Proteomic analysis and in vivo visualization of extracellular vesicles from mouse oviducts during pre-implantation embryo development.

Pre-implantation embryonic development occurs in the oviduct during the first few days of pregnancy. The presence of oviductal extracellular vesicles (oEVs, also called oviductosomes) is crucial for pre-implantation embryonic development in vivo as oEVs often contain molecular transmitters such as proteins. Therefore, evaluating oEV cargo during early pregnancy could provide insights into factors required for proper early embryonic development that are missing in the current in vitro embryo culture setting. In this study, we isolated oEVs from the oviductal fluid at estrus and different stages of early embryonic development. The 2306-3066 proteins in oEVs identified at the different time points revealed 58-60 common EV markers identified in exosome databases. Oviductal extracellular vesicle proteins from pregnant samples significantly differed from those in non-pregnant samples. In addition, superovulation changes the protein contents in oEVs compared to natural ovulation at estrus. Importantly, we have identified that embryo-protectant proteins such as high-mobility protein group B1 and serine (or cysteine) peptidase inhibitor were only enriched in the presence of embryos. We also visualized the physical interaction of EVs and the zona pellucida of 4- to 8-cell stage embryos using transmission electron microscopy as well as in vivo live imaging of epithelial cell-derived GFP-tagged CD9 mouse model. All protein data in this study are readily available to the scientific community in a searchable format at https://genes.winuthayanon.com/winuthayanon/oviduct_ev_proteins/. In conclusion, we identified oEVs proteins that could be tested to determine whether they can improve embryonic developmental outcomes in vivo and in vitro setting.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17ß-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.

Multi-omics analyses and machine learning prediction of oviductal responses in the presence of gametes and embryos.

The oviduct is the site of fertilization and preimplantation embryo development in mammals. Evidence suggests that gametes alter oviductal gene expression. To delineate the adaptive interactions between the oviduct and gamete/embryo, we performed a multi-omics characterization of oviductal tissues utilizing bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and proteomics collected from distal and proximal at various stages after mating in mice. We observed robust region-specific transcriptional signatures. Specifically, the presence of sperm induces genes involved in pro-inflammatory responses in the proximal region at 0.5 days post-coitus (dpc). Genes involved in inflammatory responses were produced specifically by secretory epithelial cells in the oviduct. At 1.5 and 2.5 dpc, genes involved in pyruvate and glycolysis were enriched in the proximal region, potentially providing metabolic support for developing embryos. Abundant proteins in the oviductal fluid were differentially observed between naturally fertilized and superovulated samples. RNA-seq data were used to identify transcription factors predicted to influence protein abundance in the proteomic data via a novel machine learning model based on transformers of integrating transcriptomics and proteomics data. The transformers identified influential transcription factors and correlated predictive protein expressions in alignment with the in vivo-derived data. In conclusion, our multi-omics characterization and subsequent in vivo confirmation of proteins/RNAs indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner.

Classical Estrogen Signaling in Ciliated Epithelial Cells of the Oviduct Is Nonessential for Fertility in Female Mice.

Ciliary action performs a critical role in the oviduct (Fallopian tube) during pregnancy establishment through sperm and egg transport. The disruption of normal ciliary function in the oviduct affects oocyte pick-up and is a contributing factor to female infertility. Estrogen is an important regulator of ciliary action in the oviduct and promotes ciliogenesis in several species. Global loss of estrogen receptor α (ESR1) leads to infertility. We have previously shown that ESR1 in the oviductal epithelial cell layer is required for female fertility. Here, we assessed the role of estrogen on transcriptional regulation of ciliated epithelial cells of the oviduct using single-cell RNA-sequencing analysis. We observed minor variations in ciliated cell genes in the proximal region (isthmus and uterotubal junction) of the oviduct. However, 17ß-estradiol treatment had little impact on the gene expression profile of ciliated epithelial cells. We also conditionally ablated Esr1 from ciliated epithelial cells of the oviduct (called ciliated Esr1d/d mice). Our studies showed that ciliated Esr1d/d females had fertility rates comparable to control females, did not display any disruptions in preimplantation embryo development or embryo transport to the uterus, and had comparable cilia formation to control females. However, we observed some incomplete deletion of Esr1 in the ciliated epithelial cells, especially in the ampulla region. Nevertheless, our data suggest that ESR1 expression in ciliated cells of the oviduct is dispensable for ciliogenesis and nonessential for female fertility in mice.

Niclosamide targets the dynamic progression of macrophages for the resolution of endometriosis in a mouse model.

Due to the vital roles of macrophages in the pathogenesis of endometriosis, targeting macrophages could be a promising therapeutic direction. Here, we investigated the efficacy of niclosamide for the resolution of a perturbed microenvironment caused by dysregulated macrophages in a mouse model of endometriosis. Single-cell transcriptomic analysis revealed the heterogeneity of macrophages including three intermediate subtypes with sharing characteristics of traditional "small" or "large" peritoneal macrophages (SPMs and LPMs) in the peritoneal cavity. Endometriosis-like lesions (ELL) enhanced the differentiation of recruited macrophages, promoted the replenishment of resident LPMs, and increased the ablation of embryo-derived LPMs, which were stepwise suppressed by niclosamide. In addition, niclosamide restored intercellular communications between macrophages and B cells. Therefore, niclosamide rescued the perturbed microenvironment in endometriosis through its fine regulations on the dynamic progression of macrophages. Validation of similar macrophage pathogenesis in patients will further promote the clinical usage of niclosamide for endometriosis treatment.

Parallel Single-Cell Multiomics Analysis of Neonatal Skin Reveals the Transitional Fibroblast States that Restrict Differentiation into Distinct Fates.

One of the keys to achieving skin regeneration lies within understanding the heterogeneity of neonatal fibroblasts, which support skin regeneration. However, the molecular underpinnings regulating the cellular states and fates of these cells are not fully understood. To investigate this, we performed a parallel multiomics analysis by processing neonatal murine skin for single-cell Assay for Transposase-Accessible Chromatin sequencing and single-cell RNA sequencing separately. Our approach revealed that fibroblast clusters could be sorted into papillary and reticular lineages on the basis of transcriptome profiling, as previously reported. However, single-cell Assay for Transposase-Accessible Chromatin sequencing analysis of neonatal fibroblast lineage markers, such as Dpp4/Cd26, Corin, and Dlk1 along with markers of myofibroblasts, revealed accessible chromatin in all fibroblast populations despite their lineage-specific transcriptome profiles. These results suggest that accessible chromatin does not always translate to gene expression and that many fibroblast lineage markers reflect a fibroblast state, which includes neonatal papillary fibroblasts, reticular fibroblasts, and myofibroblasts. This analysis also provides a possible explanation as to why these marker genes can be promiscuously expressed in different fibroblast populations under different conditions. Our single-cell Assay for Transposase-Accessible Chromatin sequencing analysis also revealed that the functional lineage restriction between dermal papilla and adipocyte fates is regulated by distinct chromatin landscapes. Finally, we have developed a webtool for our multiomics analysis: https://skinregeneration.org/scatacseq-and-scrnaseq-data-from-thompson-et-al-2021-2/.