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Xlr and Xmr are sex-specific genes which are expressed during the meiotic prophase I in the mouse. In spermatocytes, XMR concentrates on the asynapsed regions of the XY chromosomes, suggesting that XMR plays a role in sex chromosome condensation and silencing. The present study shows that in the mouse, XMR also concentrates in the nucleolus which is closely associated with the XY chromosome pair. In this species, the formation of a large fibrillo-granular nucleolus signals the activation of the ribosomal genes, but release of pre-ribosomal particles is inhibited. Using laser confocal microscopy we characterized the distribution of XMR in the XY body relative to the XY chromatin and the nucleolus. Immunoelectron microscopy showed that XMR concentrates in the fibrillo-granular component and the granular component (GC) of the nucleolus. In (T[X;16]16H) mouse spermatocytes, the nucleolus displays little or no activity and does not associate with the XY pair. XMR concentrated only on the XY chromosomes in (T[X;16]16H) mouse spermatocytes. These data suggest that XMR could play a role both in the XY pair and the nucleolus associated to the sex chromosomes.  相似文献   

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The murine Xlr (X-linked, lymphocyte-regulated) gene family was originally identified by subtractive cDNA hybridization and cloning. It was found to encode two 30-kDa nuclear proteins expressed in lymphoid cells and in primary spermatocytes in a developmentally regulated manner. Our data show that, in contrast to most X-linked genes, the Xlr family is not conserved at the DNA level between mouse and human. However, using anti-Xlr antibodies, an Xlr-immunoreactive nuclear protein of Mr 30,000 was characterized in human RAJI B-lymphoblastoid cells by flow cytofluorimetry, by immunoblotting, and by immuno-cytolabeling. An Xlr-like molecule was also found to be expressed in human activated lymphocytes and in human primary spermatocytes, with a stage specificity similar to that known in the mouse. In contrast, no Xlr-immunoreactive protein was detected in a series of human tissues including brain, skeletal muscle, colon, liver, and kidney, revealing a tissue-specific expression pattern similar to that of murine Xlr. These findings most likely identify a human equivalent of Xlr. The Xlr genes belong to a small category of X-linked genes, including STS, MIC2, CSF2RA, and KAL, that diverge at the DNA level in human and in mice. Characterization of the human XLR gene(s) should now be feasible with anti-Xlr antibodies and an expression cloning system. It should provide new insights into the evolution of mammalian X Chromosome (Chr).  相似文献   

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Gametogenesis is a complex biological process of producing cells for sexual reproduction. Xlr super family members containing a conserved COR1 domain play essential roles in gametogenesis. In the present study, we identified that Slx2, a novel member of Xlr super family, is specifically expressed in the meiotic oocytes, which is demonstrated by western blotting and immunohistochemistry studies. In the first meiotic prophase, SLX2 is unevenly distributed in the nuclei of oocytes, during which phase SLX2 is partly co-localized with SYCP3 in synaptonemal complex and γH2AX in the nucleus of oocytes. Interestingly, the localization of SLX2 was found to be switched into the cytoplasm of oocytes after prometaphase I during oocyte maturation. Furthermore, yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLX2 interacts with BLOS2, which is a novel centrosome-associated protein, and co-localized with γ-Tubulin, which is a protein marker of chromosome segregation in meiosis. These results indicated that SLX2 might get involved in chromosomes segregation during meiosis by interaction with BLOS2. In conclusion, SLX2 might be a novel gametogenesis-related protein that could play multiple roles in regulation of meiotic processes including synaptonemal complex assembly and chromosome segregation.  相似文献   

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Background

Spermatogenesis is the complex process by which diploid stem cells generate haploid germ cells in gamete production. Members of the Xlr (X-chromosome linked, lymphocyte regulated) superfamily play essential roles in spermatogenesis. The expression, localization and role in spermatogenesis of one such member, Xlr5c, has not been reported previously.

