Tex19.1 inhibits the N-end rule pathway and maintains acetylated SMC3 cohesin and sister chromatid cohesion in oocytes.

Age-dependent oocyte aneuploidy, a major cause of Down syndrome, is associated with declining sister chromatid cohesion in postnatal oocytes. Here we show that cohesion in postnatal mouse oocytes is regulated by Tex19.1. We show Tex19.1-/- oocytes have defects maintaining chiasmata, missegregate their chromosomes during meiosis, and transmit aneuploidies to the next generation. Furthermore, we show that mouse Tex19.1 inhibits N-end rule protein degradation mediated by its interacting partner UBR2, and that Ubr2 itself has a previously undescribed role in negatively regulating the acetylated SMC3 subpopulation of cohesin in mitotic somatic cells. Lastly, we show that acetylated SMC3 is associated with meiotic chromosome axes in mouse oocytes, and that this population of cohesin is specifically depleted in the absence of Tex19.1. These findings indicate that Tex19.1 regulates UBR protein activity to maintain acetylated SMC3 and sister chromatid cohesion in postnatal oocytes and prevent aneuploidy from arising in the female germline.

Tex19.1 promotes Spo11-dependent meiotic recombination in mouse spermatocytes.

Meiosis relies on the SPO11 endonuclease to generate the recombinogenic DNA double strand breaks (DSBs) required for homologous chromosome synapsis and segregation. The number of meiotic DSBs needs to be sufficient to allow chromosomes to search for and find their homologs, but not excessive to the point of causing genome instability. Here we report that the mammal-specific gene Tex19.1 promotes Spo11-dependent recombination in mouse spermatocytes. We show that the chromosome asynapsis previously reported in Tex19.1-/- spermatocytes is preceded by reduced numbers of recombination foci in leptotene and zygotene. Tex19.1 is required for normal levels of early Spo11-dependent recombination foci during leptotene, but not for upstream events such as MEI4 foci formation or accumulation of H3K4me3 at recombination hotspots. Furthermore, we show that mice carrying mutations in Ubr2, which encodes an E3 ubiquitin ligase that interacts with TEX19.1, phenocopy the Tex19.1-/- recombination defects. These data suggest that Tex19.1 and Ubr2 are required for mouse spermatocytes to accumulate sufficient Spo11-dependent recombination to ensure that the homology search is consistently successful, and reveal a hitherto unknown genetic pathway promoting meiotic recombination in mammals.

Effects of androgen receptor mutation on testicular histopathology of patient having complete androgen insensitivity.

Androgens are required for normal male sex differentiation and development of male secondary sexual characteristics. Mutations in AR gene are known to cause defects in male sexual differentiation. In current study, we enrolled a 46,XY phenotypically female patient bearing testes in inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A > G (p. Y572C) in exon 2 which is already known to cause complete androgen insensitivity syndrome (CAIS). We focused on the effects of this mutation on the testicular histopathology of the patient. Surface spreading of testicular tissues showed an absence of spermatocytes while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. This meiotic failure is likely due to the effect of the AR mutation which ultimately leads to Sertoli cell only syndrome. Tubules were stained with SOX9 and AMH which revealed Sertoli cells maturation arrest. Western blot and realtime PCR data showed that patient had higher levels of AMH, SOX9 and inhibin-B in the testis. Therefore, we suggest that the dysfunctioning of AR by mutation enhances AMH expression which ultimately leads to the failure in maturation of Sertoli cells.

Preferential Y-Y pairing and synapsis and abnormal meiotic recombination in a 47,XYY man with non obstructive azoospermia.

BACK GROUND: Men with 47, XYY syndrome are presented with varying physical attributes and degrees of infertility. Little information has been documented regarding the meiotic progression in patients with extra Y chromosome along with the synapses and recombination between the two Y chromosomes. METHODS: Spermatocyte spreading and immunostaining were applied to study the behavior of the extra Y chromosome during meiosis I in an azoospermia patient with 47, XYY syndrome and results were compared with five healthy controls with proven fertility. RESULTS: The extra Y chromosome was present in all the studied spermatocytes of the patient and preferentially paired and synapsed with the other Y chromosome. Consistently, gamma-H2AX staining completely disappeared from the synapsed regions of Y chromosomes. More interestingly, besides recombination on short arms, recombination on the long arms of Y chromosomes was also observed. No pairing and synapsis defects between homologous autosomes were detected, while significantly reduced recombination frequencies on autosomes were observed in the patient. The meiotic prophase I progression was disturbed with significantly increased proportion of leptotene, zygotene cells and decreased pachytene spermatocytes in the patient when compared with the controls. CONCLUSIONS: These findings highlight the importance of studies on meiotic behaviors in patients with an abnormal chromosomal constitution and provide an important framework for future studies, which may elucidate the impairment caused by extra Y chromosome in mammalian meiosis and fertility.

