20 research outputs found
Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential
Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12–13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis
Etudes de gènes des chromosomes sexuels au cours de la spermatogenèse chez l'homme et la souris et implication dans la fertilite masculine
Les chromosomes sexuels subissent pendant la spermatogenèse de multiples modifications qui entrainent d’importantes variations dans le niveau d’expression des gènes qu’ils portent. Notamment, ils sont inactivés au cours de la méiose et la majorité reste réprimé tout au long de la spermiogenèse. Cette étude met en évidence l’existence de transcrits alternatifs particuliers de gènes sur le chromosome X et Y, dont les profils d’expressions témoignent de leur rôle au cours de ces deux phases de la spermatogenèse. Sur le chromosome X nous avons isolé, chez l’homme et chez la souris, trois gènes ubiquitaires (Uba1x, Prdx4, Atp11c) réactivés dans les spermatides via un transcrit alternatif exprimé de façon majoritaire dans les testicules. Le gène Prdx4 code, pour deux isoformes de protéines différentes par leur domaine N-terminal. Nous avons mis au point des anticorps spécifiques de chaque isoforme et nous avons démontré que, chez la souris, le transcrit réactivé est traduit dans les spermatides et produit une protéine dans un compartiment cellulaire distinct de l’autre isofome ubiquitaire. Un total de cinq mutations, affectant ces transcrits exprimés dans les spermatides, ont été retrouvées dans les gènes UBA1X et PRDX4 chez des hommes infertiles. Sur le chromosome Y chez la souris, nous avons étudié les gènes Zfy1 et Zfy2, deux homologues testicules spécifiques codant pour des protéines à doigt de zinc avec un long domaine d’activation. Zfy2, mais pas Zfy1, promeut l’élimination apoptotique des spermatocytes contenant un chromosome X univalent. Nous avons identifié un transcrit alternatif du gène testicule spécifique Zfy1 exprimé dans les spermatocytes et les spermatides. La protéine putative issue de ce transcrit, possédant un domaine acidique réduit de moitié qui pourrait être à l’origine des différences fonctionnelles entre les gènes homologues Zfy1 et Zfy2 au cours de la méiose murine. Chez l’homme, l’orthologue de ces gènes ZFY est ubiquitaire et nous avons montré qu’il produisait un transcrit alternatif testicule spécifique, codant une protéine avec le même domaine acidique raccourci que Zfy1. Nos données indiquent que ce transcrit alternatif est prédominant dans les spermatocytes et les spermatides chez l’homme et chez la souris. Ces résultats apportent la première évidence d’une fonction du gène ZFY au cours de la spermatogenèse chez l’homme et de son implication possible dans la fertilité masculine.Sex chromosomes undergo many modifications during spermatogenesis, leading to dramatic variations in the expression levels of their genes. In particular, they are inactivated during meiosis with most genes remain silent throughout spermiogenesis. Our study describes specific alternative transcripts produced by X and Y chromosome genes, whose expression indicates roles in early spermatocytes (meiosis) and in spermatids (spermiogenesis). On the X chromosome, we have shown that three widely transcribed genes, Uba1x, Prdx4, and Atp11c, are reactivated in mouse and human spermatids via an alternative transcript that is expressed mainly in the testis. The Prdx4 gene codes two isoforms of the peroxiredoxin 4 that differ in their N-terminal domain. We have raised antibodies specific for each PRDX4 isoform and demonstrate, in mouse, that the reactivated transcript is translated in spermatids, producing a protein in a distinct cellular compartment from the ubiquitous isoform. Altogether, five mutations, affecting the spermatid-reactivated transcripts uniquely, of UBA1x and PRDX4, have been found specifically in our group of infertile men. On the mouse Y chromosome, we have studied Zfy1 and Zfy2, nearly identical testis specific zinc finger genes with long acidic (activation) domains. Zfy2, but not Zfy1, promotes the apoptotic elimination of spermatocytes with an unpaired X chromosome. We have identified an alternatively spliced transcript of Zfy1 that lacks half the acidic domain, and could explain the functional difference between Zfy1 and Zfy2. In human, the ZFY gene is widely transcribed, but we show that ZFY produces a testis specific variant transcript, encoding the same short acidic domain as Zfy1. Our data indicate that the alternative transcripts predominate in spermatocytes and spermatids, in both human and mouse. This provides the first evidence that human ZFY may play a conserved role during spermatogenesis, and contribute to human male fertility
Recombination between the mouse Y chromosome short arm and an additional Y short arm-derived chromosomal segment attached distal to the X chromosome PAR
International audienceIn a male mouse, meiosis markers of processed DNA double strand breaks (DSBs) such as DMC1 and RAD51 are regularly seen in the non-PAR region of the X chromosome; these disappear late in prophase prior to entry into the first meiotic metaphase. Marker evidence for DSBs occurring in the non-PAR region of the Y chromosome is limited. Nevertheless, historically it has been documented that recombination can occur within the mouse Y short arm (Yp) when an additional Yp segment is attached distal to the X and/or the Y pseudoautosomal region (PAR). A number of recombinants identified among offsprings involved unequal exchanges involving repeated DNA segments; however, equal exchanges will have frequently been missed because of the paucity of markers to differentiate between the two Yp segments. Here, we discuss this historical data and present extensive additional data obtained for two mouse models with Yp additions to the X PAR. PCR genotyping enabled identification of a wider range of potential recombinants; the proportions of Yp exchanges identified among the recombinants were 9.7 and 22.4 %. The frequency of these exchanges suggests that the Yp segment attached to the X PAR is subject to the elevated level of recombinational DSBs that characterizes the PAR
Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development
International audienceA 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
The expression of Y-linked Zfy2 in XY mouse oocytes leads to frequent meiosis 2 defects, a high incidence of subsequent early cleavage stage arrest and infertility.
