Jesús del Mazo

STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3-related genes flanking the Williams-Beuren syndrome deletion

Chromatin rearrangements in the meiotic prophase are characterized by the assembly and disassembly of synaptonemal complexes (SC), a protein structure that stabilizes the pairing of homologous chromosomes in prophase. We report the identification of human and mouse cDNA coding for stromalin 3 (STAG3), a new mammalian stromalin member of the synaptonemal complex. The stroma‐lins are a group of highly conserved proteins, represented in several organisms from yeast to humans. Stromalins are characterized by the stromalin conservative domain (SCD), a specific motif found in all proteins of the family described to date. STAG3 is expressed specifically in testis, and immunolocalization experiments show that STAG3 is associated to the synaptonemal complex. As the protein encoded by the homologous gene (Scc3p) in Saccharomyces cerevisiae was found to be a subunit of a cohesin complex that binds chromosomes until the onset of anaphase, our data suggest that STAG3 is involved in chromosome pairing and maintenance of synaptone‐mal complex structure during the pachytene phase of meiosis in a cohesin‐like manner. We have mapped the human STAG3 gene to the 7q22 region of chromosome 7; six human STAG3‐related genes have also been mapped: two at 7q22 near the functional gene, one at 7qll.22, and three at 7qll.23, two of them flanking the breakpoints commonly associated with the Williams‐Beuren syndrome (WBS) deletion. Since the WBS deletion occurs as a consequence of unequal meiotic crossing over, we suggest that STAG3 duplications predispose to germline chromosomal rearrangement within this region.—Pezzi, N., Prieto, I., Kremer, L., Pérez Jurado, L. A., Valero, C., del Mazo, J., Martínez‐A., C., Barbero, J. L. STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3‐related genes flanking the Williams‐Beuren syndrome deletion. FASEB J. 14, 581–592 (2000)

The expression patterns of genes involved in the RNAi pathways are tissue-dependent and differ in the germ and somatic cells of mouse testis

Different RNA interference (RNAi) components participate in post-transcriptional regulation via RNA silencing. The expression pattern of the genes Drosha and Dicer and the members of the Argonaute family Ago1, Ago2, Ago3 and Ago4, all elements participating in the RNAi pathways, were investigated in mouse somatic tissues and testis using quantitative RT-PCR. Expression patterns of different testis cells and those emerging during testis development were also investigated. The differential patterns of expression seen suggest potential pleiotropic roles for certain components of the RNAi machinery. Both spermatocytes and spermatids showed a defined gene expression pattern. The strong expression of Ago4 in germ cells suggests that this protein plays a key role in germ-cell differentiation in the seminiferous epithelium.

DNA methylation changes during mouse spermatogenesis

Genomic imprinting in mammals is thought to be mediated by differences in the methylation level of cytosine residues in the genome. These differences in DNA methylation are thought to be generated during the development of the germ line. To characterize the profile of global methylation of the mouse genome during male gametogenesis, we have quantified the relative level of methylation in individual cells during meiosis and spermatogenesis. A decrease in the level of DNA methylation is observed from meiotic cells to elongated spermatids. The erasure of the somatic pattern of methylation during spermatogenesis suggests the existence of a subsequent mechanism generating the parental specific methylation patterns leading to genomic imprinting of specific alleles.

Reprogramming of microRNAs by adenosine-to-inosine editing and the selective elimination of edited microRNA precursors in mouse oocytes and preimplantation embryos

Adenosine deaminases-acting-on-RNA (ADAR) proteins induce adenosine-to-inosine editing in double-stranded RNA molecules. This editing generates RNA diversity at the post-transcriptional level, and it has been implicated in the control of cell differentiation and development. The editing of microRNA (miRNA) precursors, along with Tudor-SN (Snd1) activity, could lead to the elimination of selected miRNAs and reprogram miRNA activity. Here, we report the dynamics of adenosine-to-inosine editing in miRNA precursors and their selected elimination during mouse preimplantation development. Adar1p110 and Snd1 were found to be strongly but differentially expressed in oocytes and zygotes with respect to later pre-implantation stages. When the biogenesis of miR-151 was assessed, the majority of miR-151 precursors was edited and subsequently eliminated during early development. Deep sequencing of this and other miRNAs confirmed that, in general, edited precursors were selectively eliminated at early post-zygotic stages. Moreover, in oocytes and throughout the zygote-to-blastocyst stages, Tudor-SN accumulated in newly discovered aggregates termed ‘T bodies’. These results provide new insight into how editing and Tudor-SN-mediated elimination of miRNA precursors is regulated during early development.

Exposure to Endocrine Disruptor Induces Transgenerational Epigenetic Deregulation of MicroRNAs in Primordial Germ Cells

In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation.

