Directional alignment of different cell types organizes planar cell polarity.

Oviductal (fallopian tube) cell cultures are widely used to improve understanding of oviductal pathophysiology and to improve sperm and embryo quality via co-culture. One type of oviductal cell culture is organoids, a three-dimensional cell culture that can mimic the oviduct in vitro long-term (months). Because organoids maintain their structure and function similar to in vivo, their secretions tend to be more similar to in vivo cellular products. One of these organoid secretions includes extracellular vesicles (EVs), which are bioactive nanoparticles secreted by cells for intercellular communication. Here we report a protocol for isolation of mouse oviducts, establishment and long-term culture of mouse oviductal organoids, and isolation and characterization of the EVs secreted by mouse oviductal organoids. This lab protocol can be used for research of oviductal physiology and pathology and improvement of assisted reproductive technologies.

In mammalian early pregnancy, maternal immune cells are critical during early pregnancy for initiating embryogenesis and maintaining implantation. However, the preimplantation immune environment within the reproductive tract is largely uncharacterized. Here, we detected a massive influx of immune cells into the lumen of the oviduct and uterus after mating in mice. In immune cells, CD11b+ cells up to about 38.1% in the oviduct and 59.5% in the uterus, of which neutrophils accounted for 81.7% and 97.7%, respectively. Neutrophils presented an antigen-presenting cells like phenotype (nAPCs), a specialized population expressing Ly6G and MHCII. We followed the dynamic changes and fate of neutrophils, and found that these cells rapidly disappeared from the lumen of the reproductive tract from 0.5days post-coitus (dpc) to 1.5 dpc. Instead of apoptosis, they migrated from the oviduct to the uterus and infiltrated the endometrium at 1.0-1.5 dpc. At 3.5 dpc, the number of immune cells decreased and natural killer (NK) cells became the predominant population. The cytokines involved in modulating immune cell responses (GM-CSF, IFN-γ and IL17A), pro-inflammation (IL-1β, IL-6, IL-12p70, TNF-α), and anti-inflammation (IL-10) were detected in luminal fluid, in which GM-CSF and IL-6 decreased, IL-10 increased in 3.5 dpc uterus. However, spontaneous abortion mice (CBA/J × DBA/2) exhibited altered immune patterns at the stage of embryogenesis and implantation. Our findings reveal a dynamic and finely tuned immune state in the lumen of the oviduct and uterus, which provide a microenvironment for early embryo development, and provide insights into pre-implantation embryo-uterine cross-talk in mice.

Abstract Background: While epithelial ovarian cancer (EOC) generally responds to frontline therapy, most tumors develop resistance to current treatments, prompting the need for innovative therapies. Homologous recombination deficient EOC with PARP inhibitor (PARPi) resistance are particularly difficult to treat. Here, we evaluate a novel theranostic approach for the treatment of drug-resistant EOC that leverages overexpression of PARP1 in EOC (even when PARPi resistant). Our platform is based on a small molecule similar to rucaparib labeled with 211At for alpha therapy ([211At]ParaThanatrace, PTT) and 18F for PET/CT ([18F]FluorThanatrace, FTT). Materials and Methods: PTT was synthesized and evaluated for cytotoxicity and binding properties in 16 human and 2 murine EOC cell lines with varying sensitivity to PARPi and chemotherapy. Maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and half minimal effective concentration (EC50) were calculated. PTT efficacy was evaluated in a syngeneic mouse model of mouse oviduct secretory epithelial derived cells (MOSEC) engineered to include loss of Trp53 and Pten. Results: PTT had a 30-fold greater in vitro cytotoxicity than untargeted 211At. Comparing EOC OVCAR8 cells with endogenous PARP1 expression versus OVCAR8-PARP1KO cells, PTT activity was dependent on PARP1 expression (EC50 0.0079 vs. 0.027 MBq/mL, respectively). Human ovarian cancer cell lines had variable sensitivity with a range of EC50’s from 0.0021-0.80 MBq/mL (mean=0.011, median=0.007), concentrations that are safely achievable in vivo. Cytotoxicity was similar between PARPi-sensitive and -resistant cells (P=0.86). The number of PTT binding sites per cell (Bmax) ranged from 6.31x105-2.14x106 and the binding affinity was 2.0-11.4 nM. EC50 values significantly correlated to binding affinity (r=0.69, P=0.009), whereas no correlation was observed with Bmax, PARP1 expression, or tumor cell doubling time. To evaluate the anti-tumor efficacy in ovarian cancer in vivo, homologous proficient MOSECs were inoculated intraperitoneally into immunocompetent C57BL/6 mice. Four days following tumor inoculation; mice were treated intravenously with four fractionated doses of PTT at the MTD (48 MB/kg/fraction) or ½ MTD (24 MB/kg/fraction) over two weeks. PTT treatment was tolerable and significantly prolonged survival compared to untreated control mice (P=0.0027). In vitro cytotoxicity determined an IC50 of 0.97 mCi/mL in MOSEC, which is considerably higher than most of the evaluated human EOC cell lines, further supporting the efficacy of PTT in vivo. Conclusions: PTT cytotoxicity is largely independent of PARPi-sensitivity but reliant on PARPi binding affinity and line-specific radiation sensitivity. In vivo data supports the safety and efficacy of PTT. Overall, this study highlights a novel role for theranostic radiopharmaceutical therapy in drug-resistant EOC. Citation Format: Sarah B. Gitto, Aladdin Riad, Alastair McArthur, Fiona Simpkins, Daniel A. Pryma, Micheal D. Farwell. PARP-targeted alpha therapy for the treatment of PARP inhibitor resistant ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A029.

