Two Y genes can replace the entire Y chromosome for assisted reproduction in the mouse

Abstract

The Y chromosome is thought to be important for male reproduction. We have previously shown that with the use of assisted reproduction, live offspring can be obtained from mice lacking the entire Y chromosome long arm. Here, we demonstrated that live mouse progeny can also be generated using germ cells from males with the Y chromosome contribution limited to only two genes, the testis determinant factor Sry and the spermatogonial proliferation factor Eif2s3y. Sry is believed to function primarily in sex determination during fetal life. Eif2s3y may be the only Y chromosome gene required to drive mouse spermatogenesis allowing formation of haploid germ cells that are functional in assisted reproduction. Our findings are relevant but not directly translatable to human male infertility cases.

The mammalian Y chromosome, once thought to be a genetic wasteland (1), is now known to encode a battery of genes, many of which are thought to be involved in male reproduction (2). A substantial amount of work has been done to define which genes are important for maintaining sperm function under normal, in vivo, conditions. In the era of assisted reproduction technologies (ART) it is now possible to bypass several steps of normal human fertilization using immotile, non-viable, or even immature sperm. We have shown that infertile male mice lacking the entire Y chromosome long arm can generate live offspring when their severely morphologically abnormal sperm are delivered into oocytes via intracytoplasmic sperm injection (ICSI) (3). In these mice the Y chromosome is reduced from 78Mb to ~2Mb, and encodes only 7 genes and 3 gene families (Fig. S1, XY*^XSxr^a).

In most mammals, including humans and mice, testis determination is regulated by Sry, which directs developing gonads to male differentiation (4–6). Upon transgenic addition of Sry, mice with one X chromosome (XOSry) develop testes which are populated with spermatogonia, male germ cells that have the potential to undergo differentiation and initiate spermatogenesis. In the absence of other Y chromosome genes these spermatogonia undergo proliferation arrest, and meiotic and post-meiotic stages of spermatogenesis are absent (7). Transgenic addition of individual missing Y genes led to the identification of Eif2s3y as the gene that restored normal spermatogonial proliferation (7). In XOSry males transgenic for Eif2s3y spermatogenesis was shown to complete meiotic prophase and the first meiotic division before the cells arrested as secondary spermatocytes, with the occasional production of spermatid-like cells (7, 8). Here, we tested whether these spermatid-like cells were functional in assisted reproduction, and what other components of the Y chromosome help to increase development of functional gametes.

We first examined mice with the Y gene complement limited to two transgenically derived genes, autosomally located Sry and X chromosome located Eif2s3y (Fig. S1, X^EOSry)(9). These mice had testes smaller than wild-type XY males (Fig. S2) but populated with germ cells. Analysis of testicular sections confirmed that spermatogenesis was ongoing allowing development of germ cells with spermatid-like morphology (Fig. 1A arrowheads), similar to those observed earlier (7, 10). The occurrence of these spermatids was low and their development was restricted to steps 5–7 of spermatid development, with the occasional presence of step 8–9 spermatids (Fig. 1A(inset)&S3). We also observed secondary changes to seminiferous tubules, with increased incidence of dying cells, multinucleate bodies, and vacuoles (Fig. 1B). These changes increased progressively as the males aged, ultimately leading to Sertoli cell only (SCO) syndrome tubules (Fig. 1C).

Fig. 1.

Testis histology, round spermatids and live offspring. (A) Males with only 2 Y genes, Eif2s3y and Sry, have meiotic and post-meiotic arrests that only occasionally allow formation of spermatids (arrowheads) that are frequently delayed and do not develop beyond St8/9. (B) Tubule degeneration with formation of 'multinucleate bodies' (long arrows), vacuoles (short arrows) and dying/apoptotic germ cells (arrowheads) is frequently observed. (C) Sertoli cell only-like phenotype in an old male. (D) Testicular suspension from wild-type males contains testicular sperm (arrows) and many round spermatids (arrowheads) with clear morphological features. (E) Males with 2 Y genes have significantly less germ cells in testicular cell suspension, with no sperm and very few round spermatids (arrowhead). These spermatids are functional in ROSI. (F) ROSI pup obtained after transfer of embryos generated with spermatids from a male with only two Y genes. (G) an adult female developed from the pup shown in (G) with her own litter. Roman and arabic numerals in (A–C) are tubule stages and steps of spermatid development (St), respectively (see Fig. S3). Scale is 50 µm (A–C) and 10 µm (D–F); insets = x3 magnification; mo = months of age.

We next tested the function of the spermatid-like cells from X^EOSry in assisted reproduction. Round, spermatid-like cells could be found in testicular cell suspensions from all males used in ART trials although these cells were rare and their morphology was often slightly abnormal (increased size, less pronounced nuclei, rough rather than smooth surface), when compared to spermatids from control XY males (Fig. 1D&E). Nevertheless, when we performed round spermatid injection (ROSI), the oocytes were successfully fertilized as evidenced by the development of two pronuclei and extrusion of the second polar body, and subsequent cleavage (Fig. S4). When the developed 2-cell embryos were transferred into the oviducts of recipient females, live offspring were obtained (Table 1 and Fig. 1F). Three out of four males, which provided spermatids for injections, successfully sired offspring. The efficiency of ROSI with X^EOSry males was significantly lower than with XY controls (Table 1, 9% vs. 26%). All the progeny had the genotypes as expected when derived from X^EOSry fathers and were healthy; those bred were fertile (Fig. 1G,S5 and supplementary online text).

Table 1.

The results of round spermatid injection (ROSI) with spermatids from males with limited Y gene complement.

Male genotype	Y gene contribution (see Fig. S1)	Live offspring % (no)#	Implants % (no)#
XEOSry	Eif2s3y & Sry	9.1 (12/132)a	29.5 (39/132)b
XEY*XSry	Eif2s3y & Sry	5.7 (13/227)b	27.3 (62/227)b
XESxrbO	Eif2s3y & Sxrb	20.0 (24/120)	45.8 (55/120)a
XESxrbY*X	Eif2s3y & Sxrb	16.0 (24/150)	37.3 (56/150)b
XYRIII control	intact Y	26.0 (19/73)	69.9 (51/73)

^#Percentage calculated from embryos transferred. Male age ranged 63–229 days.

Statistical significance (Fisher's Exact Test):

^aP<0.01,

^bP<0.001 vs. XY^RIII control.

Sxr^b Sry, Zfy2/1, H2al2y, Rbmy cluster (reduced)

An unpaired sex chromosome leads to meiotic arrest and apoptosis (11) so partial meiotic failure in X^EOSry males was not unexpected. Few spermatids that could be found in the testes could be the cells that 'leaked' through the meiotic arrest, i.e. finished meiosis and were haploid. Alternatively, these could be the cells that developed spermatid-like morphology without undergoing the second meiotic division (8, 10).

Spermatid nuclear DNA content (Fig. 2&S6) and zygotic chromosome analyses (Fig. 3) revealed that the great majority of spermatids from X^EOSry males were diploid and yielded triploid zygotes, which would explain the poor ROSI success. In order to overcome the problem of meiotic block arising from X chromosome univalence we generated males, in which a minute Y*^X chromosome (Fig. S1, Y*^X) was added to provide a second pairing region (PAR) for PAR-PAR chromosome synapsis (12). In the resulting X^EY*^XSry males (Fig. S1), successful pairing of Y*^X and X was observed in 85% of pachytene spermatocytes (Fig. S8). However, the testicular phenotype did not improve (Fig. S2&S7A–C). The proportion of live offspring obtained after injection was similarly low as with X^EOSry males (Table 1) and only five out of eight males that provided cells for ROSI sired offspring. We therefore tested for ploidy and demonstrated that most of the X^EY*^XSry spermatids were diploid (Fig. 2&S6) and most zygotes after ROSI were triploid (Fig. 3). Thus, overcoming X chromosome univalence in X^EY*^XSry males did not allow overcoming meiotic arrest and increasing ROSI success (see supplementary online text).

Fig. 2.

Incidence of haploid spermatids. Each graph bar represents an individual male providing testicular cells for analysis; the numerals above show the percentage of pups obtained after ROSI. The data in rectangular boxes represent average percentage of haploid spermatids (blue) and ROSI offspring (red) for each genotype. Control male with intact Y chromosome had 97% (67/69) of spermatids haploid. X^ESxr^bY*^X males had higher average incidence of haploid spermatids than other genotypes (P<0.01).

Fig. 3.

Zygote chromosome analysis after ROSI. (A) Normal diploid mouse zygote generated after ROSI with XY male contains 40 chromosomes. (B) Triploid zygote obtained after ROSI with spermatids from a male with two Y genes (~60 chromosomes). (C) Diploid zygote after ROSI with spermatids from male of the same genotype (~40 chromosomes). (D) Incidences of diploid zygotes generated after ROSI. cf=chromosome fragments. n= chromosome number. ^a P<0.05, ^b P<0.001 vs. XY control. Bar=50 µm.

We next asked whether other Y chromosome genes may be beneficial for spermatid function in assisted reproduction. To address this, we produced males in which the Sry transgene driving sex determination was replaced with the sex reversal factor Sxr^b (Fig. S1, X^ESxr^bO & X^ESxr^bY*^X). In these males the X chromosome carries an Eif2s3y transgene necessary for spermatogonial proliferation (7) together with the Sxr^b encoding for Zfy2/1, Sry, H2al2y, and the Rbmy gene cluster.

The testes from X^ESxr^bO and X^ESxr^bY*^X males were larger than from X^EOSry and X^EY*^XSry but smaller than in wild-type XY males (Fig. S2). The incidence of round spermatids increased only in X^ESxr^bY*^X (Fig. S3). In both male types spermatid development was more advanced with clear elongation up to step 10, occasional presence of step 11–12 spermatids, and even sporadic development to mature testicular sperm (Fig. S7D&G). There were secondary changes to the seminiferous epithelium (Fig. S7E,F,H&I), albeit in X^ESxr^bY*^X males less pronounced than in the other genotypes. Zygote chromosome analysis showed that most of the zygotes after ROSI were diploid (71–85%, Fig. 3), and the ROSI outcome was significantly improved, with all tested males (3 per genotype) yielding live offspring with rate now reaching 20% and 16% (Table 1). Interestingly, the frequency of haploid spermatids in X^ESxr^bO males remained low (Fig. 2&S6). The discrepancy between the ploidy of the spermatids and zygotes raised the possibility that the second meiotic division took place in the oocytes. This suggests that Sxr^b encodes a gene(s) that promotes the second meiotic division when all chromosomes are paired, and enables overcoming meiotic arrest of the diploid spermatid in the oocyte when an X chromosome pairing partner is missing (see supplementary online text).

We have shown that only two mouse Y chromosome genes, Sry and Eif2s3y, are necessary for the development of male haploid germ cells that are sufficient for successful reproduction yielding live offspring. This minimal Y gene complement must be enough to ensure correct male-specific methylation and proper formation of any other epigenetic modifications that are required for embryogenesis in the paternal genome.

That Sry is one of the two genes is not surprising as it drives testis determination (4–6). Mouse Sry expression and tramslation occurs very briefly during fetal development. In adult testes Sry transcripts are thought to be aberrant and not translatable (13, 14) although their role as epigenetic regulator cannot be excluded (15). Nevertheless, it is reasonable to conclude that Sry plays a role primarily during sex determination.

This suggests that the second Y gene, Eif2s3y, is the only gene necessary to drive spermatogenesis through the first meiotic division and with the occasional meiotic progression to form haploid round spermatids. Eif2s3y is a Y encoded subunit 3 of the eukaryotic translation initiation factor 2. It is ubiquitously expressed and in the testis plays a role in spermatogonial proliferation (7). The Eif2s3y gene has been conserved on the Y chromosome during eutherian evolution but there is no Y-linked copy of Eif2s3 detected in any of the simian primates including humans (16).

In men, spermatogonial proliferation arrest results most often from deletions of AZFa (azoospermia; Sertoli-cell-only syndrome) and it has been related to a loss of DDX3Y (17–19). In both men and mice the Eif2s3 and Ddx3 genes belong to the family of widely expressed genes encoding proteins involved in initiation of mRNA translation at the ribosome. These genes have X homologues that escape X-inactivation, and the Y and X copies are suspected to be at least in part interchangeable, with the Y copy conserved to provide two doses of gene product in both male and female. The loss of the mouse Y encoded Ddx3y is not detrimental for spermatogonesis because of the compensation provided by an X copy retrotransposed on an autosome (20). Analogically, in men, the presence of a retroposed X copy of EIF2S3X, in addition to EIF2S3X itself, explains why the loss of the Y copy was still permissive for spermatogenesis. Although there is no human copy of Eif2s3y, men have a Y encoded copy of the translation factor EIF1A (eukaryotic translation initiation factor 1A, Y-linked) (21), which likely acts as part of a multi-protein complex that includes EIF2S3X as well as other EIF family members. Interestingly, EIF1AY is found in the AZFb region, and its diminished expression sporadically contributes to azoospermia (22).

At present, our findings in mice do not translate directly to humans. ROSI is still considered experimental in human ART due to concerns regarding the safety of injecting immature germ cells and technical difficulties (23). In spite of this, some children have already been born (24, 25) and those were healthy. As we learn more about the effects and improve technical aspects of ROSI, this method may become more acceptable. Indeed, studies on ROSI effects in mice have been encouraging (26). Thus our study may bear importance for clinicians working in ART clinics supporting the possibility that ROSI may be a viable option for overcoming infertility in men with non-obstructive azoospermia.

Considering that we have obtained live offspring using germ cells from males with only two Y chromosome genes one could question the importance of Y chromosome in male reproduction. We believe that the answer lies in defining the need. Human Y chromosome is not on the way to oblivion, as it has been implied in the past (27), and its genetic information is undoubtedly important for many aspects of reproduction involving the development of mature sperm and its function in normal fertilization (28). Most of the mouse Y chromosome genes are involved in spermiogenesis and sperm function and as such are necessary for normal fertilization (29, 30). However, when it comes to assisted reproduction, our mouse study proves that the Y chromosome contribution can be brought to a bare minimum consisting of Sry and Eif2s3y. Indeed, it may well be possible to eliminate mouse Y chromosome altogether if appropriate replacements are made for those two genes.