Methodology/Principal Findings

Xlr5c mRNA and protein levels in murine testes and other tissues were investigated using RT-PCR and Western blotting. Xlr5c was abundantly transcribed in mouse testes, particularly during the early stages of spermatogenesis and throughout prophase I in the nuclei of spermatocytes. Xlr5c was specifically localized at synaptonemal complexes(SCs) region in preleptotene and pachytene spermatocytes, as was the homologous Xlr protein Sycp3.

Conclusions/Significance

These results suggest that Xlr5c was abundantly transcribed in germ cells, localized at SCs region, where it may play a potential role during the early stages of spermatogenesis. Identification and characterization of this novel testis protein may offer a new perspective for understanding of the molecular mechanisms involved in germ cell differentiation.  相似文献   

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Anion channels activated by increased cell volume are a nearly ubiquitous mechanism of cell volume regulation, including in early preimplantation mouse embryos. Here, we show that the swelling-activated anion current (I(Cl,swell)) in early mouse embryos is cell-cycle dependent, and also that this dependence is developmentally regulated. I(Cl,swell) is present both in first meiotic prophase (germinal vesicle stage) mouse oocytes and in unfertilized mature oocytes in second meiotic metaphase, and it persists after fertilization though the 1-cell and 2-cell stages. I(Cl,swell) was found to remain unchanged during metaphase at the end of the 1-cell stage. However, I(Cl,swell) decreased during prophase and became nearly undetectable upon entry into metaphase at the end of the 2-cell stage. Entry into prophase/metaphase was required for the decrease in I(Cl,swell) at the end of the 2-cell stage, since it persisted indefinitely in 2-cell embryos arrested in late G(2). There is considerable evidence that the channel underlying I(Cl,swell) is not only permeable to inorganic anions, but to organic osmolytes as well. We found a similar pattern of cell cycle and developmental dependence in the 1-cell and 2-cell stages for the swelling-induced increase in permeability to the organic osmolyte glycine. Thus, entry into metaphase deactivates I(Cl,swell) in embryos, but only after developmental progression through the 2-cell stage.  相似文献   

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Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. We show that Drosophila alpha-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. We found that Twine and Polo kinase levels are reduced in endos mutants, and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human alpha-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and alpha-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function.  相似文献   

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In eukaryotes, mitosis entry is induced by activation of maturation‐promoting factor (MPF), which is regulated by a network of kinases and phosphatases. It has been suggested that Greatwall (GWL) kinase was crucial for the M‐phase entry and could maintain cyclin B–Cdc2 activity through regulation of protein phosphatase 2A (PP2A), a counteracting phosphatase of MPF. Here, the role of GWL was assessed during release of mouse oocytes from prophase I arrest. GWL was crucial for meiotic maturation in mouse oocytes. As a positive regulator for meiosis resumption, GWL was continually expressed in germinal vesicle (GV) and MII stage oocytes and two‐cell stage embryos. Additionally, GWL localized to the nucleus and dispersed into cytoplasm during GV breakdown (GVBD). Furthermore, downregulation of GWL or overexpression of catalytically‐inactive GWL inhibited partial meiotic maturation. This prophase I arrest induced by GWL depletion could be rescued by the PP2A inhibition. However, both GWL‐depleted and rescued oocytes had severe spindle defects that hardly reached MII. In contrast, oocytes overexpressing wild‐type GWL resumed meiosis and progressed to MII stage. Thus, our data demonstrate that GWL acts in a pathway with PP2A which is essential for prophase I exit and metaphase I microtubule assembly in mouse oocytes.  相似文献   

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In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs-linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Galphas protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Galphas on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.  相似文献   

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Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.  相似文献   

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Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.  相似文献   

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Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyl-transferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here.  相似文献   

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When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.  相似文献   

18.
Xu B  Hua J  Zhang Y  Jiang X  Zhang H  Ma T  Zheng W  Sun R  Shen W  Sha J  Cooke HJ  Shi Q 《PloS one》2011,6(1):e16046
Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.  相似文献   

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Background  

The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5–21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I.  相似文献   

20.
Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.  相似文献   

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