Mary Ann Handel

Meiotic Prophase Arrest with Failure of Chromosome Synapsis in Mice Deficient for Dmc1, a Germline-Specific RecA Homolog

DMC1 is a meiosis-specific gene first discovered in yeast that encodes a protein with homology to RecA and may be component of recombination nodules. Yeast dmc1 mutants are defective in crossing over and synaptonemal complex (SC) formation, and arrest in late prophase of meiosis I. We have generated a null mutation in the Dmc1 gene in mice and show that homozygous mutant males and females are sterile with arrest of gametogenesis in the first meiotic prophase. Chromosomes in mutant spermatocytes fail to synapse, despite the formation of axial elements that are the precursor to the SC. The strong similarity of phenotypes in Dmc1-deficient mice and yeast suggests that meiotic mechanisms have been highly conserved through evolution.

Genetics of mammalian meiosis: regulation, dynamics and impact on fertility

Meiosis is an essential stage in gamete formation in all sexually reproducing organisms. Studies of mutations in model organisms and of human haplotype patterns are leading to a clearer understanding of how meiosis has adapted from yeast to humans, the genes that control the dynamics of chromosomes during meiosis, and how meiosis is tied to gametic success. Genetic disruptions and meiotic errors have important roles in infertility and the aetiology of developmental defects, especially aneuploidy. An understanding of the regulation of meiosis, coupled with advances in genomics, may ultimately allow us to diagnose the causes of meiosis-based infertilities, more wisely apply assisted reproductive technologies, and derive functional germ cells.

Sex chromosomes, recombination, and chromatin conformation

We review what is known about the transcriptional inactivation and condensation of heteromorphic sex chromosomes in contrast to the activation of homomorphic sex chromosomes during meiotic prephase in animals. We relate these cytological and transcriptional features to the recombination status of the sex chromosomes. We propose that sex chromosome condensation is a meiotic adaptation to prevent the initiation of potentially damaging recombination events in nonhomologous regions of the X and Y chromosome.

BRCA2 deficiency in mice leads to meiotic impairment and infertility

The role of Brca2 in gametogenesis has been obscure because of embryonic lethality of the knockout mice. We generated Brca2-null mice carrying a human BAC with the BRCA2 gene. This construct rescues embryonic lethality and the mice develop normally. However, there is poor expression of the transgene in the gonads and the mice are infertile, allowing examination of the function of BRCA2 in gametogenesis. BRCA2-deficient spermatocytes fail to progress beyond the early prophase I stage of meiosis. Observations on localization of recombination-related and spermatogenic-related proteins suggest that the spermatocytes undergo early steps of recombination (DNA double strand break formation), but fail to complete recombination or initiate spermiogenic development. In contrast to the early meiotic prophase arrest of spermatocytes, some mutant oocytes can progress through meiotic prophase I, albeit with a high frequency of nuclear abnormalities, and can be fertilized and produce embryos. Nonetheless, there is marked depletion of germ cells in adult females. These studies provide evidence for key roles of the BRCA2 protein in mammalian gametogenesis and meiotic success.

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.

SUMO modified proteins localize to the XY body of pachytene spermatocytes

The XY body is a specialized chromatin territory that forms during meiotic prophase of spermatogenesis and comprises the transcriptionally repressed sex chromosomes. Remodeling of the XY chromatin is brought about by recruitment of specific proteins to the X and Y chromosomes during meiosis, and also by post-translational modifications of histones and other chromatin-associated proteins. Here, we demonstrate that SUMO, a small ubiquitin-related modifier protein that regulates a wide variety of nuclear functions in somatic cells, dramatically localizes to the XY body. SUMO was first detected in the XY body of early pachytene spermatocytes and gradually accumulated, reaching maximal levels there during the mid to late pachytene stages. Several known SUMO substrates, including PML and DAXX, were also found to accumulate in the XY body of mid to late stage pachytene spermatocytes. These same proteins localize to PML nuclear bodies of somatic interphase nuclei. Together, these findings indicate a role for SUMO modification in regulating the structure and function of the XY body and reveal molecular similarities between the XY body and PML nuclear bodies.

