Mouse Y-Linked <i>Zfy1</i> and <i>Zfy2</i> Are Expressed during the Male-Specific Interphase between Meiosis I and Meiosis II and Promote the 2^nd Meiotic Division

<div><p>Mouse <i>Zfy1</i> and <i>Zfy2</i> encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (<i>Zfy1</i> and <i>Zfy2</i>) and at the first meiotic metaphase (<i>Zfy2</i>). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and <i>Zfy</i> gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding <i>Zfy2</i>-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified <i>Zfy1</i> and <i>Zfy2</i> as the genes responsible. <i>Zfy2</i> was substantially more effective and proved to have a much more potent transactivation domain than <i>Zfy1</i>. We previously established that only <i>Zfy2</i> is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was <i>de novo Zfy1</i> and <i>Zfy2</i> transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded <i>Zfx</i> was also expressed at this stage and <i>Zfx</i> over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of <i>Zfx</i>, <i>Zfy1</i> and <i>Zfy2</i> validate this hypothesis.</p></div

The mouse <i>Zfy</i> and <i>Zfx</i> genes are transcribed in interphasic secondary spermatocytes.

<p>Representative images of interphasic secondary spermatocyte nuclei are shown hybridized with RNA FISH probes specific for <i>Zfy1</i>, <i>Zfy2</i> or <i>Zfx</i> (arrows, top panels). Interphasic secondary spermatocytes were distinguished from diploid spermatids by staining spread spermatogenic cells from 6-week old XY males with an antibody against SYCP3 (red, top panels). The appropriate localization of the RNA FISH probe to the encoding genes was confirmed by DNA FISH (arrows, bottom panels). Nuclei are stained with DAPI (blue). X- or Y-bearing secondary spermatocytes are respectively represented by an X or a Y next to the cell.</p

<i>Zfy1</i> and <i>Zfy2</i> promote meiosis-II in the presence of the sex chromosome pairing partner Y*^X.

<p>Data collected after DNA quantitation of spermatids using DAPI fluorescence intensity measurement on SYCP3-labelled testis cell spreads. Pooled data expressed as percentages are shown for each genotype (n = 4). Key: in black, models with robust apoptotic elimination of MI spermatocytes with X univalents; in white, XO models with markedly reduced apoptotic response; striped, XY*^X models with markedly reduced apoptotic response in which the frequency was adjusted to remove spermatids derived from MI spermatocytes that had not formed an X-Y*^X bivalent by PAR-PAR synapsis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444.s006" target="_blank">Table S1</a>). A. Percentage of haploid round spermatids found in testis of 6 week old XO and XY*^X males with various Yp chromosome gene contents. The data for the two XO male genotypes derive from Vernet et al., 2012 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444-Vernet2" target="_blank">[14]</a>. The Yp-derived <i>Sxr^b</i> (which includes a <i>Zfy2/1</i> fusion gene encoding a ZFY1-like protein) and <i>Sxr^a</i> (which includes <i>Zfy1</i> and <i>Zfy2</i>) promote meiosis II in the presence of Y*^X; <i>Sxr^a</i> is substantially more effective than <i>Sxr^b</i>. B. Percentage of haploid round spermatids found in testis of 6 week old X<i>^E</i>Y*^X<i>Sry</i> males with X-linked <i>Zfy</i> transgene additions. <i>Zfy1</i>, and to a greater extent <i>Zfy2</i>, promote meiosis II. NS, Non significant; *p≤0.05; **p≤0.01; ***p≤0.001.</p

X and Y-linked ‘<i>Zf</i>’ gene expression by RNA-FISH in spread interphasic secondary spermatocytes from 6 week old XY male.

<p>X and Y-linked ‘<i>Zf</i>’ gene expression by RNA-FISH in spread interphasic secondary spermatocytes from 6 week old XY male.</p

<i>Zfy2</i> acidic domain is a much more potent transactivator than other <i>‘Zf’</i> acidic domains.

<p>Levels of β-galactosidase induced by the Gal4-DNA-binding domain on its own (pGB-CEN6; negative control) or fused to an acidic domain from one of six different ZF isoforms from human (hs) or mouse (mm). Among the mouse sex-linked genes, <i>Zfy2</i> has a substantially more potent activation domain than <i>Zfy1</i>, and <i>Zfx</i> is significantly less potent than <i>Zfy1</i>. mm ZFA derives from the autosomal <i>Zfa</i> gene that originated from a retroposed X transcript. *p≤0.05; **p≤0.01.</p

A summary of the meiotic outcome in XO and XY*^X males with varying Yp gene content.

