Specific recognition and differential affinity of meiotic X-Y pairing sites in Lucilia cuprina males (Diptera: Calliphoridae)

Meiotic pairing of X and Y chromosomes in male Lucilia cuprina was studied by cytological observation of normal, rearranged and deficient sex chromosome karyotypes in spermatogenesis. Two X-Y pairing regions located distally in each arm of the X and Y chromosomes were defined. Contrasting with findings in Drosophila melanogaster, these pairing regions show specific recognition of their partners. By studying rearranged sex chromosomes short arm pairing was localised to their distal ends, closely associated with secondary constrictions containing nucleolar organisers in both sex chromosomes. Short arm pairing is very tight and not greatly disrupted by chromosome rearrangement, deficiency for the Y chromosome long arm or the presence of supernumerary X chromosomes. The pairing region of the long arms could not be precisely localised but probably also occurs at their distal ends. Pairing between the long arm sites is much weaker and is easily disrupted by chromosome rearrangement, failing completely in flies deficient for the Y chromosome short arm. No cytologically visible pairing was seen between X chromosomes and the remainder of the Y. In males with an extra X chromosome, the ends of both X chromosomes pair to form multivalents with normal and rearranged Y chromosomes provided the Y short arm is present, otherwise an independent X chromosome bivalent is formed. The mechanism of pairing in male Lucilia sex chromosomes thus seems to depend on specific loci of distinctive structure within the X and Y heterochromatin. Comparison of cytological and genetic data shows that increasing cytological pairing failure is matched by higher genetic X-Y nondisjunction but that the former occurs at much higher levels. In some karyotypes cytologically observed X-Y pairing failure is not matched by high frequencies of nondisjunction presumably because weak pairing associations are disrupted during slide preparation.     

联会复合体——原发无精症发病中的重要角色

联会复合体(synaptonemal complex,SC)是一种减数分裂特异性超分子蛋白质结构,与减数分裂I(改罗文)中同源染色体的凝缩、配对、重组和分离密切相关。近年来,联会复合体的研究取得了一系列重要的进展,包括在其组成成分和功能上的一些新发现。在小鼠不育模型中联会复合体及其编码基因的异常可引起精子发生障碍。更重要的是,联会复合体编码基因之一SCP3单个碱基缺失导致的无精症已在人类原发不育患者中得到证实。对联会复合体基因SCP1的进一步研究也正在进行之中。      

男性育性障碍与联会复合体异常的关系

据有关资料统计,男人中大约有11%育性有障碍,其中由遗传因素引起的男性不育占居首位,包括染色体异常、微小缺失和基因突变等3类。研究表明,男性染色体畸变与精子发生失败或受孕浪费现象密切相关。联会复合体（synaptonemal complex SC）分析为揭示二者之间的关系提供了证据。本文结合近年来SC在男性不育症诊断中的应用和我们在这方面的研究结果,对男性育性障碍与SC异常的关系进行了以下5个方面的评述和讨论。1.XY-二价体与重排染色体联合,干扰或影响X染色体的正常功能,从而干扰精子发生。2. 重排染色体在断裂点处广泛的不配对,引起精子发生失败。3. SC粉碎化、侧生组分膨化、配对紊乱导致精子发生失败。 4. 重排染色体直接的异源配对导致不平衡配子的产生而出现受孕浪费。5.SC蛋白基因的突变引起SC超微结构的变化导致男性不育。     

Mammalian spermatogenesis investigated by genetic engineering.

Genes involved in mammal spermatogenesis can now be identified through mutants created by genetic engineering. Information has been obtained on male meiosis, but also on the factors regulating the proliferation, maintenance and differentiation of male germ cells. Its has also increased our knowledge of the germ cell phenotype emerging from an altered germ cell genotype. This review is focused on data from genes expressed in male germ cells and on the question of how germ cells and Sertoli cells cope with the molecular lesions induced. The conservation of a wild-type phenotype of male germ cells in mutant mice is discussed, and how the mouse genetic background can lead to different germ cell phenotypes for a given gene mutation.     

