect the frequency of X- and Y-bearing sperm present in the epididymides. Here, when spermatozoa from 2/3MSYq– males were injected into the oocytes, no sex ratio distortion toward females was observed in the offspring. This implies that the 2/3MSYq deletion does not interfere with sperm production; thus, spermatozoa carrying the Y chromosome with the 2/3MSYq deletion must have a poorer fertilizing ability than their X-bearing counterparts.
The physiological basis for the overall reduction in fertilizing capacity of sperm from males with MSYq deletions, or for the poorer fertilizing capacity of Y-bearing as compared to X-bearing sperm in 2/3MSYq– males, is not known. Both are presumably a consequence of the loss of MSYq-encoded RNAs or proteins [13]. Recently it has been shown that a downstream consequence of this loss of MSYq gene expression is the upregulation of multiple X- and Y-linked spermatid-expressed genes, which, together with the sex ratio skew in the offspring of 2/3MSYq– males, is taken as evidence for intragenomic conflict between X- and Y-linked genes [35]. Our finding that the sex ratio distortion arises during the fertilization process requires that there is some sort of imbalance of X- and/or Y-encoded products between the X- and Y-bearing sperm, and identifying the gene products responsible is a challenge for the future.
In this study we demonstrated that ICSI can be successfully used to reproduce infertile and subfertile mice with deletions of the long arm of the Y chromosome. Our results also support previous findings showing that Yq deletions affect the ability of sperm to fertilize oocytes in vitro and that this effect increases in proportion to deletion size. Thus, a normal complement of the Ssty and/or Sly gene families on mouse Yq appears to be necessary for normal sperm function. Finally, we showed that deletions on mouse Yq do not interfere with the production of Y chromosome-bearing gametes, as judged from the frequency of Y chromosome transmission to the offspring.
FOOTNOTES
1 Supported by Hawaii State Biomedical Research Infrastructure Network grant P20 RR16467 and Victoria S. and Bradley L. Geist Foundation grant 20031970 to M.A.W. 
2 Correspondence: Monika A. Ward, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 7316; mward{at}hawaii.edu
3 Monika A. Ward previously published manuscripts under the name Monika A. Szczygiel.
Received: 30 September 2005.
First decision: 11 November 2005.
Accepted: 13 December 2005.
REFERENCES
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