Follicle Online: an integrated database of follicle assembly, development and ovulation.

Folliculogenesis is an important part of ovarian function as it provides the oocytes for female reproductive life. Characterizing genes/proteins involved in folliculogenesis is fundamental for understanding the mechanisms associated with this biological function and to cure the diseases associated with folliculogenesis. A large number of genes/proteins associated with folliculogenesis have been identified from different species. However, no dedicated public resource is currently available for folliculogenesis-related genes/proteins that are validated by experiments. Here, we are reporting a database 'Follicle Online' that provides the experimentally validated gene/protein map of the folliculogenesis in a number of species. Follicle Online is a web-based database system for storing and retrieving folliculogenesis-related experimental data. It provides detailed information for 580 genes/proteins (from 23 model organisms, including Homo sapiens, Mus musculus, Rattus norvegicus, Mesocricetus auratus, Bos Taurus, Drosophila and Xenopus laevis) that have been reported to be involved in folliculogenesis, POF (premature ovarian failure) and PCOS (polycystic ovary syndrome). The literature was manually curated from more than 43,000 published articles (till 1 March 2014). The Follicle Online database is implemented in PHP + MySQL + JavaScript and this user-friendly web application provides access to the stored data. In summary, we have developed a centralized database that provides users with comprehensive information about genes/proteins involved in folliculogenesis. This database can be accessed freely and all the stored data can be viewed without any registration. Database URL: http://mcg.ustc.edu.cn/sdap1/follicle/index.php

Abnormal meiotic recombination with complex chromosomal rearrangement in an azoospermic man.

Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P < 0.001) and decrease in the pachytene spermatocytes (P < 0.001) were observed in the t(5;7;9;13) carrier compared with controls. It was further observed that 93% of octavalents were found partially asynapsed between homologous chromosomes. A significant decrease in the recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.

Decreased XY recombination and disturbed meiotic prophase I progression in an infertile 48, XYY, +sSMC man.

Small supernumerary marker chromosomes (sSMCs) are structurally abnormal rare chromosomes, difficult to characterize by karyotyping, and have been associated with minor dysmorphic features, azoospermia, and recurrent miscarriages. However, sSMC with a gonosomal trisomy has never been reported. Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis, and recombination. In all the analyzed spermatocytes of the patient, the extra Y chromosome was not detected while the sSMC was present. The recombination frequency on autosomes was not affected, while the recombination frequencies on XY chromosome was significantly lower in the patient than in the controls. The meiotic prophase I progression was disturbed with significantly increased proportion of zygotene and decreased pachytene spermatocytes in the patients as compared with the controls. These findings highlight the importance of studies on meiotic behaviors in patients with an abnormal chromosomal constitution and provide an important framework for future studies, which may elucidate the impairment caused by sSMC in mammalian meiosis and fertility.

Specific deletion of Cdh2 in Sertoli cells leads to altered meiotic progression and subfertility of mice.

CDH2 (cadherin 2, Neural-cadherin, or N-cadherin) is the predominant protein of testicular basal ectoplasmic specializations (basal ES; a testis-specific type of adhesion junction), one of the major cell junctions composing the blood-testis barrier (BTB). The BTB is found between adjacent Sertoli cells in seminiferous tubules, which divides the tubules into basal and adluminal compartments and prevents the deleterious exchange of macromolecules between blood and seminiferous tubules. However, the exact roles of basal ES protein CDH2 in BTB function and spermatogenesis is still unknown. We thus generated mice with Cdh2 specifically knocked out in Sertoli cells by crossing Cdh2 loxP mice with Amh-Cre mice. Cdh2 deletion in Sertoli cells did not affect Sertoli cell counts, but led to compromised BTB function, delayed meiotic progression from prophase to metaphase I in testes, increased germ cell apoptosis, sloughing of meiotic cells, and, subsequently, reduced sperm counts in epididymides and subfertility of mice. However, the testes with Cdh2-specific deletion in germ cells did not show any difference from the normal control testes, and phenotypes observed in Sertoli cell and germ cell Cdh2 double-knockout mice were indistinguishable from those in mice with Cdh2 specifically knocked out only in Sertoli cells. Taken together, our data demonstrate that the adhesion junction component, Cdh2, functions just in Sertoli cells, but not in germ cells during spermatogenesis, and is essential for the integrity of BTB function, its deletion in Sertoli cells would lead to the BTB damage and subsequently meiosis and spermatogenesis failure.

miR-214-mediated downregulation of RNF8 induces chromosomal instability in ovarian cancer cells.

