Proteins influencing the integrity of meiotic chromosome dynamics

Abstract

Aneuploidy (trisomy or monosomy) is the leading genetic cause of human pregnancy loss and results from aberrant meiotic chromosome segregation. Accurate segregation of meiotic chromosomes depends on extensive pairing (called synapsis) and recombination between them. The main focus of this thesis is to understand the mechanisms regulating chromosomal pairing and segregation in meiotic cells and the mechanistic failures that generate aneuploid cells.

Synapsis is aided by a meiosis-specific protein complex, the synaptonemal complex (SC), which comprises two axial elements (AEs, which colocalise with the sister chromatids of each homologue) and a central element (CE). The axial core between synapsed homologues is composed of discrete filaments that include the AE (formed by two meiosis-specific proteins, SCP2 and SCP3) and a second protein complex, composed of cohesins, which promotes sister chromatid pairing. Inactivation of SCP3 in male mice abolishes AE formation, resulting in chromosomal pairing failures. These, in turn, trigger a chromosome pairing 'checkpoint' that induces apoptosis in early meiotic (zygotene) male germ cells, rendering males infertile.

To explore the functions of the SC and the cohesin complex in mammalian male meiotic cells, we have analysed how the absence of SCP3 affects the distribution and assembly of proteins integral to meiotic chromosome pairing. We conclude that the cohesin complex can recruit recombination proteins, maintain sister chromatid pairing and promote synapsis between homologous chromosomes despite the absence of SCP3 (and a visually observable AE), leading to a new model for chromosome pairing and synapsis initiation in mammalian meiotic cells.

We find that the checkpoint that responds to improper chromosome pairing selectively eliminates male germ cells. What is the mechanism that regulates this checkpoint and thereby mediates the apoptotic response in SCP3-/- male germ cells? To investigate the involvement of p53, a known apoptotic regulator, we generated SCP3-/-Trp53-/- mice. We show that p53-deficiency fails to rescue the meiotic defects seen in SCP3-/- testes, thereby identifying a novel p53-independent pathway that monitors meiotic chromosomal pairing and asynapsis in mammals.

Mice lacking both SCP3 and E2F-1, a transcription factor implicated in p53-independent apoptosis, were also generated to further characterise this checkpoint. We report that E2F-1 does not rescue SCP3-/ spermatocytes, but rather that absence of E2F-1 aggravates testicular degeneration when combined with SCP3-deficiency. The checkpoint monitoring chromosome pairing is thus p53- and E2F-1-independent.

The death of SCP3-null spermatocytes at zygotene has made the function of SCP3 during post-zygotene prophase and meiotic divisions 1 and 11 impossible to study. SCP3-/- females, however, are fertile. We describe reduced fertility in SCP3-/- females, due to aberrant meiotic chromosome segregation in their developing oocytes. The abnormal oocyte karyotype is inherited by embryos, which die in utero. We demonstrate that SCP3 is not essential for chromosome synapsis nor the formation of chiasmata, physical links that are established between homologues following recombination. However, we show that SCP3 is required for AE formation, the incorporation of SCP2 into the AE and for normal meiotic chromatin compaction in females.

This is the first known mammalian model of inherited aneuploidy in oocytes, and thus should prove valuable for understanding the basis of chromosomal nondisjunction in human oocytes and aneuploidy-linked embryo death.

Additionally, we have developed a 'localisation proteomics' strategy, which utilises the intracellular localisation of uncharacterised proteins as a functional pointer. Protein subcellular localisation information is crucial to proteomics efforts as it provides a context for a protein's sequence, structure, and function. The approach, termed PROLOC, has resulted in the tentative classification of 25 uncharacterised proteins.