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Biol Reprod. 2009 August; 81(2): 353–361.
Published online 2009 May 6. doi:  10.1095/biolreprod.109.076307
PMCID: PMC2849819

Live Offspring from Mice Lacking the Y Chromosome Long Arm Gene Complement1

Abstract

The mouse Y chromosome long arm (Yq) comprises ~70 Mb of repetitive, male-specific DNA together with a short (0.7-Mb) pseudoautosomal region (PAR). The repetitive non-PAR region (NPYq) encodes genes whose deficiency leads to subfertility and infertility, resulting from impaired spermiogenesis. In XSxraY*X mice, the only Y-specific material is provided by the Y chromosome short arm-derived sex reversal factor Sxra, which is attached to the X chromosome PAR; these males (NPYq- males) produce sperm with severely malformed heads and are infertile. In the present study, we investigated sperm function in these mice in the context of intracytoplasmic sperm injection (ICSI). Of 261 oocytes injected, 103 reached the 2-cell stage, and 46 developed to liveborn offspring. Using Xist RT-PCR genotyping as well as gamete and somatic cell karyotyping, all six predicted genotypes were identified among ICSI-derived progeny. The sex chromosome constitution of NPYq- males does not allow production of offspring with the same genotype, but one of the expected offspring genotypes is XY*XSxra (NPYq-2), which has the same Y gene complement as NPYq-. Analysis of NPYq-2 males revealed they had normal-sized testes with ongoing spermatogenesis. Like NPYq- males, these males were infertile, and their sperm had malformed heads that nevertheless fertilized eggs via ICSI. In vitro fertilization (IVF), however, was unsuccessful. Overall, we demonstrated that a lack of NPYq-encoded genes does not interfere with the ability of sperm to fertilize oocytes via ICSI but does prevent fertilization via IVF. Thus, NPYq-encoded gene functions are not required after the sperm have entered the oocyte. The present work also led to development of a new mouse model lacking NPYq gene complement that will facilitate future studies of Y-encoded gene function.

Keywords: assisted reproductive technology, ICSI, intracytoplasmic sperm injection, IVF, in vitro fertilization, sperm, Y chromosome deletions

INTRODUCTION

The mouse Y chromosome (Fig. 1A, right) has been estimated to contain ~78 Mb of DNA [1], of which 0.7 Mb constitutes the pseudoautosomal region (PAR) at the end of the long arm [2]. The PAR is the region of homology with the X chromosome (Fig. 1A, left) that mediates pairing and recombination between the X and Y chromosomes in normal males. The remaining non-PAR (male-specific) part of the Y chromosome (NPY) contains several genes and gene families. On the short arm (NPYp) are seven known single-copy genes—Ube1y1 [3, 4], Kdm5d (previously called Jarid1d and Smcy) [5], Eif2s3y [6], Uty [7], Ddx3y (previously called Dby) [8], Usp9y [9], and Sry [10]—as well as one duplicated gene, Zfy (Zfy1/2) [11], and a multicopy gene, Rbmy1a1 [12]. The NPYq represents ~90% of NPY and contains mostly repetitive sequences [1318]. Whereas the mouse Y chromosome is still in the process of being sequenced, it is already clear that within the NPYq lie multiple copies of at least four distinct genes that are expressed in spermatids: Ssty, Sly, Asty, and Orly [1924].

FIG. 1.
Sex chromosome complement of males lacking NPYq. A) Normal male sex chromosome complement. B) Sex chromosome complement of NPYq- male. Note that the sex reversal factor Sxra is attached distal to the X PAR. C) Sex chromosome complement of NPYq-2 male ...

