Oviductal Cells Research Articles

Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle.

Simple SummaryThe oestrous cycle in canines is specifically more extended than that in other mammals. This implies that the oocytes do not reach maturity within the ovarian follicle but undergo final maturation in the oviducts. Besides oocyte maturation, the oviduct provides the necessary milieu for fertilization and preimplantation embryonic development. Consequently, the oviductal environment presumably changes in the postovulatory period and throughout the entire reproductive cycle to provide a suitable condition for supporting different functions. In this study, we evaluated the gene expression of different genes associated with oocyte-embryo development, such as progesterone receptor, cyclooxygenase-2, growth differentiation factor 9, and bone morphogenetic protein 15 in the canine oviductal cells at different phases of the oestrous cycle. Using quantitative PCR (qPCR) analysis in bitch oviductal cells, this study revealed the ovarian cycle’s influence on the oviductal essential transcripts in the bitch. It also assessed the influence of the ovulated cumulus-oocytes complexes on the expression of GDF-9 and BMP-15 genes. Thus, the oestrous-cycle-dependent gene expression pattern of PR, COX-2, GDF-9, BMP-15 in the canine oviduct was found to execute the oviductal cell interactions necessary for the development and function of the canine reproductive tract.The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.

Progesterone induces porcine sperm release from oviduct glycans in a proteasome-dependent manner.

In mammals, the oviduct retains sperm, forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and secondly, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and channeled with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein, we found that the release of sperm from an immobilized oviduct glycan, a six-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis-X trisaccharide. Thus, CatSper may be required for release of sperm from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release requires both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

1155 Are common processes involved in both implanting an embryo and ovarian cancer?

Implantation is fundamental to successful establishment of both the trophoblast (placenta) and embryo. However, there are practical, ethical and legal restrictions to studying this process in humans. Implantation of free-floating cells originating from the oviduct leads to benign ectopic lesions (endosalpingiosis, ES) and may also play a key role in the pathogenesis of gynecologic malignancies. We hypothesized that by understanding the mechanisms driving ES, we would gain crucial insight into early pregnancy implantation and failures. We aimed to develop a mouse model to interrogate the role of implantation signaling factors in promoting benign ectopic lesions such as ES, as a novel model for early pregnancy and ovarian cancer. We developed an animal model of ES using auto-fluorescing (inherent in the donor mouse cells) oviductal tissue or oviductal epithelia cells (OECs). Utilizing this ES model, donor mice were treated with hormones to induce infertility by blocking implantation or with a vehicle control (Fig. 1). Oviductal tissue from these mice was harvested and implanted into the peritoneal cavity of recipient wild type mice. Recipient mice are monitored for a month before mice are imaged for the formation of auto-fluorescing legions resulting from the donor tissue. We observed 100% successful implantation rate in our ES mouse model of oviductal tissue was administered (Fig. 2). When isolated OECs were used, they required the co-injection of endometrium to successfully implant, however only 30% successfully implanted. We successfully created a model of ES that allows us to identify growth of lesions in vivo allowing us to study the mechanisms of implantation. We anticipate that by blocking key signals of implantation the formation of oviductal lesions in the peritoneal cavity will be blocked or significantly reduced. Success with this model will allow us to not only understand ES but has applications to the molecular mechanisms of pregnancy success and failures, alongside ovarian cancers derived from oviduct and endometrial cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Avian sperm increase in vitro the release of exosomes from SST-enriched organoids.