Ring chromosome 21 in healthy persons: different consequencies in females and in males

Summary A stable ring chromosome 21 was found in an azoospermic man with an otherwise normal phenotype. Meiotic studies in another known azoospermic male with r(21) had indicated that breakdown of spermatogenesis resulted from pairing failure of chromosome 21, followed by degenerative changes in the chromosomes, before the cells had completed the first meiotic division. While primary sterility was a constant feature in the three adult males, eight healthy females with r(21) were fertile. However, they were at risk for Down syndrome and spontaneous abortions.     

Homologous pairing and chromosome dynamics in meiosis and mitosis

Pairing of homologous chromosomes is an essential feature of meiosis, acting to promote high levels of recombination and to ensure segregation of homologs. However, homologous pairing also occurs in somatic cells, most regularly in Dipterans such as Drosophila, but also to a lesser extent in other organisms, and it is not known how mitotic and meiotic pairing relate to each other. In this article, I summarize results of recent molecular studies of pairing in both mitosis and meiosis, focusing especially on studies using fluorescent in situ hybridization (FISH) and GFP-tagging of single loci, which have allowed investigators to assay the pairing status of chromosomes directly. These approaches have permitted the demonstration that pairing occurs throughout the cell cycle in mitotic cells in Drosophila, and that the transition from mitotic to meiotic pairing in spermatogenesis is accompanied by a dramatic increase in pairing frequency. Similar approaches in mammals, plants and fungi have established that with few exceptions, chromosomes enter meiosis unpaired and that chromosome movements involving the telomeric, and sometimes centromeric, regions often precede the onset of meiotic pairing. The possible roles of proteins involved in homologous recombination, synapsis and sister chromatid cohesion in homolog pairing are discussed with an emphasis on those for which mutant phenotypes have permitted an assessment of effects on homolog pairing. Finally, I consider the question of the distribution and identity of chromosomal pairing sites, using recent data to evaluate possible relationships between pairing sites and other chromosomal sites, such as centromeres, telomeres, promoters and heterochromatin. I cite evidence that may point to a relationship between matrix attachment sites and homologous pairing sites.     

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 spermatogenetic process in Barbus longiceps, Capoeta damascina and their natural sterile hybrid (Teleostei, Cyprinidae)

Testicular ultrastructure was studied in Barbus longiceps, Capoeta damascina and their natural hybrid. The testes of these teleosts belong to the unrestricted or lobular type. Germ cell morphology is similar in the parental males. In the hybrid, spermatogenesis does not extend beyond the pachytene of the first meiotic division, probably due to the unsuccessful pairing of the homologous chromosomes. Hybrid testes are occupied mainly by degenerating primary spermatocytes, at the leptotene and pachytene stages. In both parents and the hybrid, Sertoli and Leydig cells are characterized by the presence of granular endoplasmic reticulum and of mitochondria with tubular cristae. Due to the arrest of spermatogenesis, the male germ cell protective barrier is absent in the hybrid. Germ cell nuclear size was measured by a computerized analysis system, using light-microscopy images. In the parents and the hybrid, germ cells attain a uniform inter-individual nuclear size when they reach the first meiotic prophase. The nuclear size of primary spermatocytes is similar among the three groups of fish, possibly reflecting their close genetic relationship.     

Yq deletion and failure of spermatogenesis

The spermatogenesis of a sterile male carrying a Y long arm deletion was analyzed by meiotic techniques and by light and electron microscopy on testicular biopsies. R-, Q- and C- banding techniques have shown that the Y long arm deletion included the entire heterochromatin and a part of the euchromatic region, with breakpoint between q11.21 and q11.23. The seminiferous tubules showed a sharp decrease in spermatogonia, degenerative phenomena in their nuclei and a spermatogenic block. Abnormal meiotic aspects were observed: pairing failure with atypical diakinesis configurations. These findings confirm the presence of spermatogenesis controlling factors on the distal euchromatic region of the Y long arm.     