Four mouse models with NPYq-deficient genotypes have been described: XYRIIIqdel [20], XYB10.BRqdel [25], XYTdym1qdelSry [24], and XSxraY*X [26]. Both XYRIIIqdel and XYB10.BRqdel males have Y chromosomes originating from the RIII and B10.BR inbred strains, respectively, and each carry a deletion removing approximately two thirds of NPYq. These males exhibit an increased incidence of mild sperm head defects, some impairment of sperm function, and a distortion of the offspring sex ratio in favor of females [20, 25, 2734]. The XYTdym1qdelSry males have a more extensive deletion, removing approximately nine tenths of the NPYq, together with an 11-kb deletion removing the testis-determinant Sry [35] that is complemented by an autosomally located Sry transgene [12]. These males have spermatozoa with slightly or grossly distorted heads, are infertile, and have sperm that fail to fertilize eggs in vitro [24, 33]. In XSxraY*X males (hereafter called NPYq-), the only Y-specific material is provided by the YRIII short arm-derived sex reversal factor Sxra attached distal to the X PAR (Fig. 1B). The Y*X chromosome, which serves as a pairing partner during meiosis, is effectively an X chromosome with a very large deletion, removing most of the X-specific region but leaving an intact PAR and X PAR boundary, together with the X centromere [36] (P.S. Burgoyne, unpublished results). The Y*X notation is confusing, because this chromosome lacks any Y-specific sequences (Fig. 1B). The sex reversal factor Sxra encompasses all known NPYp genes except that the number of copies of Rbmy1a1 is decreased from ~50 to 7; thus, NPYq- males lack the entire gene complement of NPYq. These males still produce sperm, but sperm head morphology is even more severely affected than in XYTdym1qdelSry males. These males also are infertile [24, 26].

In our past work, we investigated the functional capacity of sperm from XYRIIIqdel and XYTdym1qdelSry males using in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We provided the first evidence that offspring can be produced by ICSI from mice with dominant infertility caused by deletion of nine tenths of NPYq. We also demonstrated that this extensive deletion does not interfere with production of Y chromosome-bearing gametes, as judged from the frequency of Y chromosome transmission to the offspring [33]. In the present study, we used ICSI and IVF to test sperm function in mice with complete absence of NPYq-encoded genes.

MATERIALS AND METHODS

Chemicals

Mineral oil was purchased from Squibb and Sons; pregnant mare serum gonadotropin (eCG) and human chorionic gonadotropin (hCG) were from Calbiochem. All other chemicals were obtained from Sigma Chemical Co., unless otherwise stated.

Animals

The B6D2F1 (C57BL/6 × DBA/2) and CD1 mice were obtained at 6 wk of age from the National Cancer Institute and Charles River Laboratories, respectively. The B6D2F1 females were used as oocyte donors and CD1 females as surrogate mothers. The mice were fed ad libitum with a standard diet and were maintained in a temperature- and light-controlled room (22°C, 14L:10D photoperiod) in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and the guidelines presented in the National Research Council's Guide for Care and Use of Laboratory Animals, published in 1996 by the Institute for Laboratory Animal Research of the National Academy of Sciences. The mice of interest in the present study were XSxraY*X males (here termed NPYq-, because they lack the non-PAR region of Yq) produced on an outbred MF1 (National Institute for Medical Research [NIMR] colony) genetic background. These mice were imported from the Burgoyne laboratory at the Medical Research Council (MRC) NIMR. Because the sex reversal factor Sxra originates from an RIII strain Y chromosome, the appropriate controls for these mice are XYRIII males carrying an intact YRIII. These controls were generated at the Institute for Biogenesis Research on the same MF1 background.

Media

Medium T6 [37] was used for IVF, and Hepes-buffered CZB medium (Hepes-CZB) [38] was used for gamete handling and ICSI. Medium CZB [38] was used for embryo culture. Both CZB and T6 were maintained in an atmosphere of 5% CO2 in air, and Hepes-CZB was maintained in air.

Testicular and Epididymal Sperm Collection for ICSI

The testes and caudae epididymides were dissected and subjected to gross morphological analyses. To obtain testicular sperm, a portion of testis was cut off and minced in ETBS buffer (50 mM EGTA, 50 mM NaCl, and 10 mM Tris-HCl buffer; overall pH 8.2–8.5 [39]) to release spermatogenic cells. To obtain epididymal sperm, the epididymal contents were expressed with needles and placed in either Hepes-CZB or ETBS medium. The samples of testicular or epididymal cell suspension were used for ICSI or for analyses immediately after dispersion or were preserved for future use.

Oocyte Collection

The oocytes from B6D2F1 females were collected for IVF and ICSI as previously described [33].

Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection was carried out as described in detail by Szczygiel and Yanagimachi [40]. Injections were performed in Hepes-CZB within 1–2 h from oocyte collection. Sperm were randomly chosen for the injections. Sperm-injected oocytes were transferred into CZB medium and cultured at 37°C. The survival of ICSI oocytes was scored at 1–2 h after the commencement of culture. The activation of ICSI oocytes was scored at 6 h after the commencement of culture; the oocytes with two well-developed pronuclei and extruded second polar body were considered to be activated. The number of 2-cell embryos (fertilized) was recorded after 24 h in culture.

In Vitro Fertilization

Sperm capacitation and IVF were performed as previously described [41]. Gametes were coincubated for 1.5 h. After gamete coincubation, 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.

Embryo Transfer

Embryos reaching the 2-cell stage were transferred to the oviducts (n = 10–14 per oviduct) of CD1 females mated during the previous night with vasectomized CD1 males. Surrogate mothers were allowed to deliver and raise their offspring or had cesarean section performed and the progeny raised by foster mothers. The progeny were genotyped after weaning (age, 21 days) and subsequently used for sperm analyses or as sperm donors for the next ICSI series.

Xist RT-PCR

The RNA was isolated using RNeasy Mini kit (catalog no. 74104; Qiagen) according to the manufacturer's instruction. The RT-PCR was performed essentially as previously described [42]. Briefly, amplification from cDNA was carried out using Xist primers Y MIX20 (5′-CGA CCT ATT CCC TTT GAC GA-3′) and JT4 (5′-GTT GAT CCT CGG GTC ATT TA-3′), with initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 96°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, and then elongation at 72°C for 5 min; the amplified product was 433 bp. Hprt amplified with primers Hprt1A (5′-CCT GCT GGA TTA CAT TAA AGC ACT-3′) and Hprt1B (5′-GTC AAG GGC ATA TCC AAC AAC AAA-3′), yielding a 352-bp cDNA product, served as the amplification control. Both Xist and Hprt primer pairs overlap two exons.

Gamete Karyotyping

Gamete chromosome preparations were done to visualize chromosomes in oocytes and sperm. To evaluate oocyte chromosomes, the metaphase II (MII) oocytes were incubated in Ca2+-free CZB containing 5 mM Sr2+ for 4 h to achieve activation. Activated oocytes (recognized as those that had extruded a second polar body and contained pronuclei) were washed and cultured in CZB until chromosome preparation. To evaluate sperm chromosomes, sperm were injected into MII oocytes. Chromosome preparation and analysis were performed as described earlier [43, 44].

Somatic Cell Karyotyping

Bone marrow metaphases were prepared by standard techniques. Briefly, cells were flushed from femurs using RPMI medium 1640 with 25 mM Hepes (Invitrogen) plus KaryoMAX Colcemid Solution (0.04%; Invitrogen), incubated for 15–60 min at 31°C, and centrifuged (1000 × g, 5 min), after which the medium was replaced with hypotonic KCl (0.56%) at room temperature for 20 min. After brief vortexing, the suspension was centrifuged (1000 × g, 5 min) and drained. The pellet was washed with fixative (3:1, methanol:glacial acetic acid) several times, and the fixed cells were resuspended and air-dried on slides and then stained with 2% Giemsa (pH 6.8, 15 min).

Testis and Sperm Analyses

For histology, testes were dissected, Bouin-fixed, paraffin-embedded, and sectioned (thickness, 5 μm). The sections were stained with hematoxylin and eosin and/or periodic acid Schiff. To analyze sperm number, motility, and morphology, sperm were released from dissected caudae epididymides into the well of an Organ Tissue Culture Dish (Falcon) containing Hepes-CZB medium under a stereomicroscope to ensure that all sperm were released successfully and incubated for at least 10 min at 37°C immediately before analysis. Sperm counts using a hemocytometer were the mean of three independent scorings per sample. For analysis of sperm morphology, the sperm suspension (diluted as necessary with 0.9% NaCl) was smeared on three slides and allowed to dry, fixed in methanol and acetic acid (3:1), and stained with silver. The slides were coded, and the head morphology was scored blind at 1000× magnification for at least 100 sperm per slide.

Testing Fecundity in Mating

Sexually mature males were mated with B6D2F1 hybrid females, which are robust and highly fertile. Males were housed with females for a period of at least 1 mo. At least two females were housed, consecutively, with each male. The females were checked for copulatory plugs as evidence of successful mating.