Introduction. The distribution of micro-organisms in healthy organisms remains a subject of debate. Emerging evidence revealed the colonization of microbial communities in multiple anatomical sites previously considered sterile under homeostatic conditions. However, the mechanistic relationship between compromised intestinal epithelial barrier integrity and subsequent translocation of gut-resident bacteria into systemic circulation has yet to be comprehensively elucidated.Hypothesis/Gap Statement. Under intestinal leakage, gut micro-organisms can break through the intestinal barrier and then translocate to other organs.Aim. This study investigates the distribution of micro-organisms in healthy organisms to determine whether gut bacteria translocate to sterile organs only under the condition of intestinal leakage using GFP-labelled Escherichia coli (GFP-E. coli) tracing.Methodology. Female C57BL/6 mice (5 weeks old) were administered either a glacial acetic acid enema [inflammatory bowel disease (IBD) group] or a sterile normal saline enema [normal control (NC) group]. All mice were subsequently gavaged with GFP-E. coli. HE staining and Alcian blue staining were performed to evaluate the colon injury. The expression levels of intestinal tight junction proteins (ZO-1 and occludin) were tested by reverse transcription quantitative PCR and immunofluorescence staining. The distribution of GFP-E. coli in multiple organs was assessed through bacterial culture, confocal microscopy and PCR.Results. In the IBD mice, mucopolysaccharide accumulation levels (P<0.01) and tight junction proteins ZO-1 (P<0.001) and occludin (P<0.01) in the colon were significantly decreased compared with the NC group. Bacterial culture showed that there was no GFP-E. coli in the blood, heart, liver, spleen, lungs, kidneys or oviducts of normal mice, while the number of GFP-E. coli colonies in the blood (219 c.f.u. ml-1), liver (2.39×105 c.f.u. ml-1) and lungs (2.50×108 c.f.u. ml-1) of the IBD mice was significantly higher than that of the NC group. The confocal microscopy and PCR results also showed that the number of GFP-E. coli in the liver and lungs of the IBD group was significantly higher than that of the NC group (P<0.001).Conclusion. Healthy mice maintain a sterile microenvironment in the blood, heart, liver, spleen, lungs, kidneys and oviducts. However, compromised intestinal barrier integrity facilitates microbial translocation from the intestinal lumen into the blood, liver and lungs. This study advances our understanding of endogenous infections caused by IBD, demonstrating the crucial role of intestinal permeability in bacterial infections.

The periconception period is critical for embryo development, pregnancy outcomes, and offspring health. During this stage, oviductal and uterine fluids facilitate embryo-maternal interactions and support early embryonic development. Using PANDORA-seq, we identify a diverse repertoire of small non-coding RNAs in female mouse oviduct fluid and uterine fluid during preimplantation, with tRNA-derived small RNAs and rRNA-derived small RNAs being predominant. Maternal high-fat diet during preimplantation period significantly alters tsRNA and rsRNA expression in oviduct fluid and uterine fluid compared to normal diet, disrupting blastocyst metabolic gene expression. While implantation remained unaffected, these alterations impair mid-gestation embryonic and placental growth, resulting in reduced birth weight and length, as well as metabolic disorders in offspring. Furthermore, transfecting embryos with uterine fluid-derived sncRNAs altered by maternal high-fat diet mimics the in vivo effects. These findings suggest that tsRNAs and rsRNAs in reproductive fluids may reflect maternal metabolic status and transmit dietary information to the early embryo, which might influence pregnancy outcomes and offspring health.

