Oviductal Epithelial Cells Research Articles

Investigation of seasonal changes in lipid synthesis and metabolism-related genes in the oviduct of Chinese brown frog (Rana dybowskii).

A peculiar physiological characteristic of the Chinese brown frog (Rana dybowskii) is that its oviduct dilates during pre-brumation rather than during the breeding season. This research aimed to examine the expression of genes connected with lipid synthesis and metabolism in the oviduct of R. dybowskii during both the breeding season and pre-brumation. We observed significant changes in the weight and size of the oviduct between the breeding season and pre-brumation. Furthermore, compared to the breeding season, pre-brumation exhibited significantly lower triglyceride content and a marked increase in free fatty acid content. Immunohistochemical results revealed the spatial distribution of triglyceride synthase (Dgat1), triglyceride hydrolase (Lpl and Hsl), fatty acid synthase (Fasn), and fatty acid oxidases (Cpt1a, Acadl, and Hadh) in oviductal glandular cells and epithelial cells during both the breeding season and pre-brumation. While the mRNA levels of triglycerides and free fatty acid synthesis genes (dgat1 and fasn) did not show a significant difference between the breeding season and pre-brumation, the mRNA levels of genes involved in triglycerides and free fatty acid metabolism (lpl, cpt1a, acadl, acox and hadh) were considerably higher during pre-brumation. Furthermore, the R. dybowskii oviduct's transcriptomic and metabolomic data confirmed differential expression of genes and metabolites enriched in lipid metabolism signaling pathways during both the breeding season and pre-brumation. Overall, these results suggest that alterations in lipid synthesis and metabolism during pre-brumation may potentially influence the expanding size of the oviduct, contributing to the successful overwintering of R. dybowskii.

88 Co-culture with bovine oviduct epithelial cells during in vitro culture affects the bioenergetic profile of bovine expanded blastocysts

88 Co-culture with bovine oviduct epithelial cells during in vitro culture affects the bioenergetic profile of bovine expanded blastocysts

Metabolic stressful environment drives epigenetic modifications in oviduct epithelial cells

The oviduct provides a suitable microenvironment from the gametes’ final maturation until initial embryo development. Dynamic functional changes are observed in the oviduct cells, mainly controlled by steroid hormones and well-orchestrated during the estrous cycle. However, based on the roles played by the oviduct, additional layers of complexity might be present in its regulatory process. There is a cellular process that includes metabolic adaptation that can guide molecular modifications. This process is known as metaboloepigenetics. Therefore, we aimed to better understand how this crosstalk occurs in oviductal epithelial cells (OEC). Due to limited in situ access to the oviduct, we used the primary in vitro cell culture as a culture model and glucose as a metabolic disturbed factor. For that, cells derived from the oviductal epithelial layer were collected from cows at either follicular or luteal stages (n = 4 animals per group). They were cultured on a monolayer culture system under normoglycemic (2.7 mM glucose) or hyperglycemic conditions (27 mM glucose). On day five of culture, attached cells were submitted to analysis of mitochondrial metabolism (mitochondrial membrane potential - MMP) and epigenetics markers (5- methylcytosine - 5 mC and histone 3 lysine 9 acetylation - H3K9ac). Moreover, the culture media were submitted to the metabolites analysis profile by Raman spectrometry. Data were analyzed considering the effect of glucose level (normoglycemic vs. hyperglycemic), stages when OEC were harvested (follicular vs. luteal), and their interaction (glucose level * cycle stage) by two-way ANOVA. As a result, the high glucose level decreased the H3K9ac and MMP levels but did not affect the 5 mC. Regardless of the metabolic profile of the culture media, the glucose level was the only factor that changed the Raman shifts abundance. Although this present study evaluated oviductal epithelial cells after being submitted to an in vitro monolayer culture system, which is known to lead to cell dedifferentiation, yet, these results provide evidence of a relationship between epigenetic reprogramming and energy metabolism under these cell culture conditions. In conclusion, the levels of metabolites in culture media may be crucial for cellular function and differentiation, meaning that it should be considered in studies culturing oviductal cells.

Abstract C038: BRCA2 haploinsufficiency driven neoplastic events in the fallopian tube epithelia

Abstract One in 70 women will develop ovarian cancer over their lifetime in the US and epithelial ovarian cancer (EOC) remains the deadliest gynecological cancer. EOC is a heterogenous disease and high-grade serous carcinoma (HGSOC) is the most common histotype. It is genetically unstable due to pathogenic variants in BRCA1, BRCA2, PALB2, ATM, and RAD51 genes involved in DNA damage response and repair which are part of the hereditary breast and ovarian cancer syndrome (HBOC). In women with a BRCA1 or BRCA2 mutation undergoing risk-reducing surgery, 3-17% have pre-neoplastic and neoplastic lesions in their fallopian tubes. These lesions, known as serous tubal intra-epithelial carcinoma (STIC), display differential loss of heterozygosity of BRCA1/2. New insights into BRCA2 function could provide novel opportunities for preventative interventions in individuals with this mutation, and perhaps new treatment opportunities for HGSC associated with BRCA2 heterozygosity. We have identified distinct metabolic and mitochondrial deficits in BRCA2 mutation carriers compared to BRCA1 carriers and non-carriers. Fallopian tube epithelial cell lines derived from BRCA2 mutation carriers showed increased levels of mitochondrial respiration and activity followed by BRCA1 mutation carriers and non-carriers. Transmission electron microscopy of BRCA2 mutation carriers showed increased disrupted mitochondrial networks when compared to wild type, suggesting that mutation status may contribute to increased mitochondrial fission. These observations reveal an entirely new aspect of BRCA2 biology and further studies will disaggregate the effects of BRCA2 on mitochondrial function, versus DNA damage repair. Ultimately, this work will provide new insight into the adaptive changes within FT cells which promote neoplastic transformation and inform how high-grade serous carcinoma (HGSC) cells distinctively respond to microenvironmental stimuli. Citation Format: Jovanka Ravix, Iru Paudel, Ramlogan Sowamber, Gauthami Pulivendala, David Lombard, Sophia George. BRCA2 haploinsufficiency driven neoplastic events in the fallopian tube epithelia [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C038.

Mutant p53 murine oviductal epithelial cells induce progression of high-grade serous carcinoma and are most sensitive to simvastatin therapy in vitro and in vivo

High-grade serous carcinoma (HGSC) is the most common and aggressive subtype of epithelial ovarian cancer, characterized by gain-of-function TP53 mutations originating in the fallopian tube epithelium. Therapeutic intervention occurs at advanced metastatic disease, due to challenges in early-stage diagnosis, with common disease recurrence and therapy resistance despite initial therapy success. The mevalonate pathway is exploited by many cancers and is potently inhibited by statin drugs. Statins have shown anti-cancer activity in many, but not all cancers. Here, we investigated the role of p53 status in relation to mevalonate pathway signaling in murine oviductal epithelial (OVE) cells and identified OVE cell sensitivity to statin inhibition. We found that p53R175H mutant and Trp53 knockout OVE cells have increased mevalonate pathway signaling compared to p53 wild-type OVE cells. Through orthotopic implantation to replicate the fallopian tube origin of HGSC, p53R175H mutant cells upregulated the mevalonate pathway to drive progression to advanced-stage ovarian cancer, and simvastatin treatment abrogated this effect. Additionally, simvastatin was more efficacious at inhibiting cell metabolic activity in OVE cells than atorvastatin, rosuvastatin and pravastatin. In vitro, simvastatin demonstrated potent effects on cell proliferation, apoptosis, invasion and migration in OVE cells regardless of p53 status. In vivo, simvastatin induced ovarian cancer disease regression through decreased primary ovarian tumor weight and increased apoptosis. Simvastatin also significantly increased cytoplasmic localization of HMG-CoA reductase in ovarian tumors. Downstream of the mevalonate pathway, simvastatin had no effect on YAP or small GTPase activity. This study suggests that simvastatin can induce anti-tumor effects and could be an important inhibitor of ovarian cancer progression.

