Synaptonemal complex analysis in spermatocytes of tilapia, Oreochromis niloticus (Pisces, Cichlidae).

Some adaptations of the synaptonemal complex (SC) whole-mounting technique first used in plants permitted its application to meiotic studies in tilapia, Oreochromis niloticus. Direct observation of the chromosome pairing process and bivalent structure during the meiotic prophase of this fish species by light and electron microscopy permitted the analysis of SCs in autosomes and the possible identification of sex chromosomes. The analysis of SCs in spermatocytes of O. niloticus revealed that all 22 bivalent chromosomes completely paired, except for the occurrence of a size heteromorphism in the terminal region of the largest bivalent associated with the presence of an incompletely paired segment during the synapsis process, which may be the cytological visualization of an XX/XY sex chromosome system in this species.     

Les formules gonosomiques dites aberrantes chez les Mammifères Euthériens

The chromosome complement of the sloth Choloepus hoffmanni Peters has been investigated in mitosis and also in male meiosis. The karyotype for both males and females is characterized by a diploid number of 49 chromosomes. In the male the Y-material is translocated on an autosome but the meiotic behavior of the gonosomes is normal and therefore the sex determining mechanism may be normal too, despite the translocation. The females have an XO sex-chromosome constitution in somatic cells. An hypothesis, based on a slight deviation of a normal phenomenon is proposed to explain as regular such a formula in normal animals. — Relating to these conclusions, other known deviations of the standard XX/XY sex chromosome constitution in placental mammals are discussed (multiple sexchromosomes, composite gonosomes and XO female formula). The general conclusion is that despite an apparent variability of sex chromosome morphology, all placental mammals seem to retain a truly XX/XY sex constitution.     

Analysis of sex differences in EGC imprinting

Sex-specific differences are apparent in the methylation patterns of H19 and Igf2 imprinted genes in embryonic germ cells (EGCs) derived from 11.5 or 12.5 days post coitum (dpc) primordial germ cells (PGCs). Here we studied whether these differences are associated either with the sex chromosome constitution of the EGCs or with the sex of the genital ridge (testis versus ovary) from which the PGCs were isolated. For this purpose we derived pluripotent EGC lines from sex-reversed embryos, either XY embryos deleted for Sry (XY(Tdym1)) or XX embryos carrying an Sry transgene. Southern blotting of the EGC DNA was used to analyze the differentially methylated regions of Igf2 and H19. The analysis revealed that both genes were more methylated in EGCs with an XY sex chromosome constitution than in those with an XX sex chromosome constitution, irrespective of the phenotypic sex of the genital ridge from which the EGCs had been derived. We conclude that the sex-specific methylation is intrinsic and cell-autonomous, and is not due to any influence of the genital ridge somatic cells upon the PGCs.     

Development of the synaptonemal complex of two types of pachytene in oocytes and spermatocytes of Carausius morosus Br. (Phasmatodea)

During oogenesis of the parthenogenetic stick insect Carausius morosus (2n =61+XXX) pachytene is followed by a duplication of the desynapsed chromosomes, which results in a second type of pachytene (tetrapachytene) consisting of paired sister chromosomes (autobivalents). Electron microscopic studies on sections revealed that synaptonemal complexes (SCs) are formed during tetrapachytene only. This means that the parthenogenetically produced progeny have the genetic constitution of the mother. During spermatogenesis of rare fatherless males (2n=61 + XX) and intersexes (2n=61 +XXX) either an incomplete chromosome doubling (demonstrated by up to 10% additional DNA synthesis) or a complete chromosome doubling takes place during zygotene. EM studies on sections and spreads of germ cells of the first type of meiosis showed that unpaired lateral components (LCs), pieces of SCs and complete SCs are formed during pachytene only, the sex chromosomes being represented by unpaired thickened LCs. The incomplete SC formation reflects the complex heterozygosity of the chromosome complement. In the duplicated type SCs are found in tetrapachytene nuclei only; they are wider than the SCs in oocytes. The sex chromosome bivalents are represented by unpaired thickened LCs or partially paired LCs, in which localized chiasma formation was found. The idea is discussed that formation of SCs does not take place as long as a germ cell has been programmed either to replicate or to be able to replicate its chromosomes and that consequently SCs can be formed only once per meiosis.     

Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan

We performed chromosomal analysis on 540 mouse embryonic stem (ES) cell lines obtained during 2001 to 2004 from 20 institutions in Japan. Overall, 66.5% of the ES cell lines showed normal chromosomal numbers, but 15.9%, 9.1%, and 2.8% showed modal chromosomal numbers of 41, 42, and 39, respectively. When we karyotyped 88 ES cell lines selected arbitrarily from the 540 lines, 53 (60.2%) showed normal diploid karyotypes; the sex chromosome constitution of 52 lines was XY, with the remaining 1 being XX. Among 35 ES cell lines showing abnormal karyotypes, trisomy of chromosome 8 (41, XY, +8) was dominant (51.4%), 14.3% had trisomy 8 with loss of one sex chromosome (40, XO, +8), and 11.4% had trisomy 8 together with trisomy 11 (42, XY, +8, +11). Karyotypic abnormalities including trisomy 8 and trisomy 11 occurred in 88.6% and 17.1% of ES cell lines, respectively. The XO sex chromosome constitution was observed in 25.7% of all abnormal ES cell lines. Of the 88 selected ES cell lines, 60 lines were established from strain 129 animals, 17 from F1 progeny of C57BL/6J x CBA (called TT2 in this study), and 11 from C57BL/6J mice. Normal diploid karyotypes were observed in 58.3% of lines derived from 129, 58.8% of those from TT2, and 72.7% of C57BL/6J. The relatively high incidence of abnormalities in chromosomal number and karyotype in ES cell lines used in Japan suggests the importance of chromosomal analysis of ES cells for successful establishment of new animal models through germline transmission.     

Synaptonemal complexes of the A- and B-chromosomes of the spermatocytes from the East Asian mouse Apodemus peninsulae

The analysis of whole-mount preparations of synaptonemal complexes (SCs) from surface-spread spermatocytes of A. peninsulae (2n = 48A + 1, 2, ... 12 B) had revealed SCs of 23 autosomal bivalents, sex bivalent XY, axial cores and SCs of the B-chromosomes. The intercellular and interindividual variability of the number of B-chromosomes varied from 1 to 12 per cell. The SCs of autosomal bivalents were shown to have a typical structure. The structure and behaviour of SCs of sex bivalent throughout meiotic prophase I appeared to be similar to those observed in other species of this order. Mainly B-univalents and less frequently B-bivalents containing SCs were found to be formed in meiotic prophase I. The full homologues appear to be rarely seen among B-chromosomes of the East-Asiatic mouse. A tendency of forming clusters of B-univalents near the sex bivalent was found, in addition to B-bivalents with lateral elements, having the form of bi- and tri-stranded elements with rare synaptic fragments. Besides this, the SCs of the autosomes of pachytene cells were found to contain structures resembling the recombination nodules.     

Meiotic studies of a human male carrier of the common translocation, t(11;22), suggests postzygotic selection rather than preferential 3:1 MI segregation as the cause of liveborn offspring with an unbalanced translocation

The t(11;22)(q23;q11) translocation is the only non-Robertsonian rearrangement for which there are a large number of unrelated families, apparently with the same breakpoints. These families most often have been ascertained through an abnormal child with the karyotype 47,XX or XY, +der(22) t(11;22)(q23;q11). To explain the high incidence of 3:1 segregants, rarely seen in offspring of carriers of other reciprocal translocations, a number of theoretical models have been suggested. We have used both electron microscope analysis of the synaptonemal complex (SC) and dual-color FISH to investigate the meiotic chromosome behavior in a male carrier of the translocation who has the karyotype 46,XY, t(11;22)(q23;q11). Chromosome synapsis, first-meiotic chiasma configuration, and segregation behavior of this translocation have been analyzed directly. Examination of SCs by electron microscopy showed pachytene-cross formation in 49/50 nuclei. Approximately 50% (26/50) revealed a classical fully synapsed quadrivalent. A proportion of these (10/26), however, showed some central asymmetry, suggesting heterologous synapsis. The remaining cells appeared to have incomplete synapsis. FISH analysis showed only quadrivalents in all 100 metaphase I nuclei. The chiasma frequency was increased within the interstitial segments, in comparison with the same region in normal bivalents. All types of segregation category were found in metaphase II nuclei. There was no indication of preferential 3:1 anaphase I segregation. We conclude that the +der(22) constitution in offspring of carriers of t(11;22)(q23;q11) is not likely to be due to meiotic 3:1 segregation being especially common. Rather, the +der(22) constitution is more likely to be the result of postzygotic selection against other unbalanced karyotypes.     