The mammalian oviduct (also called the fallopian tube) is an essential organ for natural pregnancy. As one of its major functions, the oviduct transports preimplantation embryos to the uterus for implantation. This is a critical process, and abnormalities are responsible for a range of reproductive disorders, such as tubal ectopic pregnancy and infertility, whose etiologies are unclear. For transporting embryos, the oviduct is fundamentally a tubular mechanical pump with motile cilia lining the luminal epithelium and smooth muscle surrounding the mucosa wall. Although bidirectional movement of embryos has been observed during the transport process, how the oviduct produces this type of embryo movement remains unknown. Understanding this pumping mechanism is vital to identifying the functional causes of oviduct-related reproductive disorders, but answering this question requires dynamic imaging of the transport process in its native environment, which is difficult to achieve in mammalian models. Here, we use optical coherence tomography and apply in vivo dynamic 3D imaging of the mouse oviduct to uncover the oviduct pumping mechanism in transporting preimplantation embryos toward pregnancy. By inhibiting the oviduct smooth muscle contraction, we first show that the oviduct muscular activity drives the bidirectional embryo movement. We then present a quantitative assessment of the oviduct contraction wave. This analysis, together with the embryo movement information, indicates that the forward movement of embryos is produced by peristalsis, while the backward embryo movement is generated by a suction process driven by the oviduct relaxation at earlier contraction sites, showing a leaky peristaltic pump. Finally, we reveal how the net displacement of embryos is created under this pumping mechanism, which effectively transports embryos toward the uterus. This work elucidates, for the first time, the oviduct pumping mechanism in transporting preimplantation embryos, paving the way for understanding the biomechanics of the mammalian oviduct.

The female reproductive organs present with the earliest aging characteristics, such as a decline in fertility and estrous cyclicity. While age-related changes in the ovary are well documented, it is unclear if any age-associated changes occur in the other female reproductive organs, such as the oviduct/Fallopian tube. At the distal end of aged oviducts in mice, we found vacuolated multi-ciliated cells (MCCs) with a severely apically displaced and deformed nucleus. This phenotype was unique to the distal oviduct epithelium-the infundibulum (INF) and ampulla (AMP). Ovariectomy did not affect the timeline of MCC vacuolation, suggesting little involvement of ovulation and hormonal regulation. MCC vacuolation was induced in hypoxia or hydroxyurea treatments in invitro organotypic culture of all oviduct regions, not limited to the INF/AMP epithelium. This suggests a high oxygen demand in MCCs, compared to other cell types, and a uniquely stressed INF/AMP epithelial microenvironment invivo. We found that the blood circulation of INF/AMP depended on the ovarian artery, different from the rest of the oviduct epithelium, and its circulation declined along with ovarian activities. We conclude that a decline in local blood circulation and distinct cellular identity of the INF/AMP epithelium caused age-associated MCC vacuolation, reflecting its mild, chronically stressed microenvironment.

Abstract Introduction: Addressing chemo-resistant homologous recombination-proficient (HRP) ovarian cancers and CCNE1-amplified tumors, while minimizing treatment-related side effects, remains a significant clinical challenge. Our research demonstrated that HDAC inhibition suppresses tumors in PARP inhibitor (PARPi)-resistant ovarian cancers and enhances PARPi sensitivity in preclinical models. However, pan-HDAC inhibitors (HDACis) have shown high toxicity in clinical trials. To overcome this, we focused on selectively targeting specific HDACs, particularly HDAC3 and HDAC8, which have unique structures and co-repressor complexes. HDAC3 is frequently upregulated in ovarian cancers, making it a promising therapeutic target. Here, we explore HDAC3 genetic depletion/knockout (KO) and precision targeting using HDAC-PROTACs in five cell lines. Methods: We employed siRNA-mediated depletion and CRISPR-Cas9 KO to study the impact of HDAC3 inhibition on CCNE1-amplified OVCAR3 cell proliferation, clonogenicity, and protein expression. Histone modifications were analyzed via mass spectrometry. The anti-proliferative efficacy of HDAC3 (XZ9002), HDAC8 (YL352), and dual HDAC3/8 (YX968) PROTACs was evaluated in OVCAR3, COV318, HRP and HRD mouse fallopian tube cells, and patient-derived primary cell lines. Results: HDAC3 siRNA treatment in OVCAR3 cells led to dose-dependent reductions in HDAC3 protein levels and PCNA expression. HDAC3-KO cells exhibited increased histone acetylation and decreased histone methylation. PROTACs showed differential efficacy across cell lines. In OVCAR3 cells, IC50 values were 1.82 µM for HDAC3-PROTAC, 1.98 µM for HDAC8-PROTAC, and ∼0.67 µM for HDAC3/8 dual PROTAC, indicating superior potency with dual targeting. In COV318 cells, the dual HDAC3/8 PROTAC exhibited an IC50 of 2.5 µM. HRD mouse fallopian tube cells were more sensitive to HDAC8 and HDAC3/8 PROTACs (IC50 ∼6 µM), while HRP cells were less sensitive (IC50: 8 µM for HDAC3/8 PROTAC, 10 µM for HDAC8 PROTAC, &amp;gt;10 µM for HDAC3 PROTAC). Patient-derived primary cell lines demonstrated the highest sensitivity to the dual HDAC3/8 PROTAC (IC50 ∼7 µM), with reduced response to single HDAC PROTACs. Pharmacodynamic analyses via Western blot and qPCR are ongoing. Conclusion: Selective targeting of HDAC3, HDAC8, and HDAC3/8 using PROTACs provides a promising therapeutic strategy to address toxicity limitations of conventional HDACis. These agents hold potential for low-dose applications in ovarian cancers and broader therapeutic use in other malignancies and diseases. Citation Format: Vijayalaxmi G. Gupta, Bisiayo Fashemi, Reni Akande, Preedia Babu, Yukihide Ota, Yufeng Xaio, Guangrong Zhang, Francisca Nathalia Vitorino, Benjamin Garcia, Mary Mullen, Dineo Khabele. Precision targeting of HDAC3 in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4221.