Protein profiles in the transfected oviductal secreting cells of laying hen (Gallus gallus domesticus)

Due to the intensive development of novel biopharming applications, there is a need for the in vitro verification models prior to in vivo testing. Laying hen has been already applied as an animal bioreactor to produce the therapeutical enzyme in a rare disease called lysosomal acid lipase deficiency. In this study, we aimed to verify how the proteome of the transfected oviduct epithelial cells would be affected by genetic nonviral modification with the human exogene. The study was based on a previously developed method to cultivate chicken oviduct epithelial cells (COEC). The typical characteristics of the COEC epithelial cells were retained across the experiments. The mean efficiency of nucleofection ranged from 2.6 to 19.7% depending on the cells' isolation and location in the oviduct (upper, infundibulum site, or magnum). The PCR confirmed the incorporation of human interferon alpha2a (hIFNα2a) exogene into the nucleofected COEC but, the production of hIFNα2a protein did not exceed the detection level in this study. The ovalbumin protein was detected in the nontransfected and transfected COEC, which confirmed the normal secreting functions of the cells subject to modification. Proteomic analysis revealed an increase in abundance of the cell adhesion molecules and collagen molecules after introducing gene under ovalbumin promoter. According to the bioinformatic analyses there was a limited negative impact of transfection on cells, and the normal biochemical pathways were not severely disordered. In conclusion, the observations provide new knowledge about the proteomic profile of the manipulated COEC with regard to the retained normal functionality of the cells, which can be informative for avian biopharma research.

Assessment of Fallopian Tube Epithelium Features Derived from Induced Pluripotent Stem Cells of Both Fallopian Tube and Skin Origins.

Fallopian tube epithelial cells (FTECs) play a significant role in the development of high-grade serous ovarian cancer (HGSOC), but their utilization in in vitro experiments presents challenges. To address these limitations, induced pluripotent stem cells (iPSCs) have been employed as a potential solution, driven by the hypothesis that orthologous iPSCs may offer superior differentiation capabilities compared with their non-orthologous counterparts. Our objective was to generate iPSCs from FTECs, referred to as FTEC-iPSCs, and compare their differentiation potential with iPSCs derived from skin keratinocytes (NHEK). By introducing a four-factor Sendai virus transduction system, we successfully derived iPSCs from FTECs. To assess the differentiation capacity of iPSCs, we utilized embryoid body formation, revealing positive immunohistochemical staining for markers representing the three germ layers. In vivo tumorigenesis evaluation further validated the pluripotency of iPSCs, as evidenced by the formation of tumors in immunodeficient mice, with histological analysis confirming the presence of tissues from all three germ layers. Quantitative polymerase chain reaction (qPCR) analysis illuminated a sequential shift in gene expression, encompassing pluripotent, mesodermal, and intermediate mesoderm-related genes, during the iPSC differentiation process into FTECs. Notably, the introduction of WNT3A following intermediate mesoderm differentiation steered the cells toward a FTEC phenotype, supported by the expression of FTEC-related markers and the formation of tubule-like structures. In specific culture conditions, the expression of FTEC-related genes was comparable in FTECs derived from FTEC-iPSCs compared with those derived from NHEK-iPSCs. To conclude, our study successfully generated iPSCs from FTECs, demonstrating their capacity for FTEC differentiation. Furthermore, iPSCs originating from orthologous cell sources exhibited comparable differentiation capabilities. These findings hold promise for using iPSCs in modeling and investigating diseases associated with these specific cell types.

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, 17β-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.

Advancing bovine in vitro fertilization through 3D printing: the effect of the 3D printed materials.

Nowadays there is an increasing demand for assisted reproductive technologies due to the growth of infertility problems. Naturally, fertilization occurs in the oviduct, where the oviductal epithelial cells (OECs) secrete many molecules that affect the embryo's metabolism and protect it from oxidative stress. When the OECs are grown in 3D culture systems, they maintain a great part of their functional characteristics, making them an excellent model for in vitro fertilization (IVF) studies. In this work, we aimed to evaluate the suitability of different 3D-printing processes in conjunction with the corresponding set of commercially available biomaterials: extrusion-based processing using polylactic acid (PLA) and polycaprolactone (PCL) and stereolithography or digital-light processing using polyethylene-glycol-diacrylate (PEGDA) with different stiffness (PEGDA500, PEGDA200, PEGDA PhotoInk). All the 3D-printed scaffolds were used to support IVF process in a bovine embryo assay. Following fertilization, embryo development and quality were assessed in terms of cleavage, blastocyst rate at days 7 and 8, total cell number (TCN), inner cell mass/trophectoderm ratio (ICN/TE), and apoptotic cell ratio (ACR). We found a detrimental effect on cleavage and blastocyst rates when the IVF was performed on any medium conditioned by most of the materials available for digital-light processing (PEGDA200, PEGDA500). The observed negative effect could be possibly due to some leaked compound used to print and stabilize the scaffolds, which was not so evident however with PEGDA PhotoInk. On the other hand, all the extrusion-based processable materials did not cause any detrimental effect on cleavage or blastocyst rates. The principal component analysis reveals that embryos produced in presence of 3D-printed scaffolds produced via extrusion exhibit the highest similarity with the control embryos considering cleavage, blastocyst rates, TCN, ICN/TE and ACR per embryo. Conversely, all the photo-cross linkable materials or medium conditioned by PLA, lead to the highest dissimilarities. Since the use of PCL scaffolds, as well as its conditioned medium, bring to embryos that are more similar to the control group. Our results suggest that extrusion-based 3D printing of PCL could be the best option to be used for new IVF devices, possibly including the support of OECs, to enhance bovine embryo development.

Replication stress and defective checkpoints make fallopian tube epithelial cells putative drivers of high-grade serous ovarian cancer

Clinical and molecular evidence indicates that high-grade serous ovarian cancer (HGSOC) primarily originates from the fallopian tube, not the ovarian surface. However, the reasons for this preference remain unclear. Our study highlights significant differences between fallopian tube epithelial (FTE) and ovarian surface epithelial (OSE) cells, providing the molecular basis for FTEs as site of origin of HGSOC. FTEs, unlike OSEs, exhibit heightened replication stress (RS), impaired repair of stalled forks, ineffective G2/M checkpoint, and increased tumorigenicity. BRCA1 heterozygosity exacerbates these defects, resulting in RS suppression haploinsufficiency and an aggressive tumor phenotype. Examination of human and mouse sections reveals buildup of the RS marker 53BP1 primarily in the fallopian tubes, particularly at the fimbrial ends. Furthermore, menopausal status influences RS levels. Our study provides a mechanistic rationale for FTE as the site of origin for HGSOC, investigates the impact of BRCA1 heterozygosity, and lays the groundwork for targeting early HGSOC drivers.

Electroporation-based Easi-CRISPR yields biallelic insertions of EGFP-HiBiT cassette in immortalized chicken oviduct epithelial cells

Laying hens are an excellent experimental oviduct model for studying reproduction biology. Because chicken oviduct epithelial cells (cOECs) have a crucial role in synthesizing and secreting ovalbumin, laying hens have been regarded an ideal bioreactor for producing pharmaceuticals in egg white through transgene or gene editing of the ovalbumin (OVA) gene. However, related studies in cOECs are largely limited because of the lack of immortalized model cells. In addition, the editing efficiency of conventional CRISPR-HDR knock-in in chicken cells is suboptimal (ranging from 1 to 10%) and remains elevated. Here, primary cOECs were isolated from young laying hens, then infected with a retrovirus vector of human telomerase reverse transcriptase (hTERT), and immortalized cOECs were established. Subsequently, an electroporation-based Easi-CRISPR(Efficient additions with ssDNA inserts-CRISPR) method was adopted to integrate an EGFP-HiBiT cassette into the chicken OVA locus (immediately upstream of the stop codon). The immortalized cOECs reflected the self-renewal capability and phenotype of oviduct epithelial cells. This is because these cells not only maintained stable proliferation and normal karyotype and had no potential for malignant transformation, but also expressed oviduct markers and an epithelial marker and had a morphology similar to that of primary cOECs. EGFP expression was detected in the edited cells through microscopy, flow cytometry, and HiBiT/Western blotting. The EGFP-HiBiT knock-in efficiency reached 27.9% after a single round of electroporation, which was determined through genotyping and DNA sequencing. Two single cell clones contained biallelic insertions of EGFP-HiBiT donor cassettes. In conclusion, our established immortalized cOECs could act as an in vitro cell model for gene editing in chicken, and this electroporation-based Easi-CRISPR strategy will contribute to the generation of avian bioreactors and other gene-edited (GE) birds.