Defective DNA damage response (DDR) is frequently associated with carcinogenesis. Abrogation of DDR leads to chromosomal instability, a most common characteristic of tumors. However, the molecular mechanisms underlying regulation of DDR are still elusive. The ubiquitin ligase RNF8 mediates the ubiquitination of γH2AX and recruits 53BP1 and BRCA1 to DNA damage sites which promotes DDR and inhibits chromosomal instability. Though RNF8 is a key player involved in DDR, regulation of its expression is still poorly understood. Here, we show that miR-214 could abrogate DDR by repressing RNF8 expression through direct binding to 3'-untranslated region (3' UTR) of RNF8 mRNA in human ovarian cancer cells. Antagonizing miR-214 by expressing its inhibitors in A2780 cells significantly increased RNF8 expression and thus promoted DNA damage repair. Consistent with the role of miR-214 in regulating RNF8 expression, the impaired DNA repair induced by miR-214 overexpression can be rescued by overexpressing RNF8 mRNA lacking the 3' UTR. Together, our results indicate that down-regulation of RNF8 mediated by miR-214 impedes DNA damage response to induce chromosomal instability in ovarian cancers, which may facilitate the understanding of mechanisms underlying chromosomal instability.

Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice.

Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.

Blood-testis barrier and spermatogenesis: lessons from genetically-modified mice.

The blood-testis barrier (BTB) is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions (TJs), adhesion junctions and gap junctions (GJs). Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.

microRNA 376a regulates follicle assembly by targeting Pcna in fetal and neonatal mouse ovaries.

In mammals, the primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. Dysregulation of primordial follicle assembly results in female reproductive diseases, such as premature ovarian insufficiency and infertility. Female mice lacking Dicer1 (Dicer), a gene required for biogenesis of microRNAs, show abnormal morphology of follicles and infertility. However, the contribution of individual microRNAs to primordial follicle assembly remains largely unknown. Here, we report that microRNA 376a (miR-376a) regulates primordial follicle assembly by modulating the expression of proliferating cell nuclear antigen (Pcna), a gene we previously reported to regulate primordial follicle assembly by regulating oocyte apoptosis in mouse ovaries. miR-376a was shown to be negatively correlated with Pcna mRNA expression in fetal and neonatal mouse ovaries and to directly bind to Pcna mRNA 3' untranslated region. Cultured 18.5 days postcoitum mouse ovaries transfected with miR-376a exhibited decreased Pcna expression both in protein and mRNA levels. Moreover, miR-376a overexpression significantly increased primordial follicles and reduced apoptosis of oocytes, which was very similar to those in ovaries co-transfected with miR-376a and siRNAs targeting Pcna. Taken together, our results demonstrate that miR-376a regulates primordial follicle assembly by modulating the expression of Pcna. To our knowledge, this is the first microRNA-target mRNA pair that has been reported to regulate mammalian primordial follicle assembly and further our understanding of the regulation of primordial follicle assembly.

Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells.

Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human-mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human-mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.

MicroRNA and piRNA profiles in normal human testis detected by next generation sequencing.

BACKGROUND: MicroRNAs (miRNAs) are the class of small endogenous RNAs that play an important regulatory role in cells by negatively affecting gene expression at transcriptional and post-transcriptional levels. There have been extensive studies aiming to discover miRNAs and to analyze their functions in the cells from a variety of species. However, there are no published studies of miRNA profiles in human testis using next generation sequencing (NGS) technology. RESULTS: We employed Solexa sequencing technology to profile miRNAs in normal human testis. Total 770 known and 5 novel human miRNAs, and 20121 piRNAs were detected, indicating that the human testis has a complex population of small RNAs. The expression of 15 known and 5 novel detected miRNAs was validated by qRT-PCR. We have also predicted the potential target genes of the abundant known and novel miRNAs, and subjected them to GO and pathway analysis, revealing the involvement of miRNAs in many important biological phenomenon including meiosis and p53-related pathways that are implicated in the regulation of spermatogenesis. CONCLUSIONS: This study reports the first genome-wide miRNA profiles in human testis using a NGS approach. The presence of large number of miRNAs and the nature of their target genes suggested that miRNAs play important roles in spermatogenesis. Here we provide a useful resource for further elucidation of the regulatory role of miRNAs and piRNAs in the spermatogenesis. It may also facilitate the development of prophylactic strategies for male infertility.

SYCP3-like X-linked 2 is expressed in meiotic germ cells and interacts with synaptonemal complex central element protein 2 and histone acetyltransferase TIP60.