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. Lastly, we found some differences between inflammatory responses in sperm-exposed mouse oviducts compared to hydrosalpinx Fallopian tubes from patients. 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.

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. Lastly, we found some differences between inflammatory responses in sperm-exposed mouse oviducts compared to hydrosalpinx Fallopian tubes from patients. 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.

ABSTRACTObjective:Superovulation protocol modifies the oviductal site-specific expression of some proteins regulating cell cycle and oxidative stress response.Methods:Swiss CD1 female mice (n=24) were sorted into 2 groups: one was used as control (Ctr, n=10), the other consisted of mice undergoing 8 Rounds (8R) of repeated gonadotropin stimulation (n=14). After their removal, oviducts were cut into two portions: one including Infundibulum and Ampulla (If-Am), and the rest including Isthmus (Is). Both portions were separately used to assess the expression levels of oviductal proteins regulating cell cycle and oxidative stress response. Statistical analysis employed t-test with significance at p<0.05.Results:In Ctr mice, superoxide dismutases 1 and 2 were significantly more expressed in the If-Am, while phospho-p53, glutathione peroxidase 1 and estrogen receptor beta mainly in the Is. Conversely, catalase, cleaved-caspase 3, estrogen alpha and progesterone receptors were similarly distributed across the oviduct. After 8R, glutathione peroxidase 1 and superoxide dismutase 1 increased in both segments, superoxide dismutase 2 and cleaved-caspase 3 increased mainly in If-Am, while catalase and phosphorylated p53 mainly in Is. Estrogen alpha/beta and progesterone receptors levels remained unchanged.Conclusions:Altogether, these results demonstrated that in the mouse oviduct many proteins were expressed in a site-specific manner and that repeated gonadotropin stimulation could modulate their expression levels. These data suggest that different localization of proteins between Infundibulum-Ampulla and Isthmus regions is fundamental for creating a suitable microenvironment for embryo development.

The present study was conducted to investigate the effects of levetiracetam (LEV) at a concentration of 350 mg/kg/day of body weight on oviduct tissue in female albino mice (Mus musculus). The experimental animals were divided into three groups and administered orally once daily for 14, 30, and 45 days. Each treatment group was matched to its own control group. The results of the present study showed that ciliated columnar cells transformed into cuboidal cells, with reduced cilia density, shortened secretory cells, hemorrhage, necrosis, and cell death of the mucosal folds, decreased tissue hyperplasia with the appearance of vacuoles, the presence of inflammatory cells in the lumen, and hyperplasia and hypoplasia of the tunica muscularis when compared to the control group.

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. Lastly, we found some differences between inflammatory responses in sperm-exposed mouse oviducts compared to hydrosalpinx fallopian tubes from patients. 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.

BackgroundThis study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1.MethodsHumanized fragments, consisting of the endothelial cell‐specific K18 promoter, human ST6GAL1‐encoding gene, and luciferase gene, were microinjected into the fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offspring were identified using PCR. Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation, and in vivo analysis was performed for screening. Expression of the humanized gene was tested by performing immunohistochemical (IHC) analysis. Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed.ResultsSuccessful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1. Seven mice were identified as carrying copies of the humanized gene, and the in vivo analysis indicated that hST6GAL1 gene expression in positive mice mirrored influenza virus infection characteristics. The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice. Moreover, the hematologic and biochemical parameters of the positive mice were within the normal range.ConclusionA humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 μg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 μg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 μg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 μg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

A ketogenic diet (KD) elevates blood β-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. A ketogenic diet elevates blood β-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters β-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and β-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and β-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated β-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

Expression and localization of apelin and apelin receptor protein in the oviduct of letrozole-induced hyperandrogenized mice.