Statistics

Chi-square, likelihood ratio, and Fisher exact probability tests were used for analyzing differences between groups in ICSI, IVF, and gamete genotype transmission. 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. The computations were done using KyPlot version 2.0 beta 13 software (developed by Koichi Yoshioka and available at http://www.woundedmoon.org/win32/kyplot.html).

For comparisons of the incidence of sperm head abnormalities, percentages were converted into angles, and differences between genotypes were assessed in a nested ANOVA with genotypes, males, and replicate slides as the factors using the generalized linear model provided by NCSS statistical data analysis software.

RESULTS

Live Offspring Could Be Obtained Using ICSI with Sperm from NPYq- Males

Three NPYq- males were brought to Hawaii from the MRC NIMR, and two of them were used as sperm donors for ICSI. Three independent ICSI attempts were performed with sperm from these males (Table 1). The first male (age, 6 mo) did not have sperm in the caudae epididymides, had few in the capita epididymides, and had some in the testes; testicular sperm were used for injections (ICSI 1). Although the second male (age, 5 mo) had some immotile sperm in the caudae epididymides, testicular sperm were used for ICSI on consecutive days for the right and left testis following initial semicastration (ICSI 2 and 3). The third male (age, 5 mo) had a good number of motile sperm in epididymides, and these sperm were cryopreserved for future work. All sperm used for ICSI were grossly abnormal. We have not attempted IVF with sperm from these males because of the limited amount of material. Based on unpublished data from the Burgoyne and Ward laboratories, sperm numbers in the caudae epididymides appear to fall as the males age, and in the present study, the older of the three males lacked sperm.

TABLE 1.
ICSI with spermatozoa from NPYq- males.

Of 261 oocytes injected, 183 survived, 125 became activated, and 103 cleaved. Similar results were obtained in the three independent ICSI trials except that the oocyte survival rate was slightly lower in the first attempt. This lower survival rate probably resulted from particular difficulties in finding sperm in the testicular cell suspension from this male, with a consequent increase in ICSI duration. In spite of the extremely poor quality of sperm from NPYq- males, ICSI was highly successful with respect to the live offspring rate. When 2-cell embryos were transferred into the oviducts of pseudopregnant females, all (9/9) gave birth to live offspring, with 45% (46/103) of transferred embryos becoming liveborn. The majority of live offspring survived to weaning. Further ICSI experiments with frozen-thawed sperm from NPYq- males have so far yielded a further four males and one female.

Overall, we demonstrated that ICSI is successful in generating normal, viable offspring from infertile males with a complete absence of NPYq genes.

All Expected Progeny Genotypes Were Obtained After ICSI with Sperm from NPYq- Males

When the XSxra and Y*X chromosomes are associated at metaphase I (MI) via the obligatory crossover within the PAR, XSxra, Y*X, Y*XSxra, and X gametes should be generated with the same frequencies. A high frequency (30–60% [26]) of MI spermatocytes, however, have the XSxra and Y*X chromosomes separated, raising the possibility of random segregation of the chromosomes at MI that would lead to additional XSxra and Y*X gametes, together with XSxraY*X and “O” (no sex chromosome) gametes. Although a large proportion of such MI spermatocytes with univalent sex chromosomes are eliminated by apoptosis, some do complete both meiotic divisions and go on to form sperm [45, 46]. Thus, six types of progeny, originating from six genotypes of spermatozoa, are predicted from NPYq- males: 1) XX females, 2) XY*X females, 3) XY*XSxra males, 4) XXSxra males, 5) XO females, and 6) XXSxraY*X males (Table 2). The latter two genotypes are expected to be relatively rare, with the remaining four genotypes relatively abundant and occurring in roughly equal frequencies.

TABLE 2.
Expected genotypes of NPYq- progeny and identification methods.

To identify the offspring genotypes, we first carried out RT-PCR for Xist, which is expressed when two X chromosomes are present, thus allowing us to separate out the putative XX females (Xist positive) and putative XY*XSxra males (Xist negative). We then karyotyped gametes and/or bone marrow cells (Fig. 2), which in addition to identifying diagnostic differences in chromosome number also allowed us to identify progeny carrying the minute chromosome variant Y*X ± Sxra (Fig. 1, B and C, and Table 2). As expected for males with two X chromosomes, the Xist-positive XXSxraY*X and XXSxra males had small testes (mean ± SEM, 25.29 ± 0.884 mg; range, 18–35 mg, n = 30 testes from 15 males), and no sperm were found in either testes or epididymides.

