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Generation of hormone-responsive organoids from fresh and cryopreserved equine endometrium: comparison between domestic and endangered Przewalski’s mares
Riley Thompson, a,b Aime Johnson...
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Endometrium is responsive to signals associated with reproductive cyclicity and pregnancy. Organoids, 3-dimensional cultured cell structures, mimic in vivo tissue structure and function better than other cell culture methods and may improve understanding of endometrial physiology. However, there are no reports of endometrial organoids generated from equine tissues. Our hypothesis was hormonallyresponsive organoids can be generated from fresh and cryopreserved endometrium from domestic (Equus ferus caballus) and Przewalski’s (E. f. przewalskii) mares. Our objective was to compare histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), and gene expression in organoids derived from fresh and frozen-thawed domestic horse and frozen-thawed Przewalski’s horse endometrial tissues. Domestic (n = 11) and Przewalski’s (n = 3) mare endometrial biopsies were collected, bisected, and either cryopreserved in 10% DMSO or dissociated enzymatically. Endometrial gland fragments were embedded in Matrigel and overlaid with DMEM/F12 (cell culture) plus supplements. Organoids (8 wells for each treatment) were cultured through 2 passages (6 days/passage) and then, for the third passage, incubated with no hormones (Control; C), or supplemented with: 1) 1M progesterone (P4 ) for 6 days; 2) 10 nM estradiol-17 (E2 ) for 6 days; 3) E 2 for 2 days then P 4 for 4 days; 4) C for 5 days then 10-5 M oxytocin (OT) for 24 hours; or 5) C for 5 days then 10-6 M OT for 24 hours.
At the end of culture, organoids were analyzed for histology, TEM, IHC, and gene expression. Gene expression was analyzed (R project) and Kruskal-Wallis and Dunn were used as posthocs. Organoids were derived from fresh and frozen-thawed domestic mare and frozen-thawed Przewalski’s mare endometrium with histology and TEM revealing cystic structures of epithelial cells with microvilli and intact secretory apparatus. Expression (IHC) of estrogen receptor- (ER) and progesterone receptor (PR) was restricted to the nuclei and prostaglandin endoperoxide synthase-2 (PTGS2) to the cytoplasm of endometrial cells. Expression of ER and oxytocin receptor (OXTR) decreased (p < 0.05) while PR and prostaglandin E synthase (PGES) increased (p < 0.05) in fresh-derived organoids exposed to certain treatments compared to C. ER, E-cadherin, and PTGS2 expression decreased (p < 0.05) and PGES increased (p < 0.05) in cryopreserved-derived organoids exposed to selected treatments compared to corresponding C. Subspecies comparison revealed an increase (p < 0.05) in OXTR expression in the Przewalski’s horse. This is the first report of equine endometrial organoid generation and long-term culture. This novel method of in vitro equine endometrial culture may lead to development of improved in vitro evaluation of normal reproductive physiology, pathological conditions, and potential therapies for uterine diseases in both domestic and endangered equids.
Keywords: Endometrium, organoid, mare, Przewalski’s, in vitro
This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020. Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT). This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.
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Affiliation of the authors at the time of publication
a Department of Large Animal Clinical Sciences, University of Tennessee, Knoxville, TN
b Center for Species Survival, Smithsonian Conservation Biology Institute, Front Royal, VA
c Auburn University, Auburn, AL;
d University of Kentucky, Lexington, KY
e University of Cambridge, Cambridge, UK;
f The Ohio State University, Columbus, OH
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