Evidence for Paternal Age-Related Alterations in Meiotic Chromosome Dynamics in the Mouse

Increasing age in a woman is a well-documented risk factor for meiotic errors, but the effect of paternal age is less clear. Although it is generally agreed that spermatogenesis declines with age, the mechanisms that account for this remain unclear. Because meiosis involves a complex and tightly regulated series of processes that include DNA replication, DNA repair, and cell cycle regulation, we postulated that the effects of age might be evident as an increase in the frequency of meiotic errors. Accordingly, we analyzed spermatogenesis in male mice of different ages, examining meiotic chromosome dynamics in spermatocytes at prophase, at metaphase I, and at metaphase II. Our analyses demonstrate that recombination levels are reduced in the first wave of spermatogenesis in juvenile mice but increase in older males. We also observed age-dependent increases in XY chromosome pairing failure at pachytene and in the frequency of prematurely separated autosomal homologs at metaphase I. However, we found no evidence of an age-related increase in aneuploidy at metaphase II, indicating that cells harboring meiotic errors are eliminated by cycle checkpoint mechanisms, regardless of paternal age. Taken together, our data suggest that advancing paternal age affects pairing, synapsis, and recombination between homologous chromosomes—and likely results in reduced sperm counts due to germ cell loss—but is not an important contributor to aneuploidy.     

SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice

Prior to the pairing and recombination between homologous chromosomes during meiosis, telomeres attach to the nuclear envelope and form a transient cluster. However, the protein factors mediating meiotic telomere attachment to the nuclear envelope and the requirement of this attachment for homolog pairing and synapsis have not been determined in animals. Here we show that the inner nuclear membrane protein SUN1 specifically associates with telomeres between the leptotene and diplotene stages during meiotic prophase I. Disruption of Sun1 in mice prevents telomere attachment to the nuclear envelope, efficient homolog pairing, and synapsis formation in meiosis. Massive apoptotic events are induced in the mutant gonads, leading to the abolishment of both spermatogenesis and oogenesis. This study provides genetic evidence that SUN1-telomere interaction is essential for telomere dynamic movement and is required for efficient homologous chromosome pairing/synapsis during mammalian gametogenesis.     

哺乳动物精原干细胞的研究进展

精原干细胞是雄性体内可以永久维持的成体干细胞,它具有自我更新和分化的能力,保证了雄性个体生命过程中精子发生的持续进行,从而实现将遗传信息传递给下一代。精原千细胞不仅可在体外实现长期培养或诱导分化为各级生精细胞,并且可在特定条件下将其诱导去分化成为多能性干细胞。同样,这种多能性干细胞如同胚胎干细胞,可被诱导形成造血细胞、神经元细胞、肌细胞等多种类型细胞。鉴于其独具的生物学特性,精原干细胞在揭示精子的发生机制、治疗雄性不育和转基因动物等研究中具有重要价值。该文对精原干细胞在生物学特性、纯化培养、移植、体外诱导分化及其相关调控方面的各项研究进行了小结,综述了近年来的研究历程和最新研究成果。     

精子DNA甲基化异常与男性不育

DNA甲基化／去甲基化是表观遗传学最重要的内容并可以控制基因的表达和印迹,越来越多的研究显示DNA甲基化异常与不育男性精子发生异常、特定肿瘤的发生、神经系统疾病、Rett综合征等有关。文章通过总结近来的相关研究资料来阐述精子发生过程中的DNA甲基化状态的改变,探讨精子DNA的甲基化异常与男性不育之间的联系,旨在为男性不育的治疗提供新的临床思路。     

Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

L’infertilité masculine: Nos connaissances ne doivent pas faire oublier notre ignorance

About 15% of couples worldwide are affected by reduced fertility. In 20% of cases of couple infertility, the problem can be predominantly attributed to the male. In 20% of cases, a genetic cause of male infertility can usually be identified. The main genetic causes are: autosomal and sex chromosomal abnormalities, microdeletions within regions of the Y-chromosome containing candidate gene families for spermatogenesis and mutations in theCFTR gene. However, despite enormous progress in the understanding of human reproductive physiology, the underlying cause of male infertility often cannot be elucidated. Candidate gene strategies, linkage analysis in large familial forms of male infertility, targeted mutagenesis in the mouse and studies of chromatin reorganization during spermatid maturation should provide rapid progress in our understanding of the genetic factors that contribute to male infertility, which may open up new approaches to the treatment of this condition.     