Cytogenetic analysis of somatic and germinal cells from 38,XX/38,XY phenotypically normal boars

Many chromosomal abnormalities have been reported to date in pigs. Most of them have been balanced structural rearrangements, especially reciprocal translocations. A few cases of XY/XX chimerism have also been diagnosed within the national systematic chromosomal control program of young purebred boars carried out in France. Until now, this kind of chromosomal abnormality has been mainly reported in intersex individuals. We investigated 38,XY/38,XX boars presenting apparently normal phenotypes to evaluate the potential effects of this particular chromosomal constitution on their reproductive performance. To do this, we analyzed (1) the chromosomal constitution of cells from different organs in one boar; (2) the aneuploidy rates for chromosomes X, Y, and 13 in sperm nuclei sampled from seven XY/XX boars. 2n = 38,XX cells were identified in different nonhematopoietic tissues including testis (frequency, <8%). Similar aneuploidy rates were observed in the sperm nuclei of XY/XX and normal individuals (controls). Altogether, these results suggest that the presence of XX cells had no or only a very limited effect on the reproduction abilities of the analyzed boars.     

Sex chromosome configurations in pachytene spermatocytes of an XYY mouse

Karyotypic investigation of a phenotypically normal but sterile male mouse showed the presence of an XYY sex chromosome constitution. The synaptic behaviour of the three sex chromosomes was examined in 65 pachytene cells. The sex chromosomes formed a variety of synaptic configurations: an XYY trivalent (40%); an XY bivalent and Y univalent (38.5%); an X univalent and YY bivalent (13.8%); or X, Y, Y univalence (7.7%). There was considerable variation in the extent of synapsis and some of the associations clearly involved nonhomologous pairing. These observations have been compared with previously published information on chromosome configurations at metaphase I from other XYY males.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Normal Female Germ Cell Differentiation Requires the Female X Chromosome to Autosome Ratio and Expression of Sex-Lethal in DROSOPHILA MELANOGASTER

In somatic cells of Drosophila, the ratio of X chromosomes to autosomes (X:A ratio) determines sex and dosage compensation. The present paper addresses the question of whether germ cells also use the X:A ratio for sex determination and dosage compensation. Triploid female embryos were generated which, through the loss of an unstable ring-X chromosome, contained some germ cells of 2X;3A constitution in their ovaries. Such germ cells were shown to differentiate along one of two alternative pathways: a minority developed into normal female oocytes and eggs; the majority developed into abnormal multicellular cysts. An X:A ratio of 1 is, therefore, required in female germ cell development, at least in the mature ovary after stem cell division. Abnormal development of female germ cells was also observed when 2X;2A germ cells which were homozygous or trans-heterozygous for mutant alleles at the Sex-lethal locus were transplanted into normal female host embryos at the blastoderm stage. Germ cells homozygous for amorphic alleles failed to give rise to normal eggs. Instead, they formed multicellular cysts, very similar to those formed by 2X;3A cells. Zygotic Sxl+ activity is, therefore, also necessary for the development of normal female germ cells. No abnormalities were detected in transplanted germ cells from female embryos whose mothers had been homozygous for the mutation daughterless. When normal XY germ cells were transplanted into female embryos, no traces of such cells could be found in the adult ovary. XY germ cells seem, therefore, not to develop as far as 2X;3A or Sxl homozygous cells in a female gonad. This indicates that neither 2X;3A nor Sxl homozygous germ cells are equivalent to normal XY germ cells.     

标记减数分裂遗传重组的免疫荧光染色方法

联会复合体免疫荧光技术在全基因减数分裂遗传重组研究中具有精确和直观的优势.本研究通过免疫荧光染色方法制备小鼠精母细胞联会复合体,研究其形态组成与遗传重组特征,展示雄性小鼠遗传重组图谱并分析其重组位点(MLH1位点)的分布特征.4只小鼠共145个精母细胞在平均每个细胞的MLH1位点数为23.3±2.4;在常染色体联会复合体中,未发现有3个MLH1位点的联会复合体,具有1个MLH1位点的联会复合体较多,平均为14.2;无XY联会复合体的细胞占所有细胞的4.1%,XY联会复合体上有MLH1位点的细胞占30.2%;联会复合体上有裂缝的细胞占0.7%.通过联会复合体免疫荧光染色可以清晰地分辨出联会复合体(红色)、着丝粒(蓝色)和MLH1位点(绿色),是遗传重组分析的一种强有力工具.     

