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Germline transcriptomics in Zebrafish

Natural selection during the germline stage in animals is poorly understood, but there is accumulating evidence that this may play an important role.

Most studies of selection on animals with diplontic life cycle have been focused on the diploid phase and the haploid phase has been largely neglected. There is growing evidence that the haploid life stage may also be subject to similar selection pressures, which may have profound implications for the fitness of the offspring. An extreme case of sperm mediated sexual conflict resulting in reproductive isolation was recently described in C. elegans (Ting et. al., 2014). Apert syndrome and achondroplasia in humans have been attributed to positive selection during the diploid pre-meiotic stage of spermatogenesis (Qin et el., 2007). The mechanisms of selection are a complex interplay of environmental factors, the phenotype and genotype of the organism (haploid & diploid), the epigenome, the transcriptome and subsequently the proteome.

The current research here at Immler Lab (Evolutionary Biology Centre, Uppsala University) is focused on the consequences of sexual reproduction such as sexual conflict, sexual selection and sperm competition. One aspect of our research investigates the potential effect of selection during the haploid phase in animals using Zebrafish (Danio rerio) as a model organism. Zebrafish sperm was artificially selected for pre-defined phenotypic traits and the offspring were raised under standard conditions. The selected offsprings have better fitness than wild type fish. We are investigating the underlying mechanism for this dramatic effect including sperm behaviour, changes in the sperm genome and transcriptome as well as changes during meiosis.

Cystic spermatogenesis in Zebrafish. A schematic illustration of spermatogenesis showing the most distinct stages in the development and maturation of the sperm. The change in ploidy numbers are indicated in parenthesis. Figure based on Schulz et al., 2010.

Cystic spermatogenesis in Zebrafish. A schematic illustration of spermatogenesis showing the most distinct stages in the development and maturation of the sperm. The change in ploidy numbers are indicated in parenthesis. Figure based on Schulz et al., 2010.

Sperm cells have a unique haploid genome as a consequence of meiotic division. The sperm cells also carry mRNA and non-coding RNAs, and at this point it is unclear whether these have has been carried over from pre-meiotic stage or transcribed from the post-meiotic haploid genome. In humans, mouse and C. elegans, it has been shown that histone proteins, responsible for maintaining the structural integrity of chromatin are replaced by protamines (Oliva and Dixon, 1991) during sperm development to enable higher chromatin condensation. This is suggested to completely shut down transcription. In contrast, no protamines were found in zebrafish sperm although histone variants and chromatin compaction were identified (Wu et al., 2011). Moreover, a recent study has shown evidence for post-meiotically transcribed nascent RNA in Drosophila spermatocytes (Vibranovski et. al., 2010).  Other studies show that mature sperm cells retain mRNAs and non-coding RNAs as well as proteins, which may be vital for fertilization and early embryonic development (Boerke et al., 2007; Hosken et al., 2014; Krawetz, 2005). The role of RNA in sperm may be linked to traits inherited transgenerationally (Gapp et al., 2014).

Developmental cell types during spermatogenesis is isolated using fluorescence-activated cell sorting (FACS) and micromanipulation. Total RNA is isolated from single cells or pooled samples, which will be used in RNA-Seq or qPCR. RNA-Seq is used to target the whole range of RNA species in the cell including coding mRNAs, regulatory non-coding RNAs such as microRNAs as well as to catalog novel transcripts and isoforms.

References

Boerke, a, Dieleman, S. J., & Gadella, B. M. (2007). A possible role for sperm RNA in early embryo development. Theriogenology, 68 Suppl 1, S147–55. doi:10.1016/j.theriogenology.2007.05.058
Gapp, K., Jawaid, A., Sarkies, P., Bohacek, J., Pelczar, P., Prados, J., Farinelli L., Miska, E. & Mansuy, I. M. (2014). Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nature Neuroscience, 17(5), 667–9. doi:10.1038/nn.3695
Hosken, D. J., & Hodgson, D. J. (2014). Why do sperm carry RNA? Relatedness, conflict, and control. Trends in Ecology & Evolution, 29(8), 451–455. doi:10.1016/j.tree.2014.05.006
Krawetz, S. a. (2005). Paternal contribution: new insights and future challenges. Nature Reviews. Genetics, 6(8), 633–42. doi:10.1038/nrg1654
Oliva R, Dixon GH (1991) Vertebrate protamine genes and the histone-to-protamine replacement reaction. Prog Nucleic Acid Res Mol Biol 40:25–94
Qin, J., Calabrese, P., Tiemann-Boege, I., Shinde, D. N., Yoon, S. R., Gelfand, D., … Arnheim, N. (2007). The molecular anatomy of spontaneous germline mutations in human testes. PLoS Biology, 5(9), 1912–1922. doi:10.1371/journal.pbio.0050224
Schulz, R. W., de França, L. R., Lareyre, J.-J., Le Gac, F., LeGac, F., Chiarini-Garcia, H., … Miura, T. (2010). Spermatogenesis in fish. General and Comparative Endocrinology, 165(3), 390–411. doi:10.1016/j.ygcen.2009.02.013
Ting, J. J., Woodruff, G. C., Leung, G., Shin, N.-R., Cutter, A. D., & Haag, E. S. (2014). Intense Sperm-Mediated Sexual Conflict Promotes Reproductive Isolation in Caenorhabditis Nematodes. PLoS Biology, 12(7), e1001915. doi:10.1371/journal.pbio.1001915
Vibranovski, M. D., Chalopin, D. S., Lopes, H. F., Long, M., & Karr, T. L. (2010). Direct evidence for postmeiotic transcription during Drosophila melanogaster spermatogenesis. Genetics, 186(1), 431–3. doi:10.1534/genetics.110.118919
Wu, S., Zhang, H., & Cairns, B. R. (2011). Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm. Genome Research, 21(4), 578–89. doi:10.1101/gr.113167.110

 

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