In birds, oviductal cells play a crucial role in the storage of sperm via cell-to-cell communication including extracellular vesicles (EV). We developed a culture of oviductal organoids enriched in sperm storage tubules (SSTorg) to demonstrate the release of EV. SSTorg were cultured for 24 h and added to live (LV), frozen (FZ) and lysed (LY) avian sperm, seminal plasma (SP), avian sperm conditioned medium (CM), or bovine sperm (BV). Western blot demonstrated that SSTorg contained EV protein markers, valosin-containing protein (VCP), heat shock proteins (HSP90AA1, HSPA8), and annexins (ANXA2, A4, A5). Co-culture with LV significantly decreased the intracellular level of all these proteins except HSPA8. Immunohistochemistry confirmed this result for VCP and ANXA4. LY, CM, SP and BV had no effect on the intracellular level of these proteins, whereas FZ induced a decrease in ANXA2, A4 and A5. In culture media, VCP and HSP90AA1 signals were detected in the presence of LV, FZ, BV, LY, CM and SP, but no ANXA4 signal was observed in the presence of FZ and SP. ANXA2 and A5 were only detected in the presence of LV. The most abundant EV were less than 150 nm in diameter. ANXA4 and A5 were more abundant in EV isolated from the SSTorg culture medium. This study provides a useful culture system for studying interactions between SST cells and sperm. We demonstrated the release of EV by SSTorg in vitro, and its regulation by sperm. This may be of crucial importance for sperm during storage in hens.

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Simple SummaryThe use of assisted reproductive techniques, which involve the manipulation of sperm and oocytes in the laboratory, support owner production of valuable animals’ offspring. However, several limitations remain underlining the need to further optimize existing protocols as well as to develop new strategies. For example, the required conditions to make equine spermatozoa competent to fertilize an oocyte in vitro (IVF) have not been established. Therefore, our initial goal was to optimize different conditions associated with frozen equine sperm manipulations in order to improve their quality. We observed that simple factors such as sample concentration, incubation period and centrifugation time affect the sperm motility. Since in vivo fertilization involves the interaction between spermatozoa and epithelial cells in the mare’s oviductal tract, our next goal was to mimic this environment by establishing primary cultures of oviductal cells. Using this in vitro system, we were able to select a sperm population capable of fertilization. In short, this study provides a novel protocol that improves the yield of fertilization-capable sperm obtained from equine frozen spermatozoa.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm–OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (p < 0.05), capacitated (p < 0.05), with progressive motility (p < 0.05) and with the intact acrosome sperm population was observed (p < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higher-quality sperm.

66 Effect of the co-culture system and the culture medium on invitro embryo development in alpacas (Vicugna pacos)

The aim was to evaluate 4 co-culture systems and 4 culture medium types to produce alpaca embryos by IVF. Gametes were obtained from ovaries and testes collected from a slaughterhouse. Oocytes were recovered by follicle aspiration using a 5-mL syringe. Oocytes with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured for 26h in an incubator (5% CO2 in air at 38.5°C), in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.02IU mL−1 FSH, 1µg mL−1 oestradiol 17β, 0.2mM sodium pyruvate, and 50µg mL−1 gentamicin sulphate. After maturation, oocytes were placed in FERT-TALP (fertilization- Tyrode’s medium with albumin, lactate, and pyruvate) solution for 30min before IVF with epididymal sperm. Motile sperm were selected by swim-up method. Oocytes were co-cultured with 3×106 spermatozoa mL−1 for 18 to 20h under the same atmospheric conditions mentioned above. In Experiment 1, we evaluated 4 co-culture systems: ear fibroblasts (T1, n=224), fetal fibroblasts (T2, n=154), granulosa cells (T3, n=225), oviduct cells (T4, n=169) and synthetic oviductal fluid (SOF) invitro culture (IVC) (T5, n=116). As culture base, we used 0.5mL of SOF IVC medium supplemented with 10% FCS in all treatments. In Experiment 2, we evaluated 4 culture media: TCM-199+10% FCS (T1, n=137), CR1aa+3mg of bovine serum albumin (BSA) mL−1 (T2, n=85), KSOM medium+3mg of BSA mL−1 (T3, n=110), and SOF IVC+10% FCS (T4, n=66). The volume of culture medium used was 0.5mL in 4-well Nunc plates for each treatment. The time (8 days) and culture (38.5°C, 5% CO2 in air and high humidity) conditions, and change of medium (each 24h) were the same in both experiments. Statistical significance was determined using ANOