Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development.

A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head.

Introduction

The mouse Y chromosome has two copies of a gene, Zfy, which together with the closely related X chromosome linked Zfx, encode zinc finger transcription factors [1-3]. They derive from an autosomal precursor that was added to the mammalian sex chromosomes via the pseudoautosomal region (PAR) subsequent to the separation of the marsupial and eutherian lineages [4, 5]. Postnatal expression of the mouse Y genes (Zfy1 and Zfy2) is restricted to spermatogenic cells [6-8]. Zfy2 encodes a much more potent transcription factor than Zfy1 [9, 10] and has also acquired an additional strong spermatid-specific promoter [6] suggesting an important role during spermiogenesis. Although these mouse ‘Zf’ genes were identified in 1989–1990 [1, 2, 11] only recently have any spermatogenic functions been ascribed to them [12-14].

This study is a sequel to our two earlier papers documenting roles for mouse Zfy with respect to the apoptotic elimination of spermatocytes with univalent chromosomes at the first meiotic metaphase [13] and for the completion of the second meiotic division [14]. The latter paper has an extended introduction that describes the ‘raison d’être’ for the Zfy transgene additions to Yp gene deficient mice; this will be unfamiliar to most readers and can be accessed via the link http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=24967676.

In 2012 [15] we established that in XOSry males (Fig 1D) in which the only Yp genes present are an X located Eif2s3y transgene and an autosomally located Sry transgene, there is a block at step 7 of the round spermatid stage of sperm development, whereas in XSxrO and XSxrO males (Fig 1B and 1C) there was evident sperm morphogenesis. Sxr provides a near complete Yp gene content, whereas Sxr has a very limited Yp gene content (Fig 1A–1C); however, Sxr is unique in having a Zfy2/1 fusion gene that includes the strong spermatid specific promotor from Zfy2. This led us to hypothesize that this spermatid specific promotor is of paramount importance in enabling Zfy to provide an essential function during sperm morphogenesis—in normal males this function would primarily be provided by Zfy2. In the current study we show through Zfy1 and Zfy2 transgene additions to Yp gene deficient models that this is indeed the case.

Fig 1

The XO and XY*X mouse models.

(A) XY. The Y short arm (Yp) gene complement of an XY male (represented to scale in the magnified view) comprises nine single copy genes, two duplicated genes and one multi copy gene. The pseudoautosomal region (PAR) located distally on the Y long arm mediates pairing and crossing over with the X PAR during meiosis to generate the XY sex bivalent. Centromeres are represented by a dot on the chromosome. (B–D) The diminishing Yp gene complements for the three XO male mouse models that lack the Y long arm. (B) XSxrO. The Yp-derived Sxr attached distal to the X PAR provides an almost complete Yp gene complement. (C) XSxrO. The Sxr-derived deletion variant Sxr has a 1.3 Mb deletion (Δ) removing 6 single copy genes and creating a Zfy2/1 fusion gene spanning the deletion breakpoint (†). The deleted gene Eif2s3y is necessary for normal spermatogonial proliferation, so an X-located Eif2s3y transgene has been added. (D) XOSry. This model has only two Yp genes—the testis determinant Sry provided as an autosomally located transgene and the spermatogonial proliferation factor Eif2s3y provided as the X-located transgene. E. Y*X. This mini sex-chromosome is an X chromosome with a deletion from just proximal to Amelx to within the DXHXF34 repeat adjacent to the X centromere († marks the deletion breakpoint). This X chromosome derivative has a complete PAR that can pair and crossover with the PAR of XSxr, XSxr or X to form a ‘minimal sex bivalent’. Scale bar for magnified views is 150 kb.

