Cytoskeletal variations in an asymmetric cell division support diversity in nematode sperm size and sex ratios

Asymmetric partitioning is an essential component of many developmental processes. As spermatogenesis concludes, sperm are streamlined by discarding unnecessary cellular components into cellular wastebags called residual bodies (RBs). During nematode spermatogenesis, this asymmetric partitioning event occurs shortly after anaphase II, and both microtubules and actin partition into a central RB. Here, we use fluorescence and transmission electron microscopy to elucidate and compare the intermediate steps of RB formation in Caenorhabditis elegans, Rhabditis sp. SB347 (recently named Auanema rhodensis) and related nematodes. In all cases, intact microtubules reorganize and move from centrosomal to non-centrosomal sites at the RB-sperm boundary whereas actin reorganizes through cortical ring expansion and clearance from the poles. However, in species with tiny spermatocytes, these cytoskeletal changes are restricted to one pole. Consequently, partitioning yields one functional sperm with the X-bearing chromosome complement and an RB with the other chromosome set. Unipolar partitioning may not require an unpaired X, as it also occurs in XX spermatocytes. Instead, constraints related to spermatocyte downsizing may have contributed to the evolution of a sperm cell equivalent to female polar bodies

Dynamic and ultrastructural characterization of chromosome segregation in C. elegans male meiosis

The production of germ cells is an essential process in all sexually reproducing eukaryotes. During male meiosis, four haploid sperm cells are formed from one primary spermatocyte, thereby undergoing two consecutive cell divisions after only one round of chromosome duplication. This process was studied in the nematode Caenorhabditis elegans, as this model organism offers a number of experimental advantages to simultaneously analyze spindle dynamics and ultrastructure. The worm is easy to cultivate, completely sequenced and numerous mutants are available, the worm is small and thus ideal for light and electron microscopic investigations, and the transparent body allows live-cell imaging within living animals. Importantly, meiotic spindles in C. elegans males are organized by centrosomes and show a lagging X-chromosome, which is always segregated after the autosomes have been partitioned to the newly forming secondary spermatocytes. The aim of this thesis was to systematically investigate this characteristic feature of chromosome segregation in male meiotic spindles. For that, spindle dynamics in the first and second meiotic division was analyzed with fluorescence light microscopy. Furthermore, the spindle ultrastructure was investigated in spindles of various stages of meiosis I using electron tomography. Light microscopy revealed a shortening of the distance between centrosomes and chromosomes (anaphase A) and an increase in the pole-to-pole distance (anaphase B). Moreover, spindles in male meiosis I and II showed differences in certain aspects of spindle dynamics. In addition it was demonstrated that spindles in metaphase II in the presence of a single X-chromosome were shorter compared to spindles without the X-chromosome. In addition, it was found that the process of aging had an impact on spindle length in both metaphase I and II. By manipulating the number of unpaired chromosomes, it could be demonstrated that the lagging behavior of univalent chromosomes is caused by the incapability of pairing in meiotic prophase. After performing a quantitative analysis of the light microscopic data it was further shown that a dynamic microtubule bundle is connecting the X-chromosome to the spindle poles. Using laser microsurgery it could be demonstrated that this bundle exerts a pulling force to the univalent chromosome throughout anaphase. Unexpectedly, electron tomography showed that anaphase-type movements of the autosomes were not accompanied by a shortening of the kinetochore microtubules. Instead, three findings indicated a shortening of the centrosome-chromosome distance itself: (1) upon anaphase onset, tension is released on the beforehand stretched autosomes; (2) centrosomes shrink in preparation for meiosis II and (3) the attachment angle of end-on microtubules changes. Interestingly, microtubules connecting the X-chromosome to the spindle poles showed a high curvature around the kinetochore region of the X-chromosome, suggesting an involvement of motor proteins in the process of segregation. Taken together, this thesis gives the first detailed quantitative analysis of spindle dynamics and architecture during male meiosis in the nematode C. elegans. This wild-type data will serve as a basis for future mutant analyses and should help to further understand the complex dynamic and ultrastructural aspects of spindle organization in the meiotic divisions in C. elegans males

Back to the Roots: Segregation of Univalent Sex Chromosomes in Meiosis.

In males of many taxa, univalent sex chromosomes normally segregate during the first meiotic division, and analysis of sex chromosome segregation was foundational for the chromosome theory of inheritance. Correct segregation of single or multiple univalent sex chromosomes occurs in a cellular environment where every other chromosome is a bivalent that is being partitioned into homologous chromosomes at anaphase I. The mechanics of univalent chromosome segregation vary among animal taxa. In some, univalents establish syntelic attachment of sister kinetochores to the spindle. In others, amphitelic attachment is established. Here, we review how this problem of segregation of unpaired chromosomes is solved in different animal systems. In addition, we give a short outlook of how mechanistic insights into this process could be gained by explicitly studying model organisms, such as Caenorhabditis elegans