A putative role for ALDH inhibitors and chemoprevention of BRCA-mutation-driven tumors

Aldehyde dehydrogenase (ALDH) enzymatic activity is a marker of cancer-initiating cells (CIC) in many tumor types. Our group and others have found that ALDH1A family inhibitors (ALDHi) can preferentially induce death of ovarian CIC in established ovarian cancer. We sought to determine if ALDHi, by targeting CIC at the time of tumor initiation, could function as a chemopreventive for ovarian cancer. As BRCA1/2 mutation carriers represent a population who could benefit from an ovarian cancer chemopreventive, we focused on BRCA mutation-associated tumor cell lines and murine tumor models. We found that, compared to BRCA wild-type cells, BRCA mutant ovarian cancer cells are more sensitive to the ALDHi673A. Similarly, while 673A treatment of wild-type fallopian tube epithelial (FTE) cells is non-toxic, 673A induces death in FTE cells with BRCA1 knockdown. Using a murine fallopian tube organoid model of ovarian carcinogenesis, we show that 673A reduced organoid complexity and significantly reduce colony formation of BRCA-mutant cells. Organoids that persisted after 673A treatment were predominantly BRCA1wt, but NF1 mutant, suggesting a resistance mechanism. Finally, using the BPRN (Brca1, Trp53, Rb1, Nf1 inactivated) mouse model of tubo-ovarian cancer, we evaluated the impact of intermittent 673A therapy on carcinogenesis. 673A treatment resulted in a significant reduction in serous tubal intraepithelial carcinoma (STIC) lesions and carcinomas. Collectively, the findings suggest that ALDHi, such as 673A, could serve as chemopreventive agents for BRCA1/2 mutation carriers.

HEATR3 involved in the cell proliferation, metastasis and cell cycle development of bladder cancer acts as a tumor suppressor.

The study was designed to detect the expression and clinical significance of the HEATR3 gene in bladder cancer (BCa) and to preliminarily explore whether this gene can affect the occurrence and development of BCa through the AKT/ERK signaling pathway. The expression and prognostic value of HEATR3 were explored based on The Cancer Genome Atlas (TCGA) and Genotypic Tissue Expression (GTEx) databases. Microarray immunohistochemical analysis was performed in 30 BCa cases to investigate the level of HEATR3 protein and to explore the relationship between HEATR3 and the clinicopathological features of BCa. Western Blot and qRT-PCR were used to detect HEATR3 protein and mRNA in BCa cell lines (5637, TCCSUP, SW780) and fallopian tube epithelial cell (SV-HUC-1). CCK8 method was employed to study the proliferation of BCa cells after heat treatment. Transwell assay was conducted to analyze the effect of HEATR3 on cell migration and invasion. And cell cycle and apoptosis were detected by flow cytometry. Furthermore, Western Blot assay was used to probe the effects of down-regulation of HEATR3 expression on the expression and phosphorylation levels of AKT and ERK proteins in BCa cells. Bioinformatics analysis showed that HEATR3 was significantly up-regulated in BCa, and high HEATR3 expression was associated with poor prognosis of BCa patients. In vitro experiments demonstrated that HEATR3 expression was up-regulated in BCa tissues compared with that in adjacent tissues. HEATR3 protein was also up-regulated in malignant cell lines. HEATR3 knockdown in BCa cells could inhibit cell proliferation, invasion and migration, block cell cycle and promote cell apoptosis. At the same time, HEATR3 knockdowns reduced the expression levels of p-AKT and p-ERK proteins. HEATR3 knockdown inhibits the development of BCa cells through the AKT/ERK signaling pathway. and it may become one of the most promising molecular targets for BCa treatment.