The mammalian oviduct (or fallopian tube) is a tubular organ hosting reproductive events leading to pregnancy. Dynamic 3D imaging of the mouse oviduct with optical coherence tomography (OCT) has recently emerged as a promising approach to study the hidden processes vital to elucidate the role of oviduct in mammalian reproduction and reproductive disorders. In particular, with an intravital window, in vivo OCT imaging is a powerful solution to studying how the oviduct transports preimplantation embryos towards the uterus for pregnancy, a long-standing question that is critical for uncovering the functional cause of tubal ectopic pregnancy. However, simultaneously tracking embryo movement and acquiring large-field-of-view images of oviduct activity in 3D has been challenging due to the generally limited volumetric imaging rate of OCT. A lack of OCT-based 3D velocimetry method for large, sparse particles acts as a technical hurdle for analyzing the mechanistic process of the embryo transport. Here, we report a new particle streak velocimetry method to address this hurdle. The method relies on the 3D streak of a moving particle formed during the acquisition of a single OCT volume, where double B-scans are acquired at each B-scan location to resolve ambiguity in assessing the movement of particle. We validated this method with the gold-standard, direct volumetric particle tracking in a flow phantom, and we demonstrated its in vivo applications for simultaneous velocimetry of embryos and imaging of oviduct. This work sets the stage for quantitative understanding of the oviduct transport function in vivo, and the method fills in a gap in OCT-based velocimetry, providing the potential to enable new applications in 3D flow imaging.

Abstract Objective: Ovulation is a major risk factor of epithelial ovarian cancer (EOC), presumably acts from the cancer initiation stage. High-grade serous carcinoma (HGSC) is the most prevalent and fatal EOC, mainly arising from fallopian tube fimbrial epithelium (FTE). TP53 missense mutations are considered the earliest event of carcinogenesis since they form “p53 signatures” in FTE and were found in virtually all HGSC. Previously, we discovered that a subset of ovulatory follicular fluids (FF) harbored high levels of ROS, which were both mutagenic and oncogenic, causing DNA double-strand breaks (DSB) and tumorigenesis. However, DSB is unlikely the cause of TP53 mutations which are mostly CG: TA base substitutions involving deamination. We hypothesized that during ovulation FF exposes the fallopian tube fimbria and causes TP53 mutations by activating the activation-induced cytidine deaminase (AID). Materials and Methods: By conducting an HPRT gene mutation assay, we determined the mutagenic activity of FF on FTE cells with or without AID-knockdown. AID gene activation and protein nuclear translocation were analyzed in human FTE tissues and cells with different degrees of transformation, as well as in the oviducts of mice under controlled ovulation. The binding of AID to the TP53 gene and the subsequent deamination were determined by ChIP-PCR and uracil-qPCR, respectively. Results: After FF exposure, the mutation frequency of HPRT in FTE cells increased by 4.1 folds, at levels 40% higher than the ethyl methane sulfonate control and could be diminished by antioxidants or knockdown of the AID gene. AID activation, including mRNA and protein inductions and nuclear translocation, was evident in FTE cells and tissue after FF exposure and in mouse oviducts after ovulation. Two waves of AID activations were noted, peaking at 45 minutes (early activity) and 8.5 hours (late activity) after ovulation or FF exposure. TNF-α and ROS were found to be responsible for the activities in FF. Mechanistically, the early activity worked through the MAPK, while the late activity worked through the NF-κB pathway. The induced AID in both activities physically binds to and biochemically deaminates the TP53 gene irrelative to the known mutational hotspots of HGSC. However, it did not act on PTEN, the other prevalent tumor suppressor gene in HGSC. Meanwhile, ovulatory FF also activates the putative tumor-initiating and stemness gene LGR6 in FTE at locations where AID was also activated. Conclusion: The serial studies characterized the mutagenic activities of ovulatory FF, which acts on the LGR6-expressing cells in the fallopian tube fimbria and targets the TP53 gene. The study also identified ROS as the mutagen and pointed to strategies of ovulation inhibition and antioxidation for the prevention of EOC. Citation Format: Tang-Yuan Chu, Sanchana Subramani, Hsuan-Shun Huang. ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A052.