Analysis of male meiotic "sex body" proteins during XY female meiosis provides new insights into their functions

During male meiosis in mammals the X and Y chromosomes become condensed to form the sex body (XY body), which is the morphological manifestation of the process of meiotic sex chromosome inactivation (MSCI). An increasing number of sex body located proteins are being identified, but their functions in relation to MSCI are unclear. Here we demonstrate that assaying male sex body located proteins during XY female mouse meiosis, where MSCI does not take place, is one way in which to begin to discriminate between potential functions. We show that a newly identified protein, "Asynaptin" (ASY), detected in male meiosis exclusively in association with the X and Y chromatin of the sex body, is also expressed in pachytene oocytes of XY females where it coats the chromatin of the asynapsed X in the absence of MSCI. Furthermore, in pachytene oocytes of females carrying a reciprocal autosomal translocation, ASY associates with asynapsed autosomal chromatin. Thus the location of ASY to the sex body during male meiosis is likely to be a response to the asynapsis of the non-homologous regions [outside the pseudoautosomal region (PAR)] of the heteromorphic X-Y bivalent, rather than being related to MSCI. In contrast to ASY, the previously described sex body protein XY77 proved to be male sex body specific. Potential functions for MSCI and the sex body are discussed together with the possible roles of these two proteins.     

Postmeiotic sex chromatin in the male germline of mice

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.     

Meiotic behaviour of sex chromosomes investigated by three-colour FISH on 35 142 sperm nuclei from two 47,XYY males

Meiotic segregation of sex chromosomes from two fertile 47,XYY men was analysed by a three-colour fluorescence in situ hybridisation procedure. This method allows the identification of hyperhaploidies (spermatozoa with 24 chromosomes) and diploidies (spermatozoa with 46 chromosomes), and their meiotic origin (meiosis I or II). Alpha-satellite probes specific for chromosomes X, Y and 1 were observed simultaneously in 35 142 sperm nuclei. For both 47,XYY men (24 315 sperm nuclei analysed from one male and 10 827 from the other one) the sex ratio differs from the expected 1:1 ratio (P < 0.001). The rates of disomic Y, diploid YY and diploid XY spermatozoa were increased for both 47,XYY men compared with control sperm (142 050 sperm nuclei analysed from five control men), whereas the rates of hyperhaploidy XY, disomy X and disomy 1 were not significantly different from those of control sperm. These results support the hypothesis that the extra Y chromosome is lost before meiosis with a proliferative advantage of the resulting 46,XY germ cells. Our observations also suggest that a few primary spermatocytes with two Y chromosomes are able to progress through meiosis and to produce Y-bearing sperm cells. A theoretical pairing of the three gonosomes in primary spermatocytes with an extra sex chromosome, compatible with active spermatogenesis, is proposed. Received: 12 April 1996 / Revised: 26 August 1996     

Sex Chromosome Meiotic Drive in DROSOPHILA MELANOGASTER Males

In Drosophila melanogaster males, deficiency for X heterochromatin causes high X-Y nondisjunction and skewed sex chromosome segregation ratios (meiotic drive). Y and XY classes are recovered poorly because of sperm dysfunction. In this study it was found that X heterochromatic deficiencies disrupt recovery not only of the Y chromosome but also of the X and autosomes, that both heterochromatic and euchromatic regions of chromosomes are affected and that the "sensitivity" of a chromosome to meiotic drive is a function of its length. Two models to explain these results are considered. One is a competitive model that proposes that all chromosomes must compete for a scarce chromosome-binding material in Xh(-) males. The failure to observe competitive interactions among chromosome recovery probabilities rules out this model. The second is a pairing model which holds that normal spermiogenesis requires X-Y pairing at special heterochromatic pairing sites. Unsaturated pairing sites become gametic lethals. This model fails to account for autosomal sensitivity to meiotic drive. It is also contradicted by evidence that saturation of Y-pairing sites fails to suppress meiotic drive in Xh(- ) males and that extra X-pairing sites in an otherwise normal male do not induce drive. It is argued that meiotic drive results from separation of X euchromatin from X heterochromatin.     

Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation

Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.     

The mouse Y* chromosome involves a complex rearrangement, including interstitial positioning of the pseudoautosomal region.

