Ciliary control of meiotic chromosomal pairing mechanics and germ cell morphogenesis

Avishag Mytils, Vineet Kumar, Qiu Tao, Rachael Deis, Karine Levy, Markus Masek, Hagai Eitan, Farouq Nather, Amal Shawahny, Ruxandra Bachmann-Gagescu, Sudipto Roy, Yaniv M. Elkouby

Posted on: 15 February 2021

Preprint posted on 9 February 2021

The "zygotene cilium" - a previously unrecognised cilium in oocytes.

Selected by Robert Mahen

Categories: cell biology, developmental biology, genetics, physiology

Cell division is driven by mechano-chemical macromolecular assemblies that create physical force to position the genome. During meiosis – the formation of haploid gametes – chromosomes undergo elaborate movements during prophase 1. Telomeres cluster at the nuclear envelope, forming a structure termed the chromosomal bouquet (Figure 1). The chromosomal bouquet promotes meiotic pairing of chromosome homologs and errors in its formation can result in infertility (Ding et al., 2007; Elkouby et al., 2016; Horn et al., 2013; Sato et al., 2009; Shibuya et al., 2014).

Cilia are hair-like organelles that in some cell types are considered incompatible with cell division. Mutations in ciliary genes often result in ciliopathies, disorders with a wide variety of symptoms, one of which is infertility.

In a recent preprint, Mytils, Kumar et al. report the zytgotene cilium – a cilium present during meiosis. They suggest that the zytgotene cilium is important for fertility through aiding mechanical positioning of the chromosome bouquet during gametogenesis.

Figure. 1: The chromosomal bouquet in prophase 1 of meiosis.

Key findings

Mytils, Kumar et al., used live imaging in zebrafish ovaries to record zygotene oocytes, observing that chromosomes rotate rapidly, in contrast to a positionally stable centrosome. Interestingly, the centrosome formed the base of a previously unrecognised cilium, marked by Arl13b, acetylated tubulin and glutamylated tubulin (Figure 2a). The presence of the zygotene cilia was confirmed by transmission electron microscopy and was stage specific, fully elaborated at zygotene but absent at pachytene.

Oocyte precursors develop in germline cysts – groups of developing cells that form close contacts with each other. Serial block face scanning electron microscopy showed that zygotene cilia extend throughout germline cysts and tangle in between cells (Figure 2b).

Figure. 2 Selected panels from Figure 1 of Mytilis, Kumar et al., 2021, reproduced with permission from the authors.

To understand whether zygotene cilia are important for meiosis, Mytils, Kumar et al. investigated the effects of loss of function of four different ciliary genes. cep290^-/- , cep290^-/-;kif7^-/-, and cc2d2a^-/- ciliary mutant fish were not embryonic lethal but no longer had zygotene cilia. This correlated with dysfunctional germ cell morphogenesis, ovarian development, infertility and abnormal chromosomal bouquet formation. Interestingly, cep290-/- mutants maintained a centrosome able to nucleate microtubules. Therefore, specifically the zygotene cilium may be required for bouquet function, in addition to cytoskeletal fibres from centrosomes. Finally, the authors show that the zygotene cilium is conserved not only in male meiosis in zebrafish, but also in mouse oogenesis. It is therefore not specific to one sex or one species.

Significance

Some ciliopathies are associated with infertility. This work predicts that these fertility issues could relate to dysfunction of the zygotene cilium, in addition to other known cilium-related issues such as sperm motility.

This preprint shows that cilia are not strictly incompatible with cell division, and moreover that they may play important roles during it, in agreement with other work in different systems (Riparbelli et al., 2012).

The centrosome is inherently mechanochemical, and yet the forces acting upon it are unclear. This preprint suggests that centrosome anchoring to a cilium is a method of force resistance required for chromosome movements, an exciting new theory.

References

Ding, X., Xu, R., Yu, J., Xu, T., Zhuang, Y., and Han, M. (2007). SUN1 Is Required for Telomere Attachment to Nuclear Envelope and Gametogenesis in Mice. Dev. Cell 12, 863–872.

Elkouby, Y.M., Jamieson-Lucy, A., and Mullins, M.C. (2016). Oocyte Polarization Is Coupled to the Chromosomal Bouquet, a Conserved Polarized Nuclear Configuration in Meiosis. PLOS Biol. 14, e1002335.

