Chromosome structural anomalies due to aberrant spindle forces exerted at gene editing sites in meiosis.

Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

SKAP, an outer kinetochore protein, is required for mouse germ cell development.

In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes.

Rebuilding MTOCs upon centriole loss during mouse oogenesis.

The vast majority of animal cells contain canonical centrosomes as a main microtubule-organizing center defined by a central pair of centrioles. As a rare and striking exception to this rule, vertebrate oocytes loose their centrioles at an early step of oogenesis. At the end of oogenesis, centrosomes are eventually replaced by numerous acentriolar microtubule-organizing centers (MTOCs) that shape the spindle poles during meiotic divisions. The mechanisms involved in centrosome and acentriolar MTOCs metabolism in oocytes have not been elucidated yet. In addition, little is known about microtubule organization and its impact on intracellular architecture during the oocyte growth phase following centrosome disassembly. We have investigated this question in the mouse by coupling immunofluorescence and live-imaging approaches. We show that growing oocytes contain dispersed pericentriolar material, responsible for microtubule assembly and distribution all over the cell. The gradual enlargement of PCM foci eventually leads in competent oocytes to the formation of big perinuclear MTOCs and to the assembly of large microtubule asters emanating from the close vicinity of the nucleus. Upon meiosis resumption, perinuclear MTOCs spread around the nuclear envelope, which in parallel is remodelled before breaking-down, via a MT- and dynein-dependent mechanism. Only fully competent oocytes are able to perform this dramatic reorganization at NEBD. Therefore, the MTOC-MT reorganization that we describe is one of key feature of mouse oocyte competency.