Meiosis is the process by which diploid germ cells produce haploid gametes. A key event is the formation of the synaptonemal complex. In the pachytene stage, the unpaired regions of X and Y chromosomes form a specialized structure, the XY body, within which gene expression is mostly silenced. In the present study, we showed that SYCP3-like X-linked 2 (SLX2, 1700013H16Rik), a novel member of XLR (X-linked Lymphocyte-Regulated) family, was specifically expressed in meiotic germ cells. In the spermatocyte SLX2 was distributed in the nucleus of germ cells at the preleptotene, leptotene and zygotene stages and is then restricted to the XY body at the pachytene stage. This localization change is coincident with that of phosphorylated histone H2AX (γH2AX), a well-known component of the sex body. Through yeast two-hybrid screening and coimmunoprecipitation assays, we demonstrated that SLX2 interacts with synaptonemal complex central element protein 2 (SYCE2), an important component of synaptonemal complex, and histone acetyltransferase TIP60, which has been implicated in remodeling phospho-H2AX-containing nucleosomes at sites of DNA damage. These results suggest that SLX2 might be involved in DNA recombination, synaptonemal complex formation as well as sex body maintenance during meiosis.

SpermatogenesisOnline 1.0: a resource for spermatogenesis based on manual literature curation and genome-wide data mining.

Human infertility affects 10-15% of couples, half of which is attributed to the male partner. Abnormal spermatogenesis is a major cause of male infertility. Characterizing the genes involved in spermatogenesis is fundamental to understand the mechanisms underlying this biological process and in developing treatments for male infertility. Although many genes have been implicated in spermatogenesis, no dedicated bioinformatic resource for spermatogenesis is available. We have developed such a database, SpermatogenesisOnline 1.0 (http://mcg.ustc.edu.cn/sdap1/spermgenes/), using manual curation from 30 233 articles published before 1 May 2012. It provides detailed information for 1666 genes reported to participate in spermatogenesis in 37 organisms. Based on the analysis of these genes, we developed an algorithm, Greed AUC Stepwise (GAS) model, which predicted 762 genes to participate in spermatogenesis (GAS probability >0.5) based on genome-wide transcriptional data in Mus musculus testis from the ArrayExpress database. These predicted and experimentally verified genes were annotated, with several identical spermatogenesis-related GO terms being enriched for both classes. Furthermore, protein-protein interaction analysis indicates direct interactions of predicted genes with the experimentally verified ones, which supports the reliability of GAS. The strategy (manual curation and data mining) used to develop SpermatogenesisOnline 1.0 can be easily extended to other biological processes.

Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

Rapid proliferation of daughter cells lacking particular chromosomes due to multipolar mitosis promotes clonal evolution in colorectal cancer cells.

Aneuploidy and chromosome instability (CIN) are hallmarks of the vast majority of solid tumors. However, the origins of aneuploid cells are unknown. The aim of this study is to improve our understanding of how aneuploidy and/or CIN arise and of karyotype evolution in cancer cells. By using fluorescence in situ hybridization (FISH) on cells after long-term live cell imaging, we demonstrated that most (> 90%) of the newly generated aneuploid cells resulted from multipolar divisions. Multipolar division occurred in mononucleated and binucleated parental cells, resulting in variation of chromosome compositions in daughter cells. These karyotypes can have the same chromosome number as their mother clone or lack a copy of certain chromosomes. Interestingly, daughter cells that lost a chromosome were observed to survive and form clones with shorter cell cycle duration. In our model of cancer cell evolution, the rapid proliferation of daughter cells from multipolar mitosis promotes colonal evolution in colorectal cancer cells.

CPSS: a computational platform for the analysis of small RNA deep sequencing data.

UNLABELLED: Next generation sequencing (NGS) techniques have been widely used to document the small ribonucleic acids (RNAs) implicated in a variety of biological, physiological and pathological processes. An integrated computational tool is needed for handling and analysing the enormous datasets from small RNA deep sequencing approach. Herein, we present a novel web server, CPSS (a computational platform for the analysis of small RNA deep sequencing data), designed to completely annotate and functionally analyse microRNAs (miRNAs) from NGS data on one platform with a single data submission. Small RNA NGS data can be submitted to this server with analysis results being returned in two parts: (i) annotation analysis, which provides the most comprehensive analysis for small RNA transcriptome, including length distribution and genome mapping of sequencing reads, small RNA quantification, prediction of novel miRNAs, identification of differentially expressed miRNAs, piwi-interacting RNAs and other non-coding small RNAs between paired samples and detection of miRNA editing and modifications and (ii) functional analysis, including prediction of miRNA targeted genes by multiple tools, enrichment of gene ontology terms, signalling pathway involvement and protein-protein interaction analysis for the predicted genes. CPSS, a ready-to-use web server that integrates most functions of currently available bioinformatics tools, provides all the information wanted by the majority of users from small RNA deep sequencing datasets. AVAILABILITY: CPSS is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/db/cpss/index.html or http://mcg.ustc.edu.cn/sdap1/cpss/index.html.