Background:
Australian marsupials are facing a biodiversity crisis, requiring quick and decisive action to prevent further extinction. Assisted Reproduction Technologies are a promising frontier in species conservation: however, while embryo culture methodologies have been developed for eutherian species, marsupial embryo culture has not yet been optimized. Given marsupial reliance on histotrophic nutrition for the majority of gestation, close examination of uterine fluid contents is necessary for optimizing embryo culture. Using a dasyurid animal model, this research identifies key uterine morphological details, metabolites of interest within the uterine fluid relevant to embryo culture medium composition, and has developed endometrial epithelial organoids as an in vitro model of the highly secretory marsupial uterus.
Methods:
Uteri from fat-tailed dunnarts (Sminthopsis crassicaudata) were collected at pertinent stages of the estrus cycle and across gestation. Micro-CT defined the internal structure of the uterus, and cellular detail was resolved using routine histological techniques. Uterine fluid was collected and metabolites detected by mass spectrometry. Following enzymatic digestion of uterine tissue, organoids were grown from the endometrial epithelium using previously established techniques, and immunohistochemically compared to the donor tissue in to assess cell population purity (E-Cadherin, vimentin), proliferation (PCNA), secretion (PAS), and morphology (H&E).
Results:
The dunnart uterus is highly glandular with a folded luminal surface, and contains a dynamic fluid microenvironment, with several metabolites appearing to be differentially abundant across the estrus cycle. Endometrial epithelial cells can be retrieved and cultured as organoids in previously defined medium. Organoids contain proliferating epithelial cells (PCNA+, E-Cadherin+), and exclude stromal cells (Vimentin-). Similarly to the endometrial glands, organoids accumulate PAS+ secretions.
Conclusion: We have derived the first marsupial endometrial epithelial organoids, from the highly glandular dasyurid uterus. Examination of the uterine fluid and morphology, combined with this in vitro model, provides key insights into the marsupial uterine microenvironment.