Prospects for spermatogenesis in vitro

In recent years, extraordinary progress has been made in a broad range of reproductive technologies, including spermatogonial transplantation in the male. However, effective procedures for the complete recapitulation of spermatogenesis in vitro, including meiosis, have remained elusive. Such procedures have the potential to facilitate (1) mechanistic studies of spermatogenesis, (2) directed genetic modification of the male germ line, and (3) treatment of male factor infertility. Early studies demonstrated the importance of germ cell-Sertoli association for germ cell survival in vitro. Recently, evidence for male germ cell survival and progression through meiosis has been reported for the rat, mouse, and man. We demonstrated the expression of spermatid-specific genes (protamine and transition protein 1) by alginate-encapsulate neonatal bull testis cells after 10 weeks in culture, suggesting that meiosis had occurred. Although identifiable germ cells in these cultures were very sparse, some indication of acrosome development was observed. Following round spermatid injection (ROSI) with presumptive spermatids produced in vitro, 50% of blastocysts produced were diploid and 37% were Y-chromosome positive. Improved culture conditions, which promote germ cell survival, differentiation, and proliferation, are essential for in vitro spermatogenesis (IVS) to become a useful technology. Other approaches to male germ cell manipulation and spermatid production are discussed.     

Trichlormethine hydrochloride and correlation of its mutagenic and toxic effects on male germ cells in mice.

The genetic effect of the cytostatic trichlormethine hydrochloride (TS-160 Spofa) was assessed after a 1-week administration using the dominant lethal mutation test (DLM) and the sperm abnormality test. The dosage was 0.5 mg/kg for 7 consecutive days, an equivalent of the human therapeutic dosage. Simultaneously, the cytostatic's direct toxic effect on male sex organs was assessed. TS-160 carries a genetic risk for the postmeiotic stages of spermatogenesis (DLM) and is responsible for interference in the morphology of sperm heads through its action on spermatocytes. The toxic effects of TS-160 were found to influence the body weight of mice (days 4-25 after administration), to reduce the relative weight of the testes (days 18-25 after administration), to damage spermatogenesis in the seminiferous tubules (spermatids), to be responsible for an appearance of multinucleate cells in the epididymides, and for an increased rate of abnormality of the heads of fully mature spermatozoa. Our findings stress the need to separate the cytotoxic effects from genetic effects so as to avoid false positives, especially in the test for head abnormalities, and also in the assessment of the fertility of male animals or fertilization of females mated with treated males.     

Recombination Suppression by Heterozygous Robertsonian Chromosomes in the Mouse

Robertsonian chromosomes are metacentric chromosomes formed by the joining of two telocentric chromosomes at their centromere ends. Many Robertsonian chromosomes of the mouse suppress genetic recombination near the centromere when heterozygous. We have analyzed genetic recombination and meiotic pairing in mice heterozygous for Robertsonian chromosomes and genetic markers to determine (1) the reason for this recombination suppression and (2) whether there are any consistent rules to predict which Robertsonian chromosomes will suppress recombination. Meiotic pairing was analyzed using synaptonemal complex preparations. Our data provide evidence that the underlying mechanism of recombination suppression is mechanical interference in meiotic pairing between Robertsonian chromosomes and their telocentric partners. The fact that recombination suppression is specific to individual Robertsonian chromosomes suggests that the pairing delay is caused by minor structural differences between the Robertsonian chromosomes and their telocentric homologs and that these differences arise during Robertsonian formation. Further understanding of this pairing delay is important for mouse mapping studies. In 10 mouse chromosomes (3, 4, 5, 6, 8, 9, 10, 11, 15 and 19) the distances from the centromeres to first markers may still be underestimated because they have been determined using only Robertsonian chromosomes. Our control linkage studies using C-band (heterochromatin) markers for the centromeric region provide improved estimates for the centromere-to-first-locus distance in mouse chromosomes 1, 2 and 16.