FIG. 2.
Chromosome analysis of progeny derived by ICSI with sperm from NPYq- males: A, C, and E) oocyte chromosomes; G) sperm chromosomes; and B, D, F, H, I, and J) bone marrow chromosomes. XX female had oocytes with 20 normal-sized chromosomes (A) and bone marrow ...

All six expected progeny genotypes were identified, and their frequencies are shown in Table 3. The genotype frequencies for XO, XX, XY*X, and XY*XSxra were 4%, 16%, 18%, and 27%, respectively. We were not able to separately identify XXSxra and XXSxraY*X in seven Xist RT-PCR-positive males because of accidental loss of preserved material. Karyotyping the remaining nine Xist RT-PCR-positive males revealed that seven of nine were XXSxra but two of nine were XXSxraY*X. If we assume that the distribution of XXSxra and XXSxraY*X genotypes among the nine karyotyped males and seven nonkaryotyped males was the same, then five from the latter group would be XXSxra and two would be XXSxraY*X. The calculated frequencies for these genotypes are then 27% and 9%, respectively.

TABLE 3.
NPYq- progeny genotype frequencies.a

Overall, the progeny genotype frequencies are in agreement with theoretical expectation in that the XO and XXSxraY*X progeny are substantially less abundant than the other genotypes. More importantly, these results show that all expected sperm genotypes were present in testes from males with complete absence of NPYq genes, and all were successfully transmitted to the offspring by ICSI.

NPYq-2 Male Progeny Have Phenotype Similar to That of Original NPYq- Males

The sex chromosome complement of the original NPYq- males does not allow production of offspring with the same genotype. In other words, XSxraY*X fathers cannot produce XSxraY*X sons, because all XSxra-bearing sperm generate XXSxra progeny (Table 2). One of the offspring genotypes, however, is XY*XSxra (hereafter called NPYq-2). These males also lack the entire Y chromosome long arm, and the only difference between them and the NPYq- males is that the sex reversal factor Sxra is attached to Y*X rather than to the X chromosome (Fig. 1, B and C). Because the Y gene complement of NPYq- and NPYq-2 males is the same, they are expected to have the same phenotype.

The NPYq-2 ICSI-derived males had normal-sized testes (mean ± SEM, 111.64 ± 2.756 mg; range, 88–131 mg; n = 20 testes from 10 males), similar to those reported previously for NPYq- males [26]. Spermatogenic progression was similar to that of NPYq- males, with all types of spermatogenic cells, including mature sperm, being produced. The testis tubules in both NPYq- and NPYq-2 males, however, were less populated with germ cells, especially round and elongating spermatids, compared to normal, wild-type males (Fig. 3); this is expected because of the high levels of sex chromosome univalence at MI leading to substantial MI spermatocyte apoptosis. Caudae epididymides sperm counts in NPYq-2 males were within the normal range (mean ± SEM, 16.58 × 106 ± 2.80; range, 8.4–36.0; n = 9 males). Sperm motility was noted in all males, but its quality varied.

FIG. 3.
Testis histology in NPYq- and NPYq-2 males. Stage VII/VIII tubules of NPYq- and NPYq-2 show similar spermatogenesis progression yielding mature, although clearly morphologically abnormal, sperm. Note that the tubules from males lacking the NPYq gene complement ...

Sperm morphology was analyzed for nine 5-mo-old NPYq-2 males and the two 5-mo-old NPYq- males. The NPYq-2 males were generated by ICSI using oocytes from B6D2F1 (C57BL/6 × DBA/2) females and, thus, were on a mixed genetic background (50% MF1, 25% C57BL/6, and 25% DBA/2), whereas the NPYq- males were on an outbred MF1 background (unfortunately, we found MF1 oocytes to be unsuitable for ICSI). We therefore included two XYRIII males on an MF1 genetic background and two XY B6D2F1 males in the analysis to assess if the input of the B6D2F1 genetic background was likely to have any substantial effect on sperm morphology. The sperm were classified as normal or abnormal, and abnormal sperm were further classified into one of the eight sperm head defect categories, with two of them considered to be slightly abnormal (Fig. 4, ,1S1S and and2S)2S) and six to be grossly abnormal (Fig. 4, ,33G–8G).

FIG. 4.
Specific sperm morphology defects in mice with NPYq deficiencies. Eight categories of sperm head defects (two slight, 1S–2S; six gross, G3–G8) are presented as photos and diagrammed below, with normal sperm (N) as control. Slight category ...

The incidence of morphologically abnormal sperm was slightly higher in XYRIII MF1 males (6.1%) than in the XY BD2F1 males (2.6%) (Fig. 5, A and B), but nested ANOVA of the angular transformed data revealed that the increase was nonsignificant (P = 0.2). All the sperm in the NPYq- and NPYq-2 males were abnormal, with 100% and 99.7%, respectively, being grossly abnormal (Fig. 5, C and D). In previous studies, the predominant gross abnormalities in NPYq- males fell into the classes here called 6G and 7G. In the present data, 90.0% of sperm from NPYq- males and 89.6% from NPYq-2 males fell into these classes. Nested ANOVA was carried out on the angular transformed data for classes 6G, 7G, 6G plus 7G, and for all other gross abnormalities. Significant differences were found between males within genotypes for all five comparisons (in all cases, P < 0.01), but no significant differences were found between NPYq- and NPYq-2 (in all cases, P > 0.45).

FIG. 5.
Frequencies of specific sperm morphology defects in mice with NPYq deficiencies. Graphs represent mean frequencies of specific sperm defects in XY MF1 (A), XY B6D2F1 (B), NPYq-2 (C), and NPYq- (D) males. The number of sperm scored was as follows: XX MF1, ...

Four NPYq-2 males were tested for fecundity in vivo and in IVF. All of these males mated successfully, as evidenced by the presence of copulation plugs, but none of them induced pregnancy. When sperm from these NPYq-2 males were used for IVF, none of the inseminated oocytes became fertilized (0/66, 0/107, 0/61, and 0/48 oocytes for the four males tested), whereas sperm from three B6D2F1 males tested in parallel fertilized 73% (62/85), 38% (46/120) and 58% (76/131) of the oocytes inseminated. We have not collected ICSI data comparable to those for NPYq- males (i.e., ICSI with fresh testicular sperm and oocytes from B6D2F1 females), but we have used epididymal sperm from NPYq-2 males for ICSI in an attempt to begin backcrossing of this genotype to a C57BL/6 inbred background. As expected, progeny were obtained from NPYq-2 males via ICSI; at the time of this writing, 45 live offspring have been born, with nine fourth-generation ICSI offspring among them.

Overall, we have shown that ICSI-derived NPYq-2 males had normal-sized testes; relatively normal spermatogenic progression yielding all types of spermatogenic cells, including mature sperm; and generally good epididymal sperm counts with observable motility. Nevertheless, the males were sterile, with the sperm having severely malformed heads and being unable to fertilize oocytes via IVF, though fertilization was possible via ICSI.

DISCUSSION

In the present study, we show, for the first time, that live offspring can be obtained via ICSI from males lacking the entire male-specific gene complement of the Y chromosome long arm (NPYq). Thus, genes encoded on NPYq, although essential for normal sperm function [47], are not needed for those aspects of fertilization that occur within the oocyte after sperm entry.