<p>Throughout this figure the thickness of the arrows indicates the proportion of cells progressing from one step to the next and the cheeses at the bottom represent the proportion of haploid and diploid spermatids. Size of the cheese indicates the relative success of the different models in meiosis completion. A. XO models. In X<i>^E</i>O<i>Sry</i> and X<i>^ESxr^b</i>O males the majority of spermatocytes complete meiosis I because of the reduced apoptotic response at MI due to the absence of <i>Zfy2 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444-Vernet1" target="_blank">[11]</a>. <i>Zfx</i> expression is likely responsible for the residual apoptotic response (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444.s005" target="_blank">Figure S5</a>). The majority of spermatocytes then arrest at the interphase between meiosis I and meiosis II. This could be a consequence of the prior triggering of the MI SAC by the univalent X, the reduced apoptotic response due to the absence of <i>Zfy2</i>, or the lack of a Yp gene or genes that promotes meiosis II. In X<i>Sxr^a</i>O males there is a very efficient apoptotic elimination of spermatocytes at MI so that very few complete meiosis I and this results in a 97% reduction in the number of spermatids. This precludes any firm conclusion as to a role for Yp genes for completion of meiosis II because the apoptotic elimination may have had a bias towards removing MI cells that were otherwise destined to arrest at the following interphase. B. XY*^X models. In these models the spermatocytes that form a sex bivalent circumvent the MI SAC/apoptotic response and complete meiosis I. This reveals that <i>Sxr^a</i> strongly promotes meiosis II, thus confirming that a gene or genes on Yp promotes meiosis II. Surprisingly <i>Sxr^b</i>, which did not promote meiosis II in X<i>^ESxr^b</i>O males, does so now that the apoptotic response is circumvented by formation of an X-Y*^X sex bivalent. C. The XY*^X<i>Sry ‘Zf’</i> transgene addition models. These transgene additions revealed that <i>Zfy1</i> and <i>Zfy2</i> are the genes on Yp that promote meiosis II with <i>Zfy2</i> the more effective. <i>Sxr^b</i> includes the <i>Zfy2/1</i> fusion gene that encodes a ZFY1-like protein, whereas <i>Sxr^a</i> includes <i>Zfy1</i> and <i>Zfy2</i>, thus explaining the more potent effect of <i>Sxr^a</i> in promoting meiosis II. <i>Zfx</i> over-expression also promotes meiosis II (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen-1004444-g005" target="_blank">Figure 5</a>) making it likely that the endogenous <i>Zfx</i> also does so to some degree.</p

X-Y pairing efficiency in pachytene spermatocytes.

a<p>In a few cells, the Y or the Y*^X chromosome appear to be missing. Some of them might have achieved a centromere pairing with the X chromosome or have been lost during cell spreading.</p>b<p>The average PAR-synapsis for all Y*^X bearing males (n = 15) is 74.9%.</p

<i>Zfx</i> over-expression promotes meiosis II.

<p>A. Spread cells found in testis of 6-week old X<i>^E</i>Y*^X<i>Sry</i> males without or with <i>Zfx</i> transgene. Pachytene (Pa), diploid spermatid (St d) and haploid spermatids (St h) nuclei are stained with DAPI (top panel) and higher magnifications are shown additionally labelled with γH2AFX, and SYCP3 antibodies (bottom panel). B. Percentage of haploid round spermatids found in mice from panel A (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444.s007" target="_blank">Table S2D</a>). Key: in black, X<i>^E</i>Y*^X<i>Sry</i>,<i>Zfx</i> transgenic males have a robust apoptotic elimination of the ∼25% of MI spermatocytes that have an X univalent (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444.s005" target="_blank">Figure S5</a>); striped, X<i>^E</i>Y*^X<i>Sry</i> males have a markedly reduced apoptotic response so the frequency was adjusted as detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004444#pgen.1004444.s006" target="_blank">Table S1</a>. The addition of the <i>Zfx</i> transgene significantly increases (***p≤0.00001) the proportion of haploid round spermatids.</p

The XO and XY*^X mouse models.

<p>A. XY. The Y short arm (Yp) gene complement of an XY male (represented to scale in the magnified view) comprises seven 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. B–D. The diminishing Yp gene complements for the three XO male mouse models that lack the Y long arm. B. X<i>Sxr^a</i>O. The Yp-derived <i>Sxr^a</i> sex-reversal factor, attached distal to the X PAR provides an almost complete Yp gene complement. C. X<i>Sxr^b</i>O. The <i>Sxr^a-</i>derived deletion variant <i>Sxr^b</i> has a 1.3 Mb deletion (Δ<i>^Sxr-b</i>) removing 6 single copy genes and creating a <i>Zfy2/1</i> fusion gene spanning the deletion breakpoint (†). D. XO<i>Sry</i>. This model has only one Y chromosome gene, namely the testis determinant <i>Sry</i> provided as an autosomally located transgene. E. Y*^X. This mini sex-chromosome is an X chromosome with a deletion from just proximal to <i>Amelx</i> to within the DXHXF34 repeat adjacent to the X centromere. † represents the deletion breakpoint. This X chromosome derivative has a complete PAR that can pair with the PAR of X<i>Sxr^a</i>, X<i>Sxr^b</i> or X to form a ‘minimal sex bivalent’. Scale bar for magnified views is 150 kb.</p

Efficiency of XY synapsis in the XY*^X males with varying Yp complements.

<p>Spread pachytene spermatocytes from 6 week old XY and X<i>^E</i>Y*^X<i>Sry</i> testes stained with antibodies against SYCP3 (green) and CREST (red). Frames show PAR-PAR sex chromosome synapsis in XY and X<i>^E</i>Y*^X<i>Sry</i> males; the arrow points to an unsynapsed Y*^X chromosome in an X<i>^E</i>Y*^X<i>Sry</i> male.</p