Effects of the Overexpression of Progesterone Receptors on a Precancer p53 and Rb-Defective Human Fallopian Tube Epithelial Cell Line.

This study investigated the effects of progesterone receptors A (PRA) and B (PRB) on proliferation, migration, invasion, anchorage-independent growth (AIG), and apoptosis of FE25 cells, a precancer p53- and retinoblastoma-defective human fallopian tube epithelial cell line. We observed that the transfection of PRA (FE25-PRA) or PRB (FE25-PRB) into FE25 cells significantly increased the expression of PRA or PRB at both RNA and protein levels without affecting cell morphology. The FE25-PRA cells exhibited slower proliferation, whereas FE25-PRB showed faster cell proliferation than the control cells. In contrast, the FE25-PRA cells showed the highest migration and invasion abilities, whereas the FE25-PRB cells showed the lowest migration and invasion abilities. After treatment with progesterone, all cell types showed decreased AIG levels, increased apoptotic rates in Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling assay (TUNEL) staining, and increased levels of apoptotic proteins ascertained based on cleaved caspase-3 levels. The half-maximal inhibitory concentration of carboplatin increased in FE25-PRB cells, but that of paclitaxel remained unchanged. Overall, this study suggests that PRA and PRB have distinct roles in regulating the behavior of FE25 cells, and targeting these receptors could be a potential therapeutic strategy for ovarian cancer treatment. If PRA or PRB overexpression is observed in high-grade serous carcinoma, progesterone could be considered as an adjuvant therapy for these specific cancer patients. However, further research is needed to confirm these findings and investigate the mechanisms underlying these effects.

Dysregulation of TMEM16A impairs oviductal transport of embryos.

Ectopic pregnancy is an acute abdominalgia in obstetrics and gynecology, especially in fallopian tubal pregnancy. The ion channel protein transmembrane protein 16A (TMEM16A) is widely distributed in various tissues, even in the oviduct. In this study, we showed that TMEM16A was expressed in the human fallopian tube and was upregulated in patients with tubal pregnancy. By measuring isolated fallopian tube tissues, we found that TMEM16A was involved in regulating not only the contraction of muscle strips but also the beat frequency of cilia. In addition, pharmacological activation or inhibition of TMEM16A could lead to retention of embryos in oviducts. Moreover, the embryos in oviducts were delayed in development and some of them had malformations and deletions. The total number of embryos in the oviducts and uterus was significantly less than that of the control group. Furthermore, we detected changes in the level of m6A methylation, where the relevant writers and readers were reduced in tubal tissues from tubal pregnancies. In m6A mRNA methylation, writers catalyze the addition of methyl groups to cytosine residues and readers bind to the methyl groups and affect gene translation. In human fallopian tube epithelial cell line FTE187, we found that interference with methyltransferase 3 (METTL3) expression increased TMEM16A, suggesting that TMEM16A might be regulated by m6A methylation. In general, our study revealed a novel regulatory point for embryo transport and development, introducing a new role for the diagnosis and treatment of tubal pregnancy.NEW & NOTEWORTHY The ion channel protein TMEM16A is expressed in the epithelium and smooth muscle of the human fallopian tube and is upregulated in patients with tubal pregnancy. TMEM16A is involved in regulating the smooth muscle contraction and the cilia beating. Dysregulated TMEM16A may result in embryo retention in the oviduct and delayed early embryo development. Our study reveals a new regulatory point for embryo transport and development.