Somatic and germ-cell sex in mammals

The phenotypic sex of an individual mammal is determined by the sex of its gonads, i.e. testes or ovaries. This in turn is determined by the presence or absence of a small region of the Y chromosome, located near the X-Y pairing region in man and on the short arm of the Y chromosome in the mouse. The testis-determining region of the Y appears to exert its primary effect by directing the supporting-cell lineage of the gonad to differentiate as Sertoli cells, acting at least in part cell-autonomously. The phenotypic sex of a germ cell, i.e. whether it undergoes spermatogenesis or oogenesis, is determined at least in the mouse by whether or not it enters meiotic prophase before birth. This depends not on its own sex chromosome constitution, but on its cellular environment. A germ cell in or near normal testis cords (made up mainly of Sertoli cells) is inhibited from entering meiosis until after birth; one that escapes this inhibition will develop into an oocyte even if it is in a male animal and is itself XY in chromosome constitution.     

Recombination in the pseudoautosomal region in a 47,XYY male

Males with a 47,XYY karyotype generally have chromosomally normal children, despite the high theoretical risk of aneuploidy. Studies of sperm karyotypes or FISH analysis of sperm have demonstrated that the majority of sperm are chromosomally normal in 47,XYY men. There have been a number of meiotic studies of XYY males attempting to determine whether the additional Y chromosome is eliminated during spermatogenesis, with conflicting results regarding the pairing of the sex chromosomes and the presence of an additional Y. We analyzed recombination in the pseudoautosomal region of the XY bivalent to determine whether this is perturbed in a 47,XYY male. A recombination frequency similar to normal 46,XY men would indicate normal pairing within the XY bivalent, whereas a significantly altered frequency would suggest other types of pairing such as a YY bivalent or an XYY trivalent. Two DNA markers, STS/STS pseudogene and DXYS15, were typed in sperm from a heterozygous 47,XYY male. Individual sperm (23,X or Y) were isolated into PCR tubes using a FACStarPlus flow cytometer. Hemi-nested PCR analysis of the two DNA markers was performed to determine the frequency of recombination. A total of 108 sperm was typed with a 38% recombination frequency between the two DNA markers. This is very similar to the frequency of 38.3% that we have observed in 329 sperm from a normal 46,XY male. Thus our results suggest that XY pairing and recombination occur normally in this 47,XYY male. This could occur by the production of an XY bivalent and Y univalent (which is then lost in most cells) or by loss of the additional Y chromosome in some primitive germ cells or spermatogonia and a proliferative advantage of the normal XY cells.     

The origin of an unusual sex chromosome constitution in <Emphasis Type="Italic">Acomys</Emphasis> sp. (Rodentia,Muridae) from Tanzania

This paper describes a case which presents an evident variation from the “standard” XX/XY sex chromosomal constitution in a rodent, Acomys sp. This species known to be found in three localities of East Africa has only recently been separated from A. spinosissimus, its closest relative. In our study, five specimens of Acomys sp. and eight specimens of A. spinosissimus were live-trapped in five localities. Comparisons between the two taxa assed by G- banding show a complete homology in the chromosomal shape and banding pattern for 29 pairs of chromosomes corresponding to the complete autosomal set of A. spinosissimus. However, while all the A. spinosissimus analysed have 2n = 60 and a XY-XX system, in Acomys sp. males and females constitute mosaics for sex chromosomes in the bone marrow cells. Females (2n = 59, 60) have an excess (97%) of aneuploid cells with one single giant X chromosome, and males (2n = 60, 61) show X0/XY cells occurring in somatic tissues and XY cells in the germinal lineage. In addition, an odd heterochromatic submetacentric chromosome was identified in all the cells examined in two males and a female of Acomys sp. Since this chromosome was not related to sex determination and it is not present in all the analysed specimens, it can be considered as a B chromosome. Finally, the in situ fluorescence hybridisation (FISH) with telomeric probes showed a very intense interstitial telomeric signal (ITS) at the medial part on the long heterochromatic arm of the X chromosome. This could be due to recent chromosomal rearrangement.     