Horn, H.F., Kim, D.I., Wright, G.D., Wong, E.S.M., Stewart, C.L., Burke, B., and Roux, K.J. (2013). A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton. J. Cell Biol. 202, 1023–1039.

Riparbelli, M.G., Callaini, G., and Megraw, T.L. (2012). Assembly and Persistence of Primary Cilia in Dividing Drosophila Spermatocytes. Dev. Cell 23, 425–432.

Sato, A., Isaac, B., Phillips, C.M., Rillo, R., Carlton, P.M., Wynne, D.J., Kasad, R.A., and Dernburg, A.F. (2009). Cytoskeletal Forces Span the Nuclear Envelope to Coordinate Meiotic Chromosome Pairing and Synapsis. Cell 139, 907–919.

Shibuya, H., Ishiguro, K., and Watanabe, Y. (2014). The TRF1-binding protein TERB1 promotes chromosome movement and telomere rigidity in meiosis. Nat. Cell Biol. 16, 145–156.

Tags: cilia, ciliopathy, electron microscopy, fertility, meiosis, zebrafish

doi: https://doi.org/10.1242/prelights.27402

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Yaniv Elkouby shared

Author’s response

Cilia have been reported to persist through meiotic divisions in Drosophila spermatocytes (Riparbelli et al., 2012). How does the zygotene cilium compare to this, functionally and structurally?

This is a very interesting observation, we were not aware of this study. It appears that the described cilia in this paper are very short. The authors propose that they could be unusually comprised of only elaborated transition zones, rather than fully developed cilia. They also seem to be nucleated from four centrioles and become internalized in membrane pockets. In contrast, the zygotene cilium form conventionally from a centriole and basal body, and are much longer and extend inter-cellularly. We measured that they mostly reach a length of 4-8 µm, but some can be as long as 12 µm. The cilia we identified in zebrafish spermatocytes, and in mouse oocytes, were similar to the zytgotene cilium. Unfortunately, the function of the intriguing cilia in Drosophila was not reported, so it is hard to compare. One common observation is that both the zygotene cilium in vertebrate spermatocytes and the unique cilium in Drosophila seem to be distinct from the flagella.

You suggest that the zygotene cilium could anchor a centrosome and hence counterbalance microtubule and chromosome movements. Could you test this hypothesis by somehow ectopically anchoring centrosomes which do not form the zygotene cilium? (e.g. in the cep290-/- mutant)? The prediction might be a rescue of function, if the phenotype only relates to stabilising mechanical forces.

Our next step is definitely to determine the mechanisms of action of the zygotene cilium. Other than anchoring, we could hypothesize additional functions, like signalling, that could act in concert. We have started to investigate that in different directions, attempting to tease these apart. We are investigating gonads in vivo during post-embryonic development, so these are challenging analyses. I am not sure how we can ectopically anchor the centrosome, but we are trying to take the reciprocal approach and detach it from the cilium and test the consequences. The results are very interesting, but probably too preliminary at this stage.

The potential anchoring mechanism is very interesting. There is evidence to suggest that anchoring by other means is required during homologous/analogous bouquet stages in other species. For example in S. pombe the astral microtubules that grow from the centrosome (spindle pole body) at this stage must be anchored to the cell cortex (Ding et al., 1998; Ohtaka et al., 2007; Yamashita et al., 2006). An interesting case is seen in Drosophila, where microtubules (of the analogous bouquet configuration), are not growing from the centrosome, but from the fusome MTOC (Christophorou et al., 2015). The fusome in the Drosophila cyst forms a network through cytoplasmic bridges between sister oocytes, so presumably can provide anchoring. It is very interesting to note that in this case, the centrosome is not stationary like in zebrafish and mouse, but moves around the nucleus (Christophorou et al., 2015). Perhaps the greater number of chromosomes in zebrafish and mice would require more substantial anchoring and the development of the zygotene cilium. However, we first need to definitively determine the mechanisms of function of the zygotene cilium before confidently stating either way.

Mitotic centrosomes form spindle poles, not cilia. Could you suggest why cilium related anchoring might not be required during mitosis contrastingly to your interesting data in meiosis? Less cytoskeletal generated force is present in mitosis perhaps?