An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species†.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on oviduct epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLeX), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we found that binding to suLeX diminishes the abundance of 4-hydroxybenzoic acid, the precursor to ubiquinone (also known as Coenzyme Q), 30min after addition. Ubiquinone functions as an electron acceptor in the electron transport chain (ETC). 3-O-sulfated Lewis X trisaccharide also suppressed the formation of fumarate. A component of the citric acid cycle, fumarate is synthesized by succinate-coenzyme Q reductase, which employs ubiquinone and is also known as Complex II in the ETC. Consistent with the reduced activity of the ETC, the production of harmful reactive oxygen species (ROS) was diminished. The enhanced sperm lifespan in the oviduct may be because of suppressed ROS production because high ROS concentrations have toxic effects on sperm.

Effects of extracellular vesicles derived from steroids-primed oviductal epithelial cells on porcine in vitro embryonic development

Extracellular vesicles (EVs) play an active role in regulating different physiological events, however, endocrine control of EVs cargo contents remain poorly understood. In this study, we aimed to isolate EVs from the porcine oviductal epithelial cells (POECs) that were primed with steroid hormones including estradiol (E2) and progesterone (P4), mimicking the in vivo conditions of the reproductive cycle and studied their effects on in vitro produced embryonic development. For this purpose, POECs were treated either with 0 concentration (control) or two different combinations of E2 and P4 including 50 pg/mL E2 + 0.5 ng/mL P4 (group H1), and 10 pg/mL E2 + 35 ng/mL P4 (group H2). Embryos were prepared after in vitro maturation either by parthenogenetic activation or somatic cell nuclear transfer (SCNT) technique. Treating parthenogenetic embryo with EVs, led a significantly higher rate of the blastocyst formation in the group supplemented with each EVs, compared to the control group. In addition, TUNEL assay and gene expression level analysis revealed that apoptosis was significantly reduced in the H2 EVs group. Furthermore, EVs from hormone-primed POECs improved the formation rate of porcine SCNT embryos compared to the control group. While in each EVs supplemented group (control EVs, H1 EVs, H2 EVs), the expression of cell reprogramming-related genes in cloned embryos showed a tendency of increase, the effect was stronger in H1 EVs and H2 EVs. In conclusion, EVs derived from POECs cultured in hormonal conditions simulating the in vivo environment had a positive effect on porcine blastocysts formation, which will likely facilitate in the production of cloned embryos.

Ovariole-specific Yellow-g and Yellow-g2 proteins are required for fecundity and egg chorion rigidity in the red flour beetle, Tribolium castaneum

Most insects reproduce by laying eggs that have an eggshell/chorion secreted by follicle cells, which serves as a protective barrier for developing embryos. Thus, eggshell formation is vital for reproduction. Insect yellow family genes encode for secreted extracellular proteins that perform different, context-dependent functions in different tissues at various stages of development involving, for example, cuticle/eggshell coloration and morphology, molting, courtship behavior and embryo hatching. In this study we investigated the function of two of this family's genes, yellow-g (TcY-g) and yellow-g2 (TcY-g2), on the formation and morphology of the eggshell of the red flour beetle, Tribolium castaneum. Real-time PCR analysis revealed that both TcY-g and TcY-g2 were specifically expressed in the ovarioles of adult females. Loss of function produced by injection of double-stranded RNA (dsRNA) for either TcY-g or TcY-g2 gene resulted in failure of oviposition. There was no effect on maternal survival. Ovaries dissected from those dsRNA-treated females exhibited ovarioles containing not only developing oocytes but also mature eggs in their egg chambers. However, the ovulated eggs were collapsed and ruptured, resulting in swollen lateral oviducts and calyxes. TEM analysis showed that lateral oviducts were filled with electron-dense material, presumably from some cellular content leakage out of the collapsed eggs. In addition, morphological abnormalities in lateral oviduct epithelial cells and the tubular muscle sheath were evident. These results support the hypothesis that both TcY-g and TcY-g2 proteins are required for maintaining the rigidity and integrity of the chorion, which is critical for resistance to mechanical stress and/or rehydration during ovulation and egg activation in the oviducts of T. castaneum. Because Yellow-g and Yellow-g2 are highly conserved among insect species, both genes are potential targets for development of gene-based insect pest population control methods.