Klinefelter's syndrome, mosaic 46,XX/46,XY/47,XXY/48,XXXY/48,XXYY: a case report

A 35-year-old male was investigated for primary infertility. Clinical examination showed an intelligent man with normal facial appearance and moustache and small firm testes. Testicular histopathology revealed marked atrophy of the testes with no spermatogenesis and absence of germ cells. Hormonal profile showed elevated levels of FSH,LH and low levels of testosterone. Chromosome analysis from whole blood culture showed cells with 46,XX/46,XY/47,XXY/48,XXXY/48,XXYY mosaicism. The predominant cell line was 47,XXY (87.86%). 46,XY/47,XXY mosaicism is not uncommon. However, mosaicism of multiple sex chromosome aneuploidy is rarely observed. This is the first report of mosaicism in Klinefelter's syndrome variant with five cell lines.     

Sex determination and sex reversal: genotype,phenotype, dogma and semantics

Summary The genetic terminology of sex determination and sex differentiation is examined in relation to its underlying biological basis. On the assumption that the function of the testis is to produce hormones and spermatozoa, the hypothesis of a single Y-chromosomal testis-determining gene with a dominant effect is shown to run counter to the following observed facts: a lowering in testosterone levels and an increase in the incidence of undescended testes, in addition to sterility, in males with multiple X chromosomes; abnormalities of the testes in autosomal trisomies; phenotypic abnormalities of XX males apparently increasing with decreasing amounts of Y-chromosomal material; the occurrence of patients with gonadal dysgenesis and XY males with ambiguous genitalia in the same sibship; the occurrence of identical SRY mutations in patients with gonadal dysgenesis and fertile males in the same pedigree; and the development of XY female and hermaphrodite mice having the same genetic constitution. The role of X inactivation in the production of males, females and hermaphrodites in T(X;16)16H mice has previously been suggested but not unequivocally demonstrated; moreover, X inactivation cannot account for the observed bilateral asymmetry of gonadal differentiation in XY hermaphrodites in humans and mice. There is evidence for a delay in development of the supporting cells in XY mice with ovarian formation. Once testicular differentiation and male hormone secretion have begun, other Y-chromosomal genes are required to maintain spermatogenesis and to complete spermiogenesis, but these genes do not function effectively in the presence of more than one X chromosome. The impairment of spermatogenesis by many other chromosome abnormalities seems to be more severe than that of oogenesis. It is concluded that the notion of a single testis-determining gene being responsible for male sex differentiation lacks biological validity, and that the genotype of a functional, i.e. fertile, male differs from that of a functional female by the presence of multiple Y-chromosomal genes in association with but a single X chromosome. Male sex differentiation in XY individuals can be further impaired by a euploid, but inappropriate, genetic background. The genes involved in testis development may function as growth regulators in the tissues in which they are active.     

Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat

Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase. They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length. Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods. It is not known at which step in meiotic recombination this association with the AEs is established. In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components. The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar. MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected. From preleptotene until early zygotene they were present throughout the nucleus. In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene. We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place. Received: 16 November 1999; in revised form: 29 December 1999 / Accepted: 3 January 2000     

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.     

M31, a murine homolog of Drosophila HP1, is concentrated in the XY body during spermatogenesis.

The formation of the sex vesicle, or XY body, during male meiosis and pairing of the sex chromosomes are thought to be essential for successful spermatogenesis. Despite its cytological discovery a century ago, the mechanism of XY body formation, particularly heterochromatinization of the sex chromosomes, has remained unclear. The HP1 class of chromobox genes are thought to encode proteins involved in the packaging of chromosomal DNA into repressive heterochromatin domains, as seen, for example, in position-effect variegation. Study of the distribution of a murine HP1-like chromodomain protein, M31, during spermatogenesis revealed spreading from the tip of the XY body in mid-stage pachytene spermatocytes to include the whole of the XY body in late-pachytene spermatocytes. We also demonstrate that the formation of the XY body during spermatogenic progression in neonatal mice coincides with the expression of a novel nuclear isoform of M31, M31(p21). These results support the view that a common mechanistic basis exists for heterochromatin-induced repression, homeotic gene silencing, and sex-chromosome inactivation during mammalian spermatogenesis.