The genes encoded on mouse NPYq are all multicopy, are expressed in spermatids, and have X chromosome homologues. They show a progressive reduction in transcript levels with increasing NPYq deficiency [23] and are candidates for contributing to the sperm defects associated with NPYq deletions. Ssty (spermatid-specific transcripts Y-encoded) is present on the mouse NPYq in more than 100 copies and is specifically transcribed in the testis in round spermatids. It has two distinct subfamilies, Ssty1 and Ssty2. Sly (Sycp3-like Y-linked) is testis specific and, like Ssty1, is a very abundant transcript in spermatids [23]. At least 65 Sly copies that have maintained a functional open reading frame are predicted based on Y chromosome sequence data. Sly is a member of the Xlr superfamily, the most closely related member of which is the multicopy gene Slx (formerly called Xmr). Both Sly and Slx encode abundantly expressed spermatid-specific proteins: The SLY protein is predominantly cytoplasmic, and the SLX protein being exclusively cytoplasmic [48, 49]. The products of Sly and Slx are suspected to act antagonistically during spermiogenesis [50]. Asty (amplified spermatogenic transcripts Y-encoded) is related to the multicopy X-encoded gene Astx, with which it shares 92–94% homology. Current evidence suggests that Astx is translated but that Asty is not. This, however, does not rule out a functional role for Asty, especially given the increasing literature on functional noncoding RNAs. Orly (oppositely transcribed, reassorted locus on the Y) is a chimeric locus on NPYq that is composed of partial copies of Ssty1, Sly, and Asty arranged in a sequence [21]. Orly is bidirectionally transcribed, giving rise to Orly (forward) and Orlyos (reverse) transcripts. These transcripts may potentially form double-stranded RNA in partnership with each other or with the progenitor loci Ssty1, Sly, and Asty. Orly is the most recent gene on NPYq, and its emergence triggered massive amplification of NPYq sequences. The mechanisms through which one or more of these NPYq-encoded genes affect sperm function is unknown. Recently, however, it has been established that SLY interacts in spermatids with the histone acetylase KAT5 (also known as TIP60), which is implicated in chromatin restructuring, and with the acrosomal protein DKKL1 [48]. This suggests that Sly may be a key player in restructuring the spermatid nucleus to form the sperm head and in acrosome development or, more directly, in acrosome function during sperm penetration; indeed, the sperm of XYB10.BRqdel males (fertile males with an approximately two-thirds NPYq deletion) have been found to be compromised in tests of acrosome function [28].

XSxraY*X (NPYq-) males cannot produce sons with the same genotype, but one of the expected genotypes of male offspring, XY*XSxra (NPYq-2), differs only in that the Y short arm-derived Sxra is attached to the minute Y*X chromosome rather than to the X chromosome. As expected, the NPYq-2 males also were sterile, and the effects of these two genotypes on sperm morphology were indistinguishable. The NPYq- males are difficult and costly to produce; in the Burgoyne laboratory this is currently done by breeding XYSxra males [51] with XPafY*X females [36, 52]. Only one twelfth of the viable genotypes produced in this cross are NPYq-, and the XPafY*X mothers are of poor fertility. The NPYq-2 males represent a new mouse model with complete NPYq deficiency that can continue to be efficiently reproduced by ICSI. These males are expected to generate the six progeny genotypes—XX, XY*X, and XO (rare) females as well as XXSxra, XY*XSxra, and XXY*XSxra (rare) males—and we have already confirmed that the expected XY*XSxra (NPYq-2) males, as well as the remaining three major genotypes, are produced by ICSI. Therefore, NPYq-2 males will be a valuable model for more detailed, future investigations into the consequences of lacking the NPYq gene complement.

Our study has implications for human fertility. Deletions within the male-specific region of the Y chromosome are a common genetic cause of spermatogenic failure in men, making men carrying an affected Y chromosome a target group for assisted reproduction technologies. Many children have already been born after ICSI with sperm from these men. When infertility is caused by a defect of a genes located on the Y chromosome, all male children of an affected father are affected as well, and on reaching sexual maturity, they will require the same assistance that their father needed to achieve conception, leading to a phenomenon called hereditary infertility [5355]. In our past [33] and present work, we have applied ICSI as a tool to study sperm function in NPYq-deficient mice. The analysis of ICSI-derived mice with Y chromosome aberrations, potentially over multiple ICSI-derived generations, can provide important information regarding the effects of this reproductive technology on the possible phenotypic changes associated with repeated transmission via ICSI. So far, we have not observed any enhancement in the NPYq phenotypes in mice with partial and complete NPYq deficiencies following ICSI progeny for up to three generations; however, the analyses were hampered by variable genetic background of ICSI-derived mice. Currently, we are transferring all NPYq-deficient models to the C57BL/6 background to facilitate future testing of ICSI effects in these mice.

Footnotes

1Supported by the Victoria S. and Bradley L. Geist Foundation 20071382, NIH HD058059, and NIH 1 P20 RR024206-01 (Project 2) grants to M.A.W.

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