The effects of different endocrine disruptors defining compound-specific alterations of gene expression profiles in the developing testis

Environmental contaminants considered endocrine disruptors have been shown to affect testis development and function but the mechanisms of action are not clear. We now have analyzed the effects on the transcriptome in testes of mice exposed to mono-(2-ethylhexyl)-phthalate (9.2; 46.3 or 92.7 mg/kg/d), zearalenone (1.3; 3.9 or 6.6 mg/kg/d), lindane (16.6; 32.2 or 64.4 mg/kg/d), bisphenol-A (0.16; 16 or 64 mg/kg/d) or 17β-estradiol (0.006; 0.012 or 0.048 mg/kg/d). The compounds were orally administered in the drinking water during distinct developmental periods: (A) mothers were exposed only during the two weeks before mating; (B) the exposure was continued during pregnancy until birth or (C) exposure was continued for a further four weeks after birth. Testes were studied at four weeks of age. Mono-(2-ethylhexyl)-phthalate and zearalenone, both produced specific alterations of gene signatures. Interestingly, this was irrespective of the concentration of the toxicant or the developmental period during which exposure occurred.

Fhx(Foxj2) expression is activated during spermatogenesis and very early in embryonic development

FHX (FOXJ2) is a recently characterized human fork head transcriptional activator that binds DNA with a dual sequence specificity. We have cloned the cDNA for the mouse orthologue Foxj2 and characterized its expression in the gonads and along the early pre-implantation development of the mouse. In the testis, Foxj2 is expressed from pachytene spermatocytes to round spermatids, but not in spermatogonia. In addition to the germ lineage, only Sertoli cells of the testis showed expression of Foxj2. In the ovary, only granulosa cells of the follicles express the factor. Neither mature spermatozoa nor oocytes showed expression of Foxj2. Foxj2 expression is early activated in zygotic development, being detected since as early as 8-cell stage embryos. Both cell layers of the blastocyst: the trophectoderm (TE) and the inner cell mass (ICM), express Foxj2.

Expression dynamics of microRNA biogenesis during preimplantation mouse development

The role of microRNAs (miRNAs) in early development, and particularly in the post-transcriptional regulation of maternal mRNAs remains controversial. Hence, we have assessed how miRNA processing is regulated during preimplantation mouse development, from the fully-grown oocyte to the blastocyst, quantifying the expression of genes whose proteins are involved in miRNAs biogenesis and function. The expression of the Drosha, Dgcr8, Exportin 5, Dicer, Ago1, Ago2, Ago3, Ago4 and Ago5 genes was downregulated from the zygotic cleavage stage, except for the increase of Ago1, Ago3 and Ago4 expression in the 2-cell embryo, and of Ago2 in 4- and 8-cell embryos. These findings suggest that the capacity to process miRNAs, by the considered canonical pathway, diminishes after fertilization, primarily reducing miRNA activity in the later stages of preimplantation development. However, by analyzing the different precursor and mature forms of specific miRNAs that are abundantly expressed in the blastocyst, such as miR-292-3p and miR-292-5p, we identified miRNA-duplexes and/or miRNAs bound to target mRNAs that may serve as potential stockpiles of miRNAs. In response to the demand, such stockpile could directly provide functional and mature miRNAs.

XYbp, a novel RING-finger protein, is a component of the XY body of spermatocytes and centrosomes.

RING-finger proteins participate in developmental processes, including gametogenesis. A fetal oocyte cDNA library was used to select genes expressed during male germ-cell differentiation. A novel RING-finger protein, XYbp (XY body protein), participating in mouse spermatogenesis has been identified. This novel gene generates a ubiquitously expressed transcript of 4.2 kb and a testis-specific one of 2.8 kb, processed by an alternative polyadenylation mechanism from a non-canonical polyadenylation signal. Transcription of XYbp is regulated during spermatocyte differentiation. The antiserum raised against the XYbp peptide demonstrated that XYbp is localised mainly in the XY bivalent of spermatocytes (XY body) and in the centrosomes of somatic and germ cells in all phases of the cell cycle. These studies indicate that we have identified a new member of the RING-finger family of proteins associated with the XY meiotic bivalent during spermatogenesis development and with the centrosomes of all cells.

The cytosolic aldehyde dehydrogenase gene (Aldh1) is developmentally expressed in Leydig cells

Cytosolic aldehyde dehydrogenase, ALDH1, participates in the oxidation of different aldehydes including that of all‐trans retinal to retinoic acid. The accumulation of mouse Aldh1 transcripts is characterized by having different patterns in different tissues. This paper reports the greatest expression of Aldh1 in testis and liver. It was demonstrated that in testis, Aldh1 is specifically expressed in Leydig cells and is under developmental regulation. In vitro studies of cultured Leydig TM3 cells confirmed these results though such gene expression was found not to be mediated by LH regulation. Previous investigations have associated androgen receptors, and hence the androgen insensitivity syndrome in man, with the presence of ALDH1 in genital skin fibroblasts. However, this relationship was not established in a functional cell type, as is reported here for Leydig cells. These results could suggest a model for a molecular pathway from androgen receptor to retinoic acid biogenesis in Leydig cells via the mediation of ALDH.