Continuity During Embryonic Diapause in the European Roe Deer (Capreolus Capreolus)
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Date
2023
Publication Type
Doctoral Thesis
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yes
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Abstract
Embryonic diapause in mammals describes a transitional reduction of proliferation and developmental progression occurring at the blastocyst stage. Descriptions of this form of delayed implantation occurring in the European roe deer (Capreolus capreolus) date back to the 19th century, and since then diapause has been discovered in over 130 mammalian species across different orders. Hitherto, the roe deer remains the only ungulate species known to display diapause and the molecular mechanisms of diapause in the roe deer are yet to be elucidated.
Successful implantation requires a tight communication between the developing embryo and the endometrium to enable the coordination of maternal and embryonic processes. During diapause, these processes take place at a slower pace allowing a gradual syn- chronization of maternal and embryonic cues.
In the roe deer, only few endometrial changes are known to occur. One of these changes is a gradual reduction of luminar epithelial cell height. Based on similarities to other spe- cies, these changes in cell morphology may be linked to co-occurring modifications of the cytoskeleton as well as the surrounding extracellular matrix (ECM). We employed an im- munohistochemical approach to gain insights into changes in the distribution of ECM components between early, late, and post-diapause uteri. While our findings confirmed the reduction of epithelial cell height, the selected ECM factors displayed only marginal changes in estimated quantity and localization. Major ECM remodelling events are there- fore unlikely to occur prior to implantation.
While the endometrium during diapause appears relatively static, the roe deer blastocyst continuously grows during diapause. It was unclear whether this growth occurred due to an increase in blastocoel volume accompanied by an increase in cell size or due to active proliferation. Furthermore, it remained to be clarified whether this growth was restricted to the trophectoderm (TE) or was accompanied by growth of the inner cell mass (ICM). We assessed the expression of the proliferation marker KI67 by immunofluorescence and nuclear staining and imaged the stained blastocysts using light sheet microscopy. Thereby, we found that the number of total nuclei increased almost 100-fold between September and December. The relative increase of nuclei in the ICM was only slightly lower than in the TE, whereas the fraction of Ki67 positive cells appeared lower in the TE.
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We hypothesize that this could be explained by differences in the duration of individual cell cycle phases, with TE cell having a shorter G1 phase.
In addition to proliferation, we also detected considerable morphological changes, indic- ative of ongoing differentiation and thus development within the ICM. While RNA-Seq data of embryos spanning the period of diapause and elongation was available, the first version of the roe deer transcriptome lacked annotations for a significant proportion of potential developmental marker genes. To assess developmental progression during di- apause in the roe deer, we thus developed an alternative assembly and annotation strat- egy and localized a selection of molecular markers using immunofluorescence. We con- firmed that endoderm formation occurs during diapause, while gastrulation and neurula- tion likely only occur upon elongation. The morphological changes within the ICM were reflected by correlating gene expression patterns, indicating that in the roe deer develop- mental processes are not halted during diapause.
Our studies demonstrate that while the endometrial ECM remains unchanged, the em- bryo slowly continues to proliferate and to develop. The elements that regulate this drastic reduction of developmental pace and whether the embryonic development indeed occurs fully independent of maternal signalling cues remains to be elucidated.
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published
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Examiner: Ulbrich, Susanne E.
Examiner : Schnieke, Angelika
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ETH Zurich
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Subject
DIAPAUSE (ANIMAL PHYSIOLOGY); EMBRYONIC DEVELOPMENT (ZOOLOGY); Cell cycle and cell division; REPRODUCTION (ANIMAL PHYSIOLOGY); Roe deer; Differentiation processes
Organisational unit
03999 - Ulbrich, Susanne / Ulbrich, Susanne
Notes
Funding
159734 - Embryonic Diapause in roe deer: a model for deciphering the control of developmental velocity (SNF)
185026 - Embryonic Diapause: pluripotency on hold? (SNF)
185026 - Embryonic Diapause: pluripotency on hold? (SNF)