Outbred XY(Sry-) female mice that lack Sry due to the 11 kb deletion Sry(dl1Rlb) have very limited fertility. However, five lines of outbred XY(d) females with Y chromosome deletions Y(Del(Y)1Ct)-Y(Del(Y)5Ct) that deplete the Rbmy gene cluster and repress Sry transcription were found to be of good fertility. Here we tested our expectation that the difference in fertility between XO, XY(d-1) and XY(Sry-) females would be reflected in different degrees of oocyte depletion, but this was not the case. Transgenic addition of Yp genes to XO females implicated Zfy2 as being responsible for the deleterious Y chromosomal effect on fertility. Zfy2 transcript levels were reduced in ovaries of XY(d-1) compared with XY(Sry-) females in keeping with their differing fertility. In seeking the biological basis of the impaired fertility we found that XY(Sry-), XY(d-1) and XO,Zfy2 females produce equivalent numbers of 2-cell embryos. However, in XY(Sry-) and XO,Zfy2 females the majority of embryos arrested with 2-4 cells and almost no blastocysts were produced; by contrast, XY(d-1) females produced substantially more blastocysts but fewer than XO controls. As previously documented for C57BL/6 inbred XY females, outbred XY(Sry-) and XO,Zfy2 females showed frequent failure of the second meiotic division, although this did not prevent the first cleavage. Oocyte transcriptome analysis revealed major transcriptional changes resulting from the Zfy2 transgene addition. We conclude that Zfy2-induced transcriptional changes in oocytes are sufficient to explain the more severe fertility impairment of XY as compared with XO females
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<sup>nd</sup> 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
Mapping of <i>Prssly</i> and <i>Teyorf1</i> to <i>Sxr</i><sup><i>a</i></sup> and <i>Sxr</i><sup><i>b</i></sup>.
<p><i>Prssly</i> and <i>Teyorf1</i> map to the Yp-derived <i>Sxr</i><sup><i>a</i></sup> chromosomal fragment (here attached distal to the PAR of one X of an XX<i>Sxr</i><sup><i>a</i></sup> male). As expected from the known breakpoints for the Δ<sup><i>Sxr-b</i></sup> deletion, <i>Prssly</i> and <i>Teyorf1</i> are also present in <i>Sxr</i><sup><i>b</i></sup>, whereas the <i>Tspy</i> pseudogene is absent.</p
Sperm head and tail morphogenesis in X<sup><i>E</i>,<i>Z2</i></sup>Y*<sup>X</sup><i>Sry</i> males as compared to controls.
<p><b>(A)</b> Electron micrographs of developing sperm heads from 6 week old XY, XY*<sup>X</sup><i>Sxr</i><sup><i>a</i></sup> and X<sup><i>E</i>,<i>Z2</i></sup>Y*<sup>X</sup><i>Sry</i> males. Spermatids at round to elongating transitional stage in X<sup><i>E</i>,<i>Z2</i></sup>Y*<sup>X</sup><i>Sry</i> 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 X<sup><i>E</i>,<i>Z2</i></sup>Y*<sup>X</sup><i>Sry</i> and XY*<sup>X</sup><i>Sxr</i><sup><i>a</i></sup>. It was again evident that the sperm heads in X<sup><i>E</i>,<i>Z2</i></sup>Y*<sup>X</sup><i>Sry</i> fail to elongate properly (stars). <b>(B)</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).</p
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*<sup>X</sup>.
<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*<sup>X</sup> 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*<sup>X</sup> 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*<sup>X</sup> 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<sup>b</sup></i> (which includes a <i>Zfy2/1</i> fusion gene encoding a ZFY1-like protein) and <i>Sxr<sup>a</sup></i> (which includes <i>Zfy1</i> and <i>Zfy2</i>) promote meiosis II in the presence of Y*<sup>X</sup>; <i>Sxr<sup>a</sup></i> is substantially more effective than <i>Sxr<sup>b</sup></i>. B. Percentage of haploid round spermatids found in testis of 6 week old X<i><sup>E</sup></i>Y*<sup>X</sup><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