In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs In meiosis, two rounds of chromosome segregation follow one round of replication. The first segregation, meiosis I, is reductional, as homologous chromosomes (homologs) move to opposite poles, whereas meiosis II is equational, because sister chromatids disjoin. The disjunction of homologs is prepared during the prophase of meiosis I, when homologs pair and nonsister chromatids of homologs recombine (for review, see Zickler and Kleckner 1999). The resulting crossovers and cohesion between the sister chromatids connect the homologs and ensure their proper disjunction at meiosis I. In most analyzed eukaryotes, meiotic recombination is accompanied by the close apposition of homologs by a zipper-like proteinaceous structure, the synaptonemal complex (SC). After premeiotic S-phase, the two sister chromatids of each chromosome develop a common axial structure, the axial element (AE), which consists of a linear array of protein complexes involved in sister chromatid cohesion (cohesin complexes), associated with various additional proteins (for review, see Page and Hawley 2004). Numerous transverse filaments (TFs) then connect the AEs of two homologs (synapsis) to form an SC. Within the SC, AEs are called lateral elements (LEs). Genes encoding TF proteins have been identified in mammals (Sycp1), budding yeast (ZIP1), Drosophila (c(3)G), and Caenorhabditis (Syp-1 and Syp-2). SYCP1, Zip1, and C(3)G are long coiled-coil proteins with globular domains at both ends. Within SCs, they form parallel coiled-coil homodimers, which are embedded with their C termini in the LEs, whereas the N termini of TF protein molecules from opposite LEs overlap in the narrow region between the LEs of the two homologs. Caenorhabditis Syp-1 and Syp-2 are two short coiled-coil proteins, which possibly take the place of a single longer coiled-coil protein in other species (for review, see Page and Hawley 2004).In the three species in which it has been analyzed, Drosophila, Caenorhabditis, and yeast, TF-deficient mu-
Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement.
Follicle development in the mammalian ovary requires interactions among the oocyte, granulosa cells, and theca cells, coordinating gametogenesis and steroidogenesis. Here we show that granulosa cells of growing follicles in mouse ovary act as a source of hedgehog signaling. Expression of Indian hedgehog and desert hedgehog mRNAs initiates in granulosa cells at the primary follicle stage, and we find induced expression of the hedgehog target genes Ptch1 and Gli1, in the surrounding pre-theca cell compartment. Cyclopamine, a highly specific hedgehog signaling antagonist, inhibits this induced expression of target genes in cultured neonatal mouse ovaries. The theca cell compartment remains a target of hedgehog signaling throughout follicle development, showing induced expression of the hedgehog target genes Ptch1, Ptch2, Hip1, and Gli1. In periovulatory follicles, a dynamic synchrony between loss of hedgehog expression and loss of induced target gene expression is observed. Oocytes are unable to respond to hedgehog because they lack expression of the essential signal transducer Smo (smoothened). The present results point to a prominent role of hedgehog signaling in the communication between granulosa cells and developing theca cells.
SummaryThe cytoplasmic chromatoid body (CB) organizes mRNA metabolism and small regulatory RNA pathways, in relation to haploid gene expression, in mammalian round spermatids. However, little is known about functions and fate of the CB at later steps of spermatogenesis, when elongating spermatids undergo chromatin compaction and transcriptional silencing. In mouse elongating spermatids, we detected accumulation of the testis-specific serine/threonine kinases TSSK1 and TSSK2, and the substrate TSKS, in a ring-shaped structure around the base of the flagellum and in a cytoplasmic satellite, both corresponding to structures described to originate from the CB. At later steps of spermatid differentiation, the ring is found at the caudal end of the newly formed mitochondrial sheath. Targeted deletion of the tandemly arranged genes Tssk1 and Tssk2 in mouse resulted in male infertility, with loss of the CB-derived ring structure, and with elongating spermatids possessing a collapsed mitochondrial sheath. These results reveal TSSK1-and TSSK2-dependent functions of a transformed CB in post-meiotic cytodifferentiation of spermatids.
The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.Ubiquitin is present in all cells, and the ubiquitin system is involved in different essential cellular processes such as cell division, responses to stress, and apoptosis. Protein ubiquitination occurs through the activities of ubiquitin activatingenzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-ligating enzymes (E3) (52). Polyubiquitination usually targets a substrate for degradation by a multisubunit structure called the proteasome. Ubiquitination, and in particular monoubiquitination, may also serve other purposes, such as activation or inactivation of transcription factors (12), internalization of transmembrane receptors (22, 52), and alteration of chromatin structure through stable ubiquitination of histones (13, 36). The genome encodes only very few E1 enzymes (1 or 2), and the diverse functions of the ubiquitin system are brought about by 10 to 20 different E2s and an even greater variation of E3 enzymes (52). The ubiquitin-conjugating E2 enzyme HR6B is essential for male fertility in the mouse (41).HR6B is one of the two mammalian homologs of the Saccharomyces cerevisiae E2 enzyme named RAD6/UBC2 (24). The other mammalian RAD6 homolog, HR6A, shows 96% amino acid identity to HR6B. Between mouse and human enzymes, the identity is 100%. The mouse and human HR6B genes are autosomal, whereas HR6A is located on the X chromosome in both species (24,41). RAD6 in yeast is essential for sporulation but is also involved in many other processes, as illustrated by the pleiotropic phenotype of RAD6-null mutants (27). These mutants are defective in a specific DNA damage response pathway named postreplication repair. Furthermore, there are defects in gene silencing and changes in mitotic homologous recombination frequency (10,23,44). The precise mechanism of the role of RAD6 in yeast meiosis and sporulat...
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