Genetic control of spermatogenesis in mice

In this chapter I shall review evidence for direct genetic control of spermatogenesis in mice from two perspectives. First, I shall consider genetic conditions affecting the process of spermatogenesis: both specific gene mutations and chromosome anomalies causing interruption of the spermatogenic process. Second, I shall discuss the molecular evidence for direct gene action during spermatogenesis, and for changes in the patterns of gene and chromosome activity during spermatogenesis that might be implicated in controlling events.

Analysis of expression and function of topoisomerase I and II during meiosis in male mice

Topoisomerases are nuclear enzymes that remove torsional stress in DNA. Their function is important for replication, transcription, chromosome condensation, and chromosome segregation during mitosis and meiosis. The goal of this work is to analyze both expression and function of topoisomerases during the meiotic stages of mammalian spermatogenesis. The patterns of expression of topoisomerase I and topoisomerase IIα genes were followed on Northern blots of RNA from testes of mice of different ages and from specific germ cell populations. The transcript of the topoisomerase I gene was highest in somatic cells of the testis and in the mitotically proliferating spermatogonia and meiotic prophase spermatocytes, with the level of transcript decreasing dramatically in postmeiotic spermatids. In contrast, the levels of topoisomerase IIα transcript were negligible in germcell free testes and highest in late meiotic prophase cells and round spermatids. Enzyme activity for both topoisomerase I and topoisomerase II was detected in both pachytene spermatocytes and in round spermatids; topoisomerase II exhibited a higher level of activity in meiotic spermatocytes than in round spermatids. In cultured cells, camptothecin, an inhibitor of topoisomerase I, caused some abnormalities of paired meiotic homologs, but did not inhibit the transition to metaphase. In contrast, teniposide and ICRF‐193, inhibitors of topoisomerase II, dramatically inhibited the formation of metaphase chromosomes in cells induced to progress from prophase to metaphase. However, the disassembly of the synaptonemal complex was not inhibited, indicating that this process could be uncoupled from condensation of chromatin to form chromosomes. These studies constitute evidence for a functional requirement for topoisomerase II activity in the transition from meiotic prophase to meiotic metaphase I in mammalian spermatocytes. Mol. Reprod. Dev. 46:489–498, 1997.

Tissue-Specific Functional Networks for Prioritizing Phenotype and Disease Genes

Integrated analyses of functional genomics data have enormous potential for identifying phenotype-associated genes. Tissue-specificity is an important aspect of many genetic diseases, reflecting the potentially different roles of proteins and pathways in diverse cell lineages. Accounting for tissue specificity in global integration of functional genomics data is challenging, as “functionality” and “functional relationships” are often not resolved for specific tissue types. We address this challenge by generating tissue-specific functional networks, which can effectively represent the diversity of protein function for more accurate identification of phenotype-associated genes in the laboratory mouse. Specifically, we created 107 tissue-specific functional relationship networks through integration of genomic data utilizing knowledge of tissue-specific gene expression patterns. Cross-network comparison revealed significantly changed genes enriched for functions related to specific tissue development. We then utilized these tissue-specific networks to predict genes associated with different phenotypes. Our results demonstrate that prediction performance is significantly improved through using the tissue-specific networks as compared to the global functional network. We used a testis-specific functional relationship network to predict genes associated with male fertility and spermatogenesis phenotypes, and experimentally confirmed one top prediction, Mbyl1. We then focused on a less-common genetic disease, ataxia, and identified candidates uniquely predicted by the cerebellum network, which are supported by both literature and experimental evidence. Our systems-level, tissue-specific scheme advances over traditional global integration and analyses and establishes a prototype to address the tissue-specific effects of genetic perturbations, diseases and drugs.

Ultra-High Resolution 3D Imaging of Whole Cells

Summary Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.