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.

Recombination between the mouse Y chromosome short arm and an additional Y short arm-derived chromosomal segment attached distal to the X chromosome PAR.

In 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-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division.

Mouse Zfy1 and Zfy2 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 (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). 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 Zfy 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 Zfy2-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 Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 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 de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx 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 Zfx, Zfy1 and Zfy2 validate this hypothesis.

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.

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.

HSFY genes and the P4 palindrome in the AZFb interval of the human Y chromosome are not required for spermatocyte maturation.

BACKGROUND: Recurrent AZFb deletions on the human Y chromosome are associated with an absence of ejaculated spermatozoa consequent to a meiotic maturation arrest that prevents the progression of germ cells to haploid stages. The extreme rarity of partial deletions has hampered the identification of the AZFb genes required for normal meiotic stages. The critical interval, refined by two overlapping deletions associated with full spermatogenesis (AZFc and b1/b3), measures over 4 Mb and contains 13 coding genes: CDY2, XKRY, HSFY1, HSFY2, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2 and four copies of RBMY. METHODS AND RESULTS: We screened 1186 men from infertile couples for Y chromosome deletions, and identified three unrelated oligozoospermic men and one azoospermic man who carry an identical 768 kb deletion resulting in loss of the entire P4 palindrome, including both HSFY genes, the only coding genes within the deletion interval. This 768 kb deletion was not found in 1179 control men. The deletion breakpoints share only 4 bp of nucleotide identity, revealing that the deletions are not recurrent, but are descendants of a founding deletion. Confirming this, we find that all four men carry a Y chromosome of the same highly defined haplogroup (R1b1b1a1b) (incidence 30% in Southern France), although further haplotype analyses showed that they were not closely related. CONCLUSIONS: Although the HSFY deletion is restricted to our infertile group, it has been transmitted naturally over many generations, indicating that HSFY genes make only a slight contribution to male fertility. Importantly, our study formally excludes HSFY genes as the AZFb factor required for progression through meiosis.