My guess would be that during mitosis this function is probably provided by astral microtubules, so a cilium is not needed. Astral microtubules are involved in anchoring the spindle poles in different systems to orient, position, or scale the spindle. I am not sure how to compare the forces between mitosis and meiosis. However, in mitosis each spindle pole pools 2n of chromosomes unidirectionally, while in meiosis 4n are shuffled through the rotations of telomeres around the nuclear envelope, so differences exist.

You suggest a wider paradigm – that ciliary dynamics might influence nuclear chromosomal events. Could you expand on this interesting theory?

This could apply to different contexts. For example, in different cells the position of the nucleus is regulated, and usually controlled by Sun/KASH proteins associated with microtubules. It is plausible that a cilium could contribute to nuclear positioning through its connections with the cytoskeleton.

Another interesting case is an organization similar to the bouquet that forms in somatic interphase cells, called the Rabl configuration, where centromeres are loaded on the nuclear envelope and cluster. The Rabl configuration was first identified in 1885 in salamander cells, and has been mostly studied in yeast, plants and Drosophila. It was suggested that in many cases it could be very transient, thus limiting detection in other systems. Here, Sun/KASH proteins and microtubules are similarly involved in clustering centromeres, so it is easy to imagine that a cilium could be involved as well, like in the bouquet.

All in all, KASH proteins (Nesprin family of proteins) widely mediate nuclear-cytoplasmic dynamics and regulation, by specifically interacting with various cytoskeletal components. These cytoskeleton components could in turn interact with or be regulated by ciliary mechanics and signalling to influence nuclear organization. Such mechanisms could contribute to functional nuclear features, from regulating nuclear positioning and morphology, to the regulation of chromosomal territories and association of chromosomes with the nuclear lamina and nuclear pore complexes during regulation of gene expression. Our observations offer new perspectives to these examples, which are common to many cell types, and in many coincide with a presence of a cilium.

Do mutations in kif7 or cep290 similarly cause infertility in humans? Would it be possible to look in human ciliopathy primary tissue to understand whether the zygotene cilium truly has a role in infertility in a human ciliopathy?

So far, infertility in ciliopathies was considered to be a result of loss of cilia in fallopian tubules and efferent ducts, or from loss of function of sperm flagella. With the conservation of the zygotene cilium between zebrafish and mouse, it is reasonable to assume that the zygotene cilium also forms in human oogenesis. The equivalent zygotene stages in human oogenesis, occur within the developing ovaries of the female foetus, approximately during weeks 15-20 of pregnancy. Obvious ethical and technical concerns make it difficult to identify and investigate the zygotene cilium in human foetal ovaries. However, we have been in touch with physicians to start looking and are open for collaborations. It would be insightful to examine ovaries of ciliopathic foetuses in order to better understand theses diseases, but I am not sure how possible this is.

Trying to find connections between ciliopathies and potential ovarian dysgenesis and female infertility, I have had discussions with physicians that see ciliopathic patients. It seems that the fertility and ovarian status of these patients is not usually addressed or recorded in their medical files. Some of them unfortunately are very young and suffer from conditions that require more immediate attention. In others, such phenotypes could be partially penetrant, which would be challenging to detect in human reproduction. In the soma, partial and complex pleiotropic phenotypes are common in ciliopathies. If there is any record of ovarian dysgenesis and/or female fertility in ciliopathies, I would be very curious to see it.

References

Christophorou, N., Rubin, T., Bonnet, I., Piolot, T., Arnaud, M., & Huynh, J. R. (2015). Microtubule-driven nuclear rotations promote meiotic chromosome dynamics. Nat Cell Biol, 17(11), 1388-1400. doi:10.1038/ncb3249

Ding, D. Q., Chikashige, Y., Haraguchi, T., & Hiraoka, Y. (1998). Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells. J Cell Sci, 111 ( Pt 6), 701-712.

Ohtaka, A., Saito, T. T., Okuzaki, D., & Nojima, H. (2007). Meiosis specific coiled-coil proteins in Shizosaccharomyces pombe. Cell Division, 2(1), 14. doi:10.1186/1747-1028-2-14

Yamashita, A., & Yamamoto, M. (2006). Fission Yeast Num1p Is a Cortical Factor Anchoring Dynein and Is Essential for the Horse-Tail Nuclear Movement During Meiotic Prophase. Genetics, 173(3), 1187-1196. doi:10.1534/genetics.105.050062

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Ciliary control of meiotic chromosomal pairing mechanics and germ cell morphogenesis

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