Gastric teratoma presenting as melena in the newborn: A case report.

Melena is reported in 1 of 350 to 400 new-borns. Significant upper gastrointestinal bleeding in a neonate with an antenatally diagnosed abdominal mass has not been reported before. This case highlights an unusual presentation of a gastric teratoma and proposes a probable embryological explanation for the site of occurrence.

Mammalian meiotic silencing exhibits sexually dimorphic features.

During mammalian meiotic prophase I, surveillance mechanisms exist to ensure that germ cells with defective synapsis or recombination are eliminated, thereby preventing the generation of aneuploid gametes and embryos. Meiosis in females is more error-prone than in males, and this is in part because the prophase I surveillance mechanisms are less efficient in females. A mechanistic understanding of this sexual dimorphism is currently lacking. In both sexes, asynapsed chromosomes are transcriptionally inactivated by ATR-dependent phosphorylation of histone H2AFX. This process, termed meiotic silencing, has been proposed to perform an important prophase I surveillance role. While the transcriptional effects of meiotic silencing at individual genes are well described in the male germ line, analogous studies in the female germ line have not been performed. Here we apply single- and multigene RNA fluorescence in situ hybridization (RNA FISH) to oocytes from chromosomally abnormal mouse models to uncover potential sex differences in the silencing response. Notably, we find that meiotic silencing in females is less efficient than in males. Within individual oocytes, genes located on the same asynapsed chromosome are silenced to differing extents, thereby generating mosaicism in gene expression profiles across oocyte populations. Analysis of sex-reversed XY female mice reveals that the sexual dimorphism in silencing is determined by gonadal sex rather than sex chromosome constitution. We propose that sex differences in meiotic silencing impact on the sexually dimorphic prophase I response to asynapsis.

Does Rbmy have a role in sperm development in mice?

The Y(d1) deletion in mice removes most of the multi-copy Rbmy gene cluster that is located adjacent to the centromere on the Y short arm (Yp). XY(d1) mice develop as females because Sry is inactivated, probably because it is now juxtaposed to centromeric heterochromatin. We have previously produced XY(d1)Sry transgenic males and found that they have a substantially increased frequency of abnormal sperm. Staining of testis sections with a polyclonal anti-RBMY antibody appeared to show a marked decrease of RBMY protein in the spermatids of XY(d1)Sry males compared to control males, which led us to suggest that this may be responsible for the increase in sperm anomalies. In the current study we sought to determine whether augmenting Rbmy expression specifically in the spermatids of XY(d1)Sry males would ameliorate the sperm defects. An expressing Rbmy transgene driven by the spermatid-specific mouse protamine 1 promotor (mP1Rbmy) was therefore introduced into XY(d1)Sry males. This failed to reduce the frequency of abnormal sperm. In the course of this study, a new RBMY antibody was generated that, in contrast to the original antibody, failed to detect RBMY in spermatid stages by immunostaining. The lack of RBMY was confirmed by western blotting of lysates from purified round spermatids and elongating spermatids. The implications of these results for the proposed role for RBMY in sperm development are discussed.

A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis.

In mouse and man, deletions of specific regions of the Y chromosome have been linked to early failure of spermatogenesis and consequent sterility; the Y chromosomal gene(s) with this essential early role in spermatogenesis have not been identified. The partial deletion of the mouse Y short arm (the Sxrb deletion) that occurred when Tp(Y)1CtSxr-b (hereafter Sxrb) arose from Tp(Y)1CTSxr-b (hereafter Sxra) defines Spy, a Y chromosomal factor essential for normal spermatogonial proliferation. Molecular analysis has identified six genes that lie within the deletion: Ube1y1 (refs. 4,5), Smcy, Uty, Usp9y (also known as Dffry), Eif2s3y (also known as Eif-2gammay) and Dby10; all have closely similar X-encoded homologs. Of the Y-encoded genes, Ube1y1 and Dby have been considered strong candidates for mouse Spy function, whereas Smcy has been effectively ruled out as a candidate. There is no Ube1y1 homolog in man, and DBY, either alone or in conjunction with USP9Y, is the favored candidate for an early spermatogenic role. Here we show that introduction of Ube1y1 and Dby as transgenes into Sxrb-deletion mice fails to overcome the spermatogenic block. However, the introduction of Eif2s3y restores normal spermatogonial proliferation and progression through meiotic prophase. Therefore, Eif2s3y, which encodes a subunit of the eukaryotic translation initiation factor Eif2, is Spy.