Results

For our published study showing that Zfy1 and Zfy2 have an important role in promoting completion of the second meiotic division to form haploid spermatids [14] we added a minute sex chromosome (Y*X) [16, 17]) to the three XO models. Y*X (Fig 1E) comprises a complete pseudoautosomal region (PAR), a short ‘X telomeric region’, some repeated sequences mapping adjacent to the X centromere, and a presumed X-derived centromere [18-20]. Y*X was previously shown to enable the formation of a sex bivalent, thus circumventing the MI SAC [21]. We abbreviate the three XY*X models as XY*XSxr, XY*XSxr and XY*XSry; the X-located Eif2s3y transgene is denoted X. Importantly in the context of the present study, we found that the addition of Y*X had no effect on the frequency of haploid spermatids in the context of Sry, but it markedly increased the haploid frequency in the context of Sxr (Figure 3A in reference [14]). Therefore, before assessing the effects of Zfy transgene additions on sperm morphogenesis we sought to establish: (1) whether the Y*X addition per se had any discernible effect on spermiogenic progression by comparing XOSry with XY*XSry; and (2) whether the markedly increased haploid frequency associated with the Y*X addition in the context of Sxr had any discernible effect on spermiogenic progression by comparing XSxrO with XY*XSxr.

The addition of Y*X does not enhance spermatid elongation or the associated sperm morphogenesis

Fig 2 provides a summary diagram of spermiogenic progression in normal XY males based on the classical descriptions of the spermiogenic cycle in relation to testis tubule stage assessed from tubule sections. For the purposes of this study the key spermiogenic steps are 1–12 that are present in tubule stages I-XII. Haploid round spermatids first appear at stage I as a consequence of the two meiotic divisions occurring in the preceding stage XII. During stages I-VII the round spermatids form an acrosomal cap covering one side of the nucleus. During stages VIII-XII the main features of sperm morphogenesis take place (formation of the sperm tail; restructuring and condensation of the spermatid nucleus to form the sperm head). Spermatid development in all three XO models (Fig 1B and 1D) is aberrant and delayed to varying degrees [15], so assessment of tubule stage has to be based entirely on the spermatogonial and meiotic stages (see Materials and Methods).

Fig 2

The normal steps of spermiogenesis in relation to testis tubule stages in an XY mouse.

This figure presents a schematic depiction of the 16 steps of mouse spermiogenesis based on the stage of the spermatogenic cycle (stages I–XII). Photos of step 1–12 spermatid nuclei are shown with PAS staining at the foot of the diagram. The transition from round to elongating spermatids takes place during stage VIII; this marks the beginning of sperm morphogenesis. The shaping of the sperm head and formation of the sperm tail are essentially complete by step 12. However, the sperm are not shed until the following stages VIII-IX; consequently two generations of spermatids are present in stages I-VIII.

The spermiogenic progression in XY*XSry males proved to be equivalent to that in XOSry males (Fig 3A). In epithelial stage VIII the spermatids fail to re-orientate the acrosome to face the basal membrane, which normally precedes spermatid elongation. Moreover, the spermatids do not elongate and remodel the chromatin to form the sperm head (even by stage XII), and in both models the arrested, still round, spermatids are eliminated by stages II-IV of the following cycle. Thus the Y*X addition per se has no effect on spermiogenic progression.

Fig 3

Addition of Y*X to XOSry or XSxrO models does not improve spermiogenic progression.

Periodic acid Schiff/hematoxylin stained testis sections illustrating the extent of spermiogenic progression. Roman numerals denote estimated tubule stages. (A) In XOSry, the predominantly diploid spermatids do not elongate and the acrosomes (stained dark pink) remain randomly orientated relative to the basement membrane of the tubule (insets in VIII and XII). The spermatid nuclei show signs of pycnosis by stage XII (inset) and the cells have been eliminated by stages II-IV. The abundant round cells at stages II-IV are the new generation of round spermatids with early stages of acrosome development (dark pink ‘acrosomal granules’ in inset) [for more details see Vernet et al 2012]. In XY*XSry the block to spermiogenesis remains with elimination of the arrested cells once again evident by stages II-IV (see inset). (B) In XSxrO, at stage VIII the spermatids have not elongated and they are randomly orientated relative to the tubule basement membrane. However, as previously reported (Vernet et al 2012), spermatid elongation is delayed rather than absent, and is apparent by stage XII. Nuclear condensation is also delayed as it is not evident at stage XII, but many of the elongating spermatids survive to stages II-IV at which point nuclear condensation is now evident. In XY*XSxr spermatid elongation and nuclear condensation is similarly delayed, but there appear to be fewer elongating spermatids surviving to stages II-IV [note the now evident haploid (h) as well as diploid (d) spermatids]. Scale bar is 40 μm (insets are x3 magnification).

In agreement with our previous findings, XSxrO males differ markedly from XOSry males (Fig 3B). In XSxrO males spermatids do re-orientate such that the acrosomes come to face the basal membrane and spermatid elongation and subsequent nuclear condensation do take place. However, this spermiogenic progression is delayed relative to XY controls [15]; thus re-orientation does not occur during stage VIII, and elongation and some shaping of the sperm head with associated acrosome relocation are only evident by stage XII instead of stage IX. Correctly orientated spermatids with condensed nuclei are apparent by stages II-IV instead of X-XI. A similar pattern of spermiogenic progression can be seen in XY*XSxr males (Fig 3B) with elongation and condensation of the spermatid nuclei and the correct orientation of elongating spermatids being most obvious in stage XII tubules and beyond.

Addition of Zfy2 (but not Zfy1) to XY*XSry males promotes substantial sperm morphogenesis

The effects of the Zfy1 or Zfy2 transgene additions were assessed in XY*XSry males in which the only Y genes present are Sry and the X-located Eif2s3y; the Zfy transgenes used were single copy and also located on the X chromosome, and we denote the resulting males as XY*XSry and XY*XSry. In agreement with our expectation, addition of Zfy1 had no discernible effect on spermiogenic progression, with the spermatids failing to re-orientate or elongate. In marked contrast, the Zfy2 addition did promote spermiogenic progression (Fig 4A). Indeed, tubule sections of XY*XSry males in stages X to VII all showed elongating spermatids with condensed nuclei and a tail being formed.

Fig 4

Addition of Zfy2 (but not Zfy1) to XY*XSry enables re-orientation of spermatids, together with clear head and tail morphogenesis; however the Zfy2 transgene is not as effective as Sxr in supporting sperm morphogenesis.

Hematoxylin and eosin (H & E) stained testis tubule sections. (A) By comparison with XY*XSry, it is clear that the Zfy1 transgene addition has no beneficial effect on spermiogenic progression whereas the Zfy2 addition leads to the formation of correctly orientated elongated spermatids which have condensed sperm heads and some tail formation. (B) By comparison of XY*XSry with XY*XSxr (and XY controls) it is clear that Sxr is more effective than the Zfy2 transgene in supporting the sperm morphogenesis. In agreement with the illustration in Fig 2, in XY males at stage XI sperm head morphogenesis has progressed to the early ‘hooked tip’ stage and nuclear condensation is evident. As previously reported, in XY*XSxr at this stage some of the sperm from the previous cycle have not yet been shed (stained dark blue); the spermatids from the new cycle are retarded with respect to elongation and nuclear condensation is not evident. In XY*XSry spermatid elongation is further retarded. Nevertheless, by stage I-III many spermatids with condensed chromatin are found in XY, XY*XSxr and XY*XSry. (C) Images of testicular sperm found in silver stained testicular cell smears. All three genotypes proved to have some sperm present in testicular cell smears. In XY*XSxr, as previously reported for epididymal sperm and testicular sperm, the sperm heads rarely have a hooked tip. In XY*XSry the developing sperm heads show limited elongation. In all three genotypes the tails were well developed. (D) Hematoxylin/eosin stained epididymal tubule sections. In XY there are abundant sperm present with the heads showing the characteristic hooked tip. In XY*XSxr the sperm heads rarely have a hooked tip. In XY*XSry males no sperm could be identified; instead degenerating round spermatids are found suggesting that there has been some shedding of round spermatid stages despite the addition of Zfy2. Scale bars: A = 30 μm, B = 40 μm, C = 20 μm, D = 100 μm. Insets in D = 3x magnifications.

We next wished to assess whether the Zfy2 addition reinstated spermiogenic progression to the same extent as that supported by the near complete Yp gene complement present in Sxr. For this we compared spermiogenesis in XY*XSry males with that in XY*XSxr males. The latter have Zfy1 and Zfy2 encoded within Sxr but, like XY*XSry males, lack genetic information encoded by the mouse Y long arm that is important for sperm head shaping [22-25]. We also included wild type XY controls in order to check at what tubule stage the abnormal sperm head shaping in XY*XSxr became evident. Detailed analysis of staged seminiferous epithelium revealed spermiogenic anomalies in XY*XSry males relative to both XY*XSxr and wild type control genotypes (Fig 4B). At stage XI, there was a marked delay in spermatid elongation and nuclear condensation, and by stages I-III the spermatids looked unhealthy with darkly stained nuclei. Silver stained testicular cell smears showed that these abnormal spermatids were nevertheless capable of developing tails (Fig 4C). We then examined epididymal tubule sections in order to assess the relative numbers of testicular sperm passing from the testis into the epididymis (Fig 4D). As previously established [21], in XY*XSxr males substantial numbers of sperm reach the epididymis although these sperm have abnormally shaped heads that rarely have a hooked tip. However, no sperm were seen in epididymal sections from XY*XSry males. Instead, there were many round spermatids, indicating that substantial numbers of these cells slough off from the seminiferous epithelium in the Zfy2 transgenics.

Finally we assessed sperm morphogenesis by electron microscopy. Sperm heads from XY*XSxr males elongate similarly to those of wild type males except for some acrosome invagination. In contrast, XY*XSry males have compromised sperm head morphogenesis with signs of necrosis, acrosome invagination, incomplete elongation, and formation of cytoplasmic vacuoles (Fig 5A). Degenerating condensed spermatids were often present in large cytoplasmic inclusions within the epithelium, indicative of removal by apoptosis (not shown). However, cross sections of developing sperm tails appeared grossly normal with the classic axoneme structure composed of a central microtubule pair and nine outer doublets (9x2+2) (Fig 5B).

Fig 5

Sperm head and tail morphogenesis in XY*XSry males as compared to controls.

(A) Electron micrographs of developing sperm heads from 6 week old XY, XY*XSxr and XY*XSry males. Spermatids at round to elongating transitional stage in XY*XSry present no apparent ultrastructural defects (bottom left picture). However, vacuoles (V) appear in the cytoplasm of elongating and condensing spermatids. Irregular spread of the acrosomal cap (arrows) distorting the spermatid nuclei is observed in XY*XSry and XY*XSxr. It was again evident that the sperm heads in XY*XSry fail to elongate properly (stars). (B) Electron micrographs of sperm tail sections showing a normal 9x2+2 axoneme pattern with a central microtubule pair (p) in addition to the nine outer doublets (d) in all three genotypes. Scale bars: A = 1 μm, B = 0.5 μm (Insets = 3x magnification).

Clearly, although addition of the Zfy2 transgene overcomes the step 7 round spermatid arrest to allow substantial sperm morphogenesis, qualitatively and quantitatively it does not match that achieved with the near complete Yp gene complement present in XY*XSxr males.

Discussion

The starting point for the present study was our finding that in XOSry males (Y gene complement Eif2s3y and Sry) there is a clear-cut and unique arrest of spermiogenesis at step 7 of spermatid development [15]. In the present study we showed that this arrest is also seen in XY*XSry males that have the same Y gene complement, but also have the minute Y*X chromosome that enables the formation of a sex bivalent, thus avoiding MI SAC responses. The transition from spermatid step 7 to step 8 is pivotal in spermiogenesis: at step 7 the spermatids are round and randomly oriented with respect to the basal membrane, whereas at step 8 the spermatids have re-orientated so that the developing acrosomal cap is now facing the tubule periphery. This re-orientation heralds the dramatic restructuring of the round spermatids to form spermatozoa (‘sperm morphogenesis’). We hypothesized that the arrest at step 7 was a consequence of a lack of Zfy2-dependent transcriptional changes, and that the expression of Zfy2 at the critical stage was dependent on the alternative, strong, Cypt-derived, spermatid-specific promotor acquired by Zfy2 during the evolution of mice (muridae) [15]. Our present results clearly demonstrate that Zfy2 supports sperm morphogenesis, while Zfy1, which lacks this promotor, does not.

The transcription factors encoded by Zfy1 and Zfy2, together with their X-linked homologue Zfx, are predicted to bind the same DNA sequence [26-28]. In most eutherian mammals Zfx and Zfy are widely expressed, and Zfx is exempt from X dosage compensation, suggesting a constraining dosage requirement in somatic tissues [29, 30]. However, in the myomorph rodent lineage, Zfx became subject to X-dosage compensation and the Zfy-encoded proteins diverged [1, 29, 31], and mice (muridae) ended up with two copies of Zfy with postnatal transcription restricted to spermatogenic cells [6-8]. We have previously discussed the evolutionary pressures that may have led to ZFY2 having a more potent transactivation domain than ZFY1, and ZFY1 frequently lacking the transactivation domain as a consequence of alternative splicing [14]. Here we consider the acquisition of the additional Cypt-derived promotor in the muridae [6, 9]. It is clear from recent studies that the mouse Y chromosome gene complement has been markedly affected by a post-meiotic X-Y genomic conflict, with the round spermatid specific genes Sly and Slx being key protagonists [22, 32–36]. As a consequence of this conflict there has been co-amplification of Sly and Slx (50–100 copies located on the mouse Y long arm [35]) with Sly expression serving to damp down transcription of the X and Y chromosomes in spermatids [22]. Progressive reduction of Zfx and Zfy gene transcription in spermatids as the genomic conflict continued will have provided a strong selective force for the recruitment of the strong Cypt-derived spermatid-specific promotor to Zfy2, in order to maintain sperm morphogenesis.

Our original focus on the alternative Cypt-derived Zfy2 promotor was triggered by our finding of substantial sperm morphogenesis in XY*XSxr males in which a 1.3 Mb Yp deletion (Δ–see Fig 1C) has created a Zfy2/1 fusion gene that includes the same promotor. We have developed a new PCR assay that specifically amplifies Cypt promotor dependent transcripts with exon 6 that encodes the transactivation domain (S1 Appendix and S1 Fig). Our primary interest was to see if the Cypt promotor generated comparable levels of full length transcripts from the Sxr-located Zfy2/1 fusion gene in XY*XSxr males as compared to those from the Zfy2 transgene in XY*X males. Quantification of the Cypt-driven transcript bands indicates that this is indeed the case as can be seen from the last two pairs of columns of Fig 6A. It is these transcripts that we contend are supporting the sperm morphogenesis evident in the two models. From Fig 6B it can be seen that the Zfy2/1 fusion gene present in XEif2s3yY*XSxrb is expressed at twice the level of the XYSxrb control; furthermore, the Zfy2 transgene in XY*X is also expressed at a substantially higher level than the endogenous Zfy2 gene of the XYSxr and XY controls. This elevated expression of Zfy2 is to be expected in the genotypes lacking the Y long arm gene complement because they lack the repressive effect of the estimated 50–100 copies of Sly that are involved in the post-meiotic genomic conflict. In whole testis samples from XY males with a targeted knockdown of Sly transcripts there is widespread up-regulation of spermatid expressed X and Y genes and this includes Zfy2 (see [22], Fig 4B). This up-regulation of Zfy2 is also seen with purified round spermatids (Julie Cocquet, personal communication).

Fig 6

Levels of Cypt-dependent transcripts for XYSxr, XY, XY*XSry and XY*XSxr.

The RT-PCR bands quantified are those from the RT-PCR assay in S1 Fig. The transcripts expected for each genotype (n = 2 represented by two bars on the chart) are indicated above each genotype label (2/1 denotes Cypt-dependent Zfy2/1 transcripts and 2 denotes Cypt-dependent Zfy2 transcripts). (A) The PCR primers for this assay amplified Zfy2/1 and Zfy2 transcripts; only XYSxr has both transcripts. It can be seen that the level of Cypt-Zfy2/1 transcripts in XY*XSxr is comparable to the level of Cypt-Zfy2 transgene transcripts in XY*XSry. (B) For the left panel the PCR primers are specific for Zfy2/1; the second and third genotypes lack the Zfy2/1 fusion gene so the low level signal represents ‘background’. For the right panel the PCR primers are specific for Zfy2; in this case it is the fourth genotype that lacks Zfy2. The genotypes lacking the Y long arm (Yq-) have substantially higher transcript levels than the genotypes with a complete Y (Yq+).

Our finding that sperm morphogenesis in XY*XSry males is inferior to that in XY*XSxr males points to there being other Yp genes that contribute to sperm morphogenesis. We have generated transgenic lines that express other Yp-located genes (Ube1y1/Uba1y, Smcy/Kdm5d, Uty, Dby/Ddx3y, Usp9y, H2al2y) that may have functions during spermiogenesis. Since our current study was completed two more male germ-line expressed genes, Prssly and Teyorf1, have been mapped to Yp [36]; these are present in Sxr (Fig 7), so they are also candidates for the improvement in sperm morphogenesis in XY*XSxr males relative to XY*XSry males. However, the breeding required to generate XY*X males with four or more transgenes is now prohibitively expensive. It is therefore encouraging that recent advances in Y gene targeting [37-39] promise simpler and more rapid advances in understanding mouse Yp gene functions than our onerous strategy of Yp transgene addition.

Fig 7

Mapping of Prssly and Teyorf1 to Sxr and Sxr.

Prssly and Teyorf1 map to the Yp-derived Sxr chromosomal fragment (here attached distal to the PAR of one X of an XXSxr male). As expected from the known breakpoints for the Δ deletion, Prssly and Teyorf1 are also present in Sxr, whereas the Tspy pseudogene is absent.

The present findings with respect to the pivotal role of Zfy2 (but not in enabling the transition from randomly orientated round spermatids to spermatids undergoing sperm morphogenesis, are an important addition to our recent findings documenting sometimes overlapping but nevertheless distinct roles for Zfy1 and Zfy2 during earlier stages of spermatogenesis. It will be a substantial challenge for the future to document the direct and indirect transcriptional changes triggered by these transcription factors, which underlie their distinct roles.

Materials and Methods

Animals

The mice utilized in this study had a limited Y gene complement (see below and Fig 1). Details of their production are provided in Vernet et al., 2014 [14]. All males were examined at 6 weeks of age.

XOSry (abbreviated as XOSry) males carry an X-located transgene encoding the spermatogonial proliferation factor Eif2s3y [40] and an autosomally-located transgene of testis determinant Sry [41, 42].

XY*XSry (abbreviated as XY*XSry) males have the same Y gene complement as XOSry but carry a minute X chromosome derivative (Y*X) with a complete pseudoautosomal region (PAR) [18-20].

XSxrbO (abbreviated as XSxrbO) males have the X chromosome carrying an Eif2s3y transgene [40] together with Tp(Y)1Ct attached distal to the X PAR. Tp(Y)1Ct is an Sxra derivative with a 1.3 Mb deletion that has removed the majority of the Yp gene complement and created a Zfy2/1 fusion gene [9, 43].

XY*XSxrb (abbreviated as XY*XSxrb) have the same Y gene complement as XSxrbO but also carry Y*X.

