# Organoids simulating the bovine oviduct mediate the embryo–maternal interface via extracellular vesicle-transmitted signaling

## TL;DR

This study uses 3D organoids to simulate the bovine oviduct and produce extracellular vesicles that improve embryo development under stress in a lab setting.

## Contribution

The study introduces a novel 3D organoid system that produces extracellular vesicles mimicking maternal-embryo communication in vitro.

## Key findings

- 3D oviductal organoids produce extracellular vesicles (oEVs) that enhance embryo quality under heat stress.
- oEVs from organoids and in vivo oviduct fluid improve embryonic development and gene expression profiles.
- The study reveals parallels in miRNA packaging between organoid-derived and in vivo-derived oEVs.

## Abstract

Does the implementation of a three-dimensional (3D) organoid model system that stably emulates some key functional, structural, and biological complexities of the oviduct provide a favorable apical environment for the production of extracellular vesicles (EVs) that exert an influence on early embryo development in vitro?

Our findings show that in vitro, epithelium dependably propagates highly differentiated oviductal organoids containing both networks of ciliated and secretory cells capable of producing in vivo-like, cargo-specific oviductal extracellular vesicles (oEVs) with the capacity to improve the quality of in vitro-produced embryos under conditions of heat stress (HS).

Recapitulating the maternal contribution that persists during preimplantation embryonic development in vitro is a substantial scientific challenge due to both technical limitations and the significant gaps in our scientific knowledge concerning the maternal–embryonic cellular and molecular dialogue. As a result of the limited access to suitable model systems and the inability to directly observe this process in vivo, this early stage of embryonic development has often been described as particularly elusive and an enigmatic stage of development. Irrespectively, oEVs have recently been identified as key players in mediating the biological information transfer of the embryo–oviduct interactions, which beneficially contributes to the early development of embryos in vitro.

Over a 2-year period, resected ovaries from intact reproductive tracts (n = 10; a pool of two bovine animals per replicate) containing both complete contralateral and ipsilateral oviducts from assessed stage II, diestrus tracts were processed for the generation of oviductal organoids. Afterward, enriched oEVs from 3D organoids and in vivo-collected oviductal fluid (OF) were co-cultured with bovine presumptive zygotes from Day 1 to Day 3 and continued until the blastocyst stage for further evaluation.

Organoids were characterized by light microscopy, gene expression, immunofluorescence, and 3D reconstruction, as well as histological two-dimensional (2D) cross-sectioning. Enriched oEVs from conditioned organoid culture media and OF were characterized using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Following the establishment of a stable oEV production system, bovine zygotes were divided into five groups [38.5°C Control, 41°C Control, 41°C N-EVs (oEVs derived from organoids cultured under thermoneutral conditions), 41°C S-EVs (oEVs derived from organoids cultured HS conditions), 41°C Ovi-EVs (oEVs collected from diestrus OF)] and cultured until the blastocyst stage. Following the presence or absence of oEVs during Day 1 to Day 3 of in vitro culture, the resulting cleavage and blastocyst developmental rates were recorded. We also conducted co-immunostaining for trophectoderm (CDX2) and inner cell mass (SOX2) pluripotency marker proteins, detected global DNA damage (phospho-γH2A.X), and performed real-time quantitative PCR assays in individual embryos for candidate embryo quality genes CDX2, SOX2, POU5F1, NANOG, and critical stress-regulating genes BAX, BCL-2, PRDX1, SOD1, HSP70, and HSP90. Additionally, the influence of oEVs on the epigenetic landscapes of developing embryos was analyzed through their perturbations to H3K9ac, and competitive marks H3K27ac and H3K27me3, in association to their relative expressions of hallmark DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) among individual embryos.

Here, we employed a 3D culture system to generate oviductal organoids to mimic the maternal environment’s response to HS and for the production of in vivo-like oEVs, which were used to enhance the survival and viability of in vitro-produced embryos under conditions of stress. Interestingly, our findings also effectively demonstrate the first attempt at underpinning emerging parallels in EV-packaged miRNAs released from 3D oviductal organoids, 2D oviductal epithelial cells, and in vivo-collected oEVs persistently present within OF. The aim of this approach sustains a mechanistic alternative in robustly generating physiologically relevant oEVs to improve the current in vitro culture system, which traditionally bypasses the oviduct. This model system also innovatively enhances our knowledge of the EV-mediated, maternal-embryonic communication occurring in vivo.

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This was an in vitro study in which conditions of the organoid cultures may not exactly mirror the in vivo environment in terms of the oviducts’ extracellular matrix and complex vascularization. Additionally, given the polarity of the 3D organoids utilized within this study, the population of enriched oEVs largely represents basolateral secretions versus the conventional apical secretions in vivo.

These results provide an uncharted attempt at recapitulating embryo–maternal nano-communication through the means of oEVs secreted from 3D oviductal organoids cultured ex vivo. Thus, our model establishes a foundation for incorporating oviductal cues that modulate embryonic development in vitro, providing a dynamic system to further investigate mechanisms by which the maternal environment may contribute to the early successes of embryonic development and, offering valuable insights that could facilitate advancements in current in vitro embryo production technologies.

This study was supported by the United States Department of Agriculture through a NIFA-AFRI Predoctoral Fellowship awarded to N.G.M. (Grant Number 2023-67011-40511), as well as funds from the College Research Council, Office of the Vice President for Research at Colorado State University. The authors attest that there are no competing interests that could have influenced the conduct or outcomes of this research.