Cytological analysis of the mouse Y* chromosome revealed a complex rearrangement involving acquisition of a functional centromere and centromeric heterochromatin and attachment of this chromosomal segment to the distal end of a normal Y* chromosome. This rearrangement positioned the Y* short-arm region at the distal end of the Y* chromosome and the pseudoautosomal region interstitially, just distal to the newly acquired centromere. In addition, the majority of the pseudoautosomal region was inverted. Recombination between the X and the Y* chromosomes generates two new sex chromosomes: (1) a large chromosome comprised of the X chromosome attached at its distal end to all of the Y* chromosome but missing the centromeric region (XY*) and (2) a small chromosome containing the centromeric portion of the Y* chromosome attached to G-band-negative material from the X chromosome (YX). Mice that inherit the XY* chromosome develop as sterile males, whereas mice that inherit the Y*X chromosome develop as fertile females. Recovery of equal numbers of recombinant and nonrecombinant offspring from XY* males supports the hypothesis that recombination between the mammalian X and Y chromosomes is necessary for primary spermatocytes to successfully complete spermatogenesis and form functional sperm.     

Evidence that meiotic sex chromosome inactivation is essential for male fertility

The mammalian X and Y chromosomes share little homology and are largely unsynapsed during normal meiosis. This asynapsis triggers inactivation of X- and Y-linked genes, or meiotic sex chromosome inactivation (MSCI). Whether MSCI is essential for male meiosis is unclear. Pachytene arrest and apoptosis is observed in mouse mutants in which MSCI fails, e.g., Brca1(-/-), H2afx(-/-), Sycp1(-/-), and Msh5(-/-). However, these also harbor defects in synapsis and/or recombination and as such may activate a putative pachytene checkpoint. Here we present evidence that MSCI failure is sufficient to cause pachytene arrest. XYY males exhibit Y-Y synapsis and Y chromosomal escape from MSCI without accompanying synapsis/recombination defects. We find that XYY males, like synapsis/recombination mutants, display pachytene arrest and that this can be circumvented by preventing Y-Y synapsis and associated Y gene expression. Pachytene expression of individual Y genes inserted as transgenes on autosomes shows that expression of the Zfy 1/2 paralogs in XY males is sufficient to phenocopy the pachytene arrest phenotype; insertion of Zfy 1/2 on the X chromosome where they are subject to MSCI prevents this response. Our findings show that MSCI is essential for male meiosis and, as such, provide insight into the differential severity of meiotic mutations' effects on male and female meiosis.     

Chromosome banding in Amphibia

The mitotic and meiotic chromosomes of the marsupial frog Gastrotheca riobambae were analysed with various banding techniques. The karyotype of this species is distinguished by considerable amounts of constitutive heterochromatin and unusual, heteromorphic XY sex chromosomes. The Y chromosome is considerably larger than the X chromosome and almost completely heterochromatic. The analysis of the banding patterns obtained with GC- and AT-base-pair-specific fluorochromes shows that the constitutive heterochromatin in the Y chromosome consists of at least three different structural categories. The only nucleolus organizer region (NOR) of the karyotype is localized in the short arm of the X chromosome. This causes a sex-specific difference in the number of NOR: female animals have two NORs in diploid cells, male animals one. No cytological indications were found for the inactivation of one of the two X chromosomes in the female cells. In male meiosis, the heteromorphic sex chromosomes form a characteristic sex-bivalent by pairing their telomeres in an end-to-end arrangement. The significance of the XY/XX sex chromosomes of G. riobambae for the study of X-linked genes in Amphibia, the evolution of sex chromosomes and their specific DNA sequences, and the significance of the meiotic process of sex chromosomes are discussed.     

Male meiotic segregation of gonosomes analysed by two colour FISH in human interphase spermatozoa

Human meiotic segregation of X and Y chromosomes was simultaneously analysed by dual fluorescence in situ hybridization (FISH) on 10638 interphase spermatozoa from the same donor. A modified method for sperm decondensation ensured access of both X and Y probes to the sperm chromatin and a 99% hybridization efficiency. Expected sex ratios were obtained (49.30% haploidy X and 49.22% haploidy Y). The frequencies of meiotic II non-disjunctions for X and Y chromosomes (0.05%) were similar to those observed in sperm karyotypes after heterospecific fertilization of hamster eggs. In contrast, the frequency of XY bearing cells was significantly higher (0.42%). However, XY cells detected by FISH could either be diploid somatic cells, diploid germinal cells or hyperhaploid XY spermatozoa, the latter resulting from meiotic I non-disjunctions.