P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct to investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid model was established from either primary human oviductal tissues or immortalized human OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir in oviduct is formed to maintain the fertilizing capacity of spermatozoa until ovulation by the binding of sperm to the epithelial lining of the oviduct, which is also significant to select sperm with normal chromatin structure and superior fertilization ability. The regulation and mechanism of sperm reservoir formation in human is still unknown, mainly due to the ethical issue and technical difficulty in obtaining enough human oviduct. We previously demonstrated that sperm fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein on spermatozoa, is responsible for spermatozoa-oviduct interaction in humans. Study design, size, duration Human oviductal epithelial cell line immortalized by HPV 16 E6/E7 open reading frame by retroviral expression system was employed to establish the co-culture model. Fresh human samples (human spermatozoa,N = 30; 1-2 cm primary oviductal tissue,N = 15) were collected in hospital with signed consent. Fluorescent pre-labelled sperm were co-cultured with organoids for 2 hours. At least three replicates were conducted for each assay. Participants/materials, setting, methods Morphological characterization and markers expressions of the organoid were determined by immunohistochemistry and confocal microscopy analysis. The sperm binding capacity of the oviductal organoid was studied by co-culture system. Sperm chromatin structure of the organoid-bound sperm was assessed by flow cytometry-based TUNEL assay. Affinity chromatography followed by nano-liquid chromatography–mass spectrometry was used to identify the sFUT5-binding proteins on ovidutal epithelial cells (OECs) and their expressions on OECs were validated by immunostaining/flow cytometry. Main results and the role of chance The organoid exhibited a hollow structure surrounded by a single layer of OECs. Immunohistochemistry analysis revealed the high expressions of epithelial marker E-cadherin (basolateral membrane), secretory marker pair-box 8 (PAX8) and proliferative marker Ki67. Expression of ciliated cell marker, acetylated tubulin (AcTUB) can also be detected. The modification of culture environment successfully reversed the OEC organoids polarity as demonstrated by immunostaining using antibodies against ZO-1 (apical marker) and E-cadherin (basolateral marker), making the organoid accessible during the co-culture. These apical-out OEC organoids have significantly higher sperm binding capacities than their basal-out counterparts. Furthermore, OEC organoid-bound spermatozoa possessed more intact DNA than the unbound spermatozoa. The results indicate the relevance of using OEC organoid-spermatozoa co-culture model as a tool to assess spermatozoa-oviduct interaction in humans. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was then identified as a potential sFUT5-interacting protein on OECs. This result was further supported by flow cytometry and immunofluorescent staining. 3-D oviductal organoid model demonstrates profound potential for in vitro investigation of human oviduct physiology. Limitations, reasons for caution Our 3D model may not fully represent the entire cellular complexity and tubular architecture of the oviductal epithelium. Further investigation on the sperm morphology and DNA integrity and analysis on organoid transcriptome will enhance our understanding on oviduct in human reproduction. Wider implications of the findings The results from co-culture may give a clue to future study on whether oviductal organoid can be applied clinically for functional sperm selection in in vitro fertilization. Our apical-out organoid model may also be applied to study tubal ectopic pregnency. Trial registration number not applicable

Effects of Follicular Fluid on Physiological Characteristics and Differentiation of Fallopian Tube Epithelial Cells Implicating for Ovarian Cancer Pathogenesis.

The fallopian tube (FT) is an important reproductive organ in females. Ample evidence suggests that the distal end of FT is the original site of high-grade serous ovarian carcinoma (HGSC). FT may suffer from repeated injury and repair stimulated by follicular fluid (FF); however, this hypothesis has not been examined. In fact, the molecular mechanism of homeostasis, differentiation, and the transformation of fallopian tube epithelial cells (FTECs) resulting from the stimulation of FF are still enigmatic. In this study, we examined the effects of FF along with factors present in the FF on a variety of FTEC models, including primary cell culture, ALI (air-liquid interface) culture, and 3D organ spheroid culture. We found that FF plays a similar role to estrogen in promoting cell differentiation and organoid formation. Moreover, FF significantly promotes cell proliferation and induces cell injury and apoptosis in high concentrations. These observations may help us to investigate the mechanisms of the initiation of HGSC.