Recombinational DNA double-strand breaks in mice precede synapsis.

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.

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.

An analysis of meiotic impairment and of sex chromosome associations throughout meiosis in XYY mice.

The existing XYY meiotic data for mice present a very heterogeneous picture with respect to the relative frequencies of different sex chromosome associations, both at pachytene and diakinesis/metaphase I. Furthermore, where both pachytene and diakinesis/MI data are available for the same males, the frequencies of the different configurations at the two stages are very different. In the present paper we utilise "XYY" and "XY/XYY" mosaic mice with cytologically distinguishable Y chromosomes to investigate the factors responsible for this heterogeneity between different males and between the two meiotic stages. It is concluded (1) that the initial pattern of synapsis is driven by the relatedness of the three pseudoautosomal regions (PARs); (2) that the order and extent of PAR synapsis within radial trivalents are also affected by PAR relatedness and that this leads to chiasmata being preferentially formed between closely related PARs; (3) that trivalents with a single chiasma resolve into a bivalent + univalent by the diakinesis stage; (4) that although many spermatocytes with asynapsed sex chromosomes are eliminated between pachytene and diakinesis, those that survive this phase of elimination progress to the first meiotic metaphase (MI) and accumulate in large numbers, leading to an over-representation of those with univalents as compared to radial trivalents; and (5) that the arrested MI cells are eventually eliminated, so that very few "XYY" cells contribute products to MII.

The Y* rearrangement in mice: new insights into a perplexing PAR.

In essence, the Y* rearrangement in the mouse is a Y chromosome that has been hijacked by a non-Y centromere attached distal to the pseudoautosomal region (PAR). All the Y-unique material is thought to be unaltered, but the recombinatory behaviour of the Y* with the X during male meioisis led to the conclusion that part of the PAR is inverted. In the course of a cross set up to introduce the X-linked mutation Patchy fur (Paf) into XY* males, the Y* chromosome was found to carry the wild type allele of Paf. Paf maps close to the X PAR boundary, so we hypothesised that the inverted region of the Y* PAR originated from an X chromosome that provided not only an inverted copy of proximal PAR, but also an X PAR boundary together with some adjacent X-unique material that included the Paf locus. This hypothesis was validated by Southern analysis using an X PAR boundary probe to show that Y* has an X PAR boundary. Thus the Y* PAR has resulted from an end to end fusion of an X and a Y PAR. Furthermore, it was shown that in conjunction with this PAR-PAR fusion, there has been deletion of both copies of the distally located pseudoautosomal gene Steroid sulfatase (Sts).

Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities.

An RNA-binding motif (RBM) gene family has been identified on the human Y chromosome that maps to the same deletion interval as the 'azoospermia factor' (AZF). We have identified the homologous gene family (Rbm) on the mouse Y with a view to investigating the proposal that this gene family plays a role in spermatogenesis. At least 25 and probably >50 copies of Rbm are present on the mouse Y chromosome short arm located between Sry and the centromere. As in the human, a role in spermatogenesis is indicated by a germ cell-specific pattern of expression in the testis, but there are distinct differences in the pattern of expression between the two species. Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are female due to a position effect resulting in non-expression of Sry ; sex-reversing such mice with an Sry transgene produces males with a high incidence of abnormal sperm, making this the third deletion interval on the mouse Y that affects some aspect of spermatogenesis. Most of the copies of Rbm map to this deletion interval, and the Yd1males have markedly reduced Rbm expression, suggesting that RBM deficiency may be responsible for, or contribute to, the abnormal sperm development. In man, deletion of the functional copies of RBM is associated with meiotic arrest rather than sperm anomalies; however, the different effects of deletion are consistent with the differences in expression between the two species.

Transcriptional analysis of the candidate spermatogenesis gene Ube1y and of the closely related Ube1x shows that they are coexpressed in spermatogonia and spermatids but are repressed in pachytene spermatocytes.

Ube1y is a Y-linked gene transcribed in the testis, which maps to a region of the mouse Y required for normal spermatogonial proliferation. Ube1y, together with a ubiquitously expressed homologue on the X chromosome (Ube1x), encodes ubiquitin-activating enzyme E1, an enzyme essential for eukaryotic cell proliferation. Ube1y is thus a strong candidate for the Y function in spermatogonial proliferation. Using probes specific for the two genes, we have used Northern analysis and RNase protection to assess transcript levels throughout testis development and, by using germ cell-deficient XXSxr(a) testes and purified cell fractions, we have defined the testicular cell types in which transcription occurs. Ube1y transcripts are already detectable in the fetal testis at 12.5 dpc, with higher levels at 14.5 dpc and then falling to low levels by the time of birth. Postnatally levels rise sharply, peaking at 10 dpp. Analysis of XXSxr(a) testes indicates that the bulk of the Ube1y transcription is in germ cells. The analysis of purified cell fractions shows that X- and Y-encoded transcripts are present in A spermatogonia, both are at very low levels (or perhaps absent) in pachytene spermatocytes and then return to high levels in round spermatids. The reactivation of transcription in round spermatids implies a requirement for the ubiquitination pathway at this time. The presence of Ube1x transcripts in A spermatogonia raises the question as to why Ube1y transcripts are required. This question is discussed in relation to the spermatogenic failure in XSxr(b)O mice which are deleted for Ube1y and it is argued that Ube1y serves to increase UBE1 production at a time of high demand. Ube1y transcripts were also detected in XXY and XY ovaries.

Y chromosome short arm-Sxr recombination in XSxr/Y males causes deletion of Rbm and XY female sex reversal.

We earlier described three lines of sex-reversed XY female mice deleted for sequences believed close to the testes-determining gene (Sry) on the Y chromosome short arm (Yp). The original sex-reversed females appeared among the offspring of XY males that carried the Yp duplication Sxr on their X chromosome. Earlier cytogenetic observations had suggested that the deletions resulted from asymmetrical meiotic recombination between the Y and the homologous Sxr region, but no direct evidence for this hypothesis was available. We have now analyzed the offspring of XSxr/Y males carrying an evolutionarily divergent Mus musculus domesticus Y chromosome, which permits detection and characterization of such recombination events. This analysis has enabled the derivation of a recombination map of Yp and Sxr, also demonstrating the orientation of Yp with respect to the Y centromere. The mapping data have established that Rbm, the murine homologue of a gene family cloned from the human Y chromosome, lies between Sry and the centromere. Analysis of two additional XY female lines shows that asymmetrical Yp-Sxr recombination leading to XY female sex reversal results in deletion of Rbm sequences. The deletions bring Sry closer to Y centromere, consistent with the hypothesis that position-effect inactivation of Sry is the basis for the sex reversal.

Y353/B: a candidate multiple-copy spermiogenesis gene on the mouse Y chromosome.

There is evidence from Y Chromosome (Chr) deletion mapping that there is a gene on the long arm of the mouse Y Chr that is needed for the normal development of the sperm head. Since mice with partial Y long arm deletions show incomplete penetrance of the sperm head defect, whereas mice with no Y long arm show complete penetrance, it has been suggested that the 'spermiogenesis' gene may be present in multiple copies. A Y-specific genomic DNA sequence (Y353/B) has previously been described that is present in multiple copies on the long arm of the mouse Y and identifies testis-specific transcripts. We have suggested that Y353/B could be the proposed multiple copy 'spermiogenesis' gene. In support of this suggestion, we show here that mice with a partial Y long arm deletion associated with a 3.5-fold increase in the frequency of abnormal sperm heads have a marked reduction in genomic Y353/B copies and a corresponding reduction in Y353/B-related transcripts. Thus, the incompletely penetrant phenotype correlates with a reduction in Y353/B-related transcription. Furthermore, by in situ hybridization with a Y353/B riboprobe to testis sections, we show that the Y353/B-related transcripts are confined to the round spermatid stage of spermiogenesis, just prior to the shaping of the sperm head. The transcripts sediment with the fraction of cytoplasmic RNA in adult testis that is loaded on polysomes, suggesting that the transcripts are actively translated.

Tdy-negative XY, XXY and XYY female mice: breeding data and synaptonemal complex analysis.

In this paper we have compared the breeding performance of Tdy-negative XY, XXY and XYY females to assess the relative importance of the lack of a second X chromosome compared with the presence of a Y chromosome, in reducing fertility. The XY females were of poor fertility, although five of twelve produced at least one offspring. The XXY females had larger, more frequent litters, and a longer reproductive lifespan, implicating the lack of a second X as the major cause of the poor fertility of XY females. Nevertheless, XYY females appeared to be more seriously affected than the XY females, suggesting that the presence of the Y may be a contributory factor. Pachytene analysis demonstrated that the Y is a very inefficient pairing partner for the X during female meiosis. In XY females only 11% of pachytene cells had the X and Y paired; in XXY females the two X chromosomes paired and the Y was almost always a univalent, while in XYY females the X paired with a Y in only 15% of pachytene cells. The presence of unpaired sex chromosomes has previously been implicated as a cause of oocyte loss during pachytene, and the proportion of cells with unsynapsed sex chromosomes decreased as pachytene proceeded, suggesting that they were progressively eliminated. Significantly, protection against elimination was afforded not only by synapsis between sex chromosomes, but also by self-synapsis if a sex chromosome remained as a univalent. It is concluded that sex chromosome univalence leading to pachytene oocyte failure is responsible for the postnatal oocyte deficiency seen in XY females. A separate study has shown that XXY females have a similar level of oocyte deficiency. It is suggested that the presence of a second X chromosome improves the fertility of XXY females, compared with XY females, by improving oocyte quality and by eliminating the production of lethal XY and OY zygotes. The genotype frequencies for the offspring of XY and XYY females differed from those predicted from the pachytene data. The XY females showed a marked deficiency of XO offspring compared with XXY and XYY aneuploid offspring, whereas the XXY females had fewer than expected XXY and XYY aneuploid offspring.

Fertility in mice requires X-Y pairing and a Y-chromosomal "spermiogenesis" gene mapping to the long arm.

There is accumulating evidence that the mammalian Y chromosome, in addition to its testis-determining function, may have other male limited functions, particularly in spermatogenesis. We have previously shown that the short arm of the mouse Y carries information needed for spermatogonial proliferation. This information, together with the testis-determining gene Sry, is contained within the Y-derived sex reversal factor Sxra. XO males carrying a copy of Sxra attached to the X chromosome are nevertheless sterile owing to an almost complete arrest during the meiotic metaphase stages. Here we show that this meiotic block can be overcome by providing a meiotic pairing partner (with no Y-specific DNA) for the XSxra chromosome. However, this does not restore fertility because the sperm produced all have abnormal heads. It is concluded that the Y-specific region of the mouse Y chromosome long arm includes information essential for the normal development of the sperm head.

The role of unpaired sex chromosomes in spermatogenic failure.

In 1974 Miklos reviewed evidence suggesting an association between sex chromosome pairing failure and spermatogenic arrest. He proposed that distributed over all the chromosomes there are 'meiotic pairing sites' which must be 'saturated' by homologous pairing during pachytene. In unpaired regions these sites are 'activated', and set in motion a process which leads ultimately to the death of the cell. In the present 'extended abstract' we summarize studies we have carried out on XSxraO male mice, that substantiate the main tenets of Miklos' model. Miklos' model is then used as a basis for explaining data we have collected on a large series of XYY male mice.

Unpaired chromosomes at meiosis: cause or effect of gametogenic insufficiency?

Pairing failure at meiosis has been postulated as a cause of gametogenic arrest in both heterozygous translocation carriers and males whose spermatocytes exhibit univalent X and Y chromosomes. The present investigation is a survey of pachytene translocation configurations, at the electron microscopic level, in six stocks of mice, comprising a total of 464 spermatocytes and 343 oocytes. Univalence of the X and Y chromosomes was studied in the same stocks, as well as in three additional homozygous translocation stocks. Fully paired as well as asynaptic configurations were found in all translocation stocks, and the proportions of each configuration differed considerably between spermatocytes and oocytes of mice carrying the same translocation. In both spermatocytes and oocytes, other pairing anomalies were more frequent in cells with asynapsed than with fully synapsed configurations, and spermatocytes with univalent sex chromosomes had a higher proportion of autosomal anomalies than did spermatocytes with XY bivalents. It is concluded that pairing failure at meiosis is primarily a symptom, rather than a cause, of gametogenic arrest, and that chromosome rearrangements, even if they appear to be balanced, may affect the rate of atresia by interfering with the normal rate of meiotic progression. Once pairing failure is established, it could secondarily increase the probability of gametogenic failure.

Pachytene pairing and oocyte numbers in mice with two single Robertsonian translocations and the male-sterile compound with monobrachial homology.

Oocyte numbers and synaptonemal complexes were studied in two Robertsonian translocations, Rb(6.15)1Ald and Rb(4.6)2Bnr, and their male-sterile compound. Oocyte numbers in the compound were lower than those of either parent, and there was a marked difference between reciprocal crosses. Synaptonemal complexes of homozygous females appeared as 19 bivalents, those of single heterozygotes as 18 bivalents and a trivalent, and those of compound heterozygotes as 17 bivalents and a quadrivalent. Most trivalents were fully paired, whereas the majority of quadrivalents exhibited terminal asynapsis. About one-half of all oocytes had other pairing abnormalities, probably reflecting reduced survivability. Whereas all fully paired quadrivalents were present in cells not showing any pairing anomalies, one-half of the quadrivalents with terminal asynapsis were seen in oocytes with other anomalies. It is suggested that in oocytes destined for atresia, there is a predisposition to synaptic failure of translocation configurations. Additional oocytes are likely to break down because of the deleterious effect of the compound translocation on gametogenesis. This effect seems to be more pronounced in Rb1Ald/Rb2Bnr spermatocytes than in oocytes.

XYY spermatogenesis in XO/XY/XYY mosaic mice.

The relative frequencies of XYY and XY cells in XO/XY/XYY mosaic mice were compared between somatic cells (bone marrow) and spermatogonia, and between spermatogonia and pachytene or MI spermatocytes. The results indicated there was no selection either for or against XYY spermatogonia. There was, however, a strong selection against XYY spermatocytes during pachytene, with their almost total elimination by the first meiotic metaphase. At pachytene, most XYY cells had trivalent or X univalent/YY bivalent configurations. These findings are contrasted with previous studies of XYY spermatogenesis in mice and are discussed with respect to a model that invokes sex-chromosome univalence as the cause of XYY spermatogenic failure.

Pachytene pairing and sperm counts in mice with single Robertsonian translocations and monobrachial compounds.

Data on testis weights, sperm counts, and synaptonemal complexes are presented for mice carrying the following Robertsonian translocations: Rb(6.15)lAld; Rb(4.6)2Bnr; Rb(4.15)4Rma; Rb(6.15)lAld/Rb(4.6))2Bnr, which is male-sterile; and Rb(6.15)lAld/Rb(4.15)4Rma, which is male-fertile. In RblAld/Rb2Bnr sperm were absent or sparse, whereas the sperm count in RblAld/Rb4Rma was just over 50% of the parental value. The translocated chromosomes appeared as fully paired bivalents in homozygotes, as trivalents in single heterozygotes, and as quadrivalents in compounds. About 20-40% of trivalents had unsynapsed ends. The proportion of quadrivalents with unsynapsed ends was about 85% in the male-sterile compound, compared with 75% in the male-fertile compound. The proportion of quadrivalents associated with XY was about 70% in both. Testis weights, but not sperm counts, were found to differ in two of three reciprocal crosses. It is concluded that, in addition to pairing failure and autosome/XY association, the effect of translocations on spermatogenesis is affected by other factors, including genetic background, inbreeding, and perhaps environmental factors. It remains to be elucidated whether pairing failure and XY association are primary or secondary effects.

Univalent sex chromosomes in spermatocytes of Sxr-carrying mice.

Pachytene configurations of the sex chromosomes were studied in whole-mount, silver-stained preparations of spermatocytes in mice with XY,Sxr, XX,Sxr, XO,Sxr, XO,Sxr + 5(12) and T(X;4)37H,YSxr chromosomes, and non-Sxr-carrying controls. XY,Sxr males showed an increased number of X and Y univalents and of self-synapsed Y chromosomes. In T(X;4)37H,YSxr males an increased proportion of trivalent + Y configurations was also accompanied by higher numbers of self-paired Y univalents; the proportion of trivalent + X4 was not increased, but that of self-synapsed X4 univalents was. There was more self-synapsis in cells containing one univalent than in cells containing two univalents. Spermatocytes of XX,Sxr mice contained a single univalent X, which was never seen to be self-synapsed, but self-synapsis of the X occurred in a proportion of cells in XO,Sxr males. There were no self-paired X chromosomes in the XO,Sxr + 5(12) mouse although low-level pairing of the 5(12) chromosome occurred. All four XX,Sxr and XO,Sxr males contained testicular sperm, and testicular sperm were also present in one T(X;4)37H male, while another such male had sperm in the caput. It is concluded that (1) self-synapsis of univalents is affected by variable conditions in the cell as well as by the DNA sequences of the chromosome, and (2) that the level of achievable spermatogenesis is not always rigidly predetermined by a chromosome anomaly but can be modulated by the genetic background.