XSxrO have the X chromosome carrying the Yp derivative Tp(Y)1Ct [44] attached distal to the X PAR.

XY*XSxra males have the same Y gene complement as XSxrO but also carry Y*X.

XY*XSry (abbreviated as XY*XSry) males are genotype 2 males to which a single copy, X chromosome-located, Zfy1-Uba1y BAC (RP24-327G6) transgene [12] has been added.

XY*XSry (abbreviated as XE,Z2Y*XSry) males are genotype 2 to which a single copy Zfy2 BAC transgene inserted by cassette mediated exchange (CME) into the Hprt locus on the X chromosome [12, 13] has been added.

The mice were fed ad libitum with a standard diet and maintained in a temperature and light-controlled room (22°C, 14h light/10h dark). The protocols for animal handling and treatment procedures were reviewed and approved by the Crick Biological Research Facility Strategic Oversight Committee (BRF-SOC) and Animal Welfare and Ethical Review Body(AWERB), in accordance with the United Kingdom Animal Scientific Procedures Act 1986 (Procedure Project Licence: 80/2186).

Histology and analysis of sperm head morphology

For histology analysis, part of the testes were fixed in Bouin overnight and then stored in 70% ethanol prior to embedding in paraffin wax, sectioning at 5 μm, and staining with hematoxylin-eosin (H&E) or periodic acid Schiff and hematoxylin (PAS-H). The stages of seminiferous tubules were identified based on the composition of cells near the basal membrane according to the method described by Ahmed [45]. This was necessary because of meiotic and post-meiotic arrests present in males with limited Y gene complement, which prevented staging based on the changes of acrosome and nuclear morphology of spermatids. As a consequence, some tubule stages cannot be distinguished reliably so a range has to be given.

Sperm smears obtained from the testis were silver stained and analyzed as described previously [41].

Transmission electron microscopy

Mice were perfused, through the left ventricle [46], with ice-cold 2.5% glutaraldehyde fixative diluted in PBS. The testes were dissected, left for 1h in the fixative, and cut into small blocks that were kept at 4°C in the same fixative until embedding. Testes were post-fixed for 1h in 1% osmium tetroxide, stained ‘en bloc’ for 1h with 1% aqueous uranyl acetate, dehydrated with graded alcohol series and embedded in Epon. Ultrathin sections (60nm) were contrasted 7min with uranyl acetate and lead citrate and then examined using JEOL 1200EX electron microscope.

RT-PCR band quantitation for Fig 6

Band intensity was quantified using ImageJ, background subtracted and intensity normalised to Lemd1 in each sample. For further details see S1 Appendix, S1 and S2 Figs.

PCR analysis for Fig 7

Standard PCR on genomic DNA was used to map the Y chromosome genes Prssly and Teyorf1. The controls are Tspy-ps, present in the Sxrb deletion interval, and the autosomal gene Atr. For primers and annealing temperatures see S1 Table.

Duplex RT-PCR assays.

(DOCX)

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Duplex RT-PCR assays for Cypt-driven transcript.

(A) Genomic structure of the mouse Zfy1, Zfy2 and Zfy2/1 fusion genes are shown. Exons are represented by boxes (black for Zfy2 and white for Zfy1) and are not to scale. The position of the primers used for amplifying Cypt promotor dependent transcripts with exon 6 is depicted below each gene (see S1 Table for the primer sequence). (B) Gel picture of the RT-PCR assays showing amplification Cypt-dependent transcripts for XYSxr, XY, XY*XSry and XY*XSxr. Lemd1 is a round spermatids expressed gene used as a control.

(TIF)

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RT-PCR analysis of staged mouse testes.

Lemd1 transcription begins between 20 and 27 dpp. RT-PCR for Lemd1 and loading control Hmbs were performed separately with standard Taq polymerase and mixed for migration. Primers used are in S1 Table.

(TIF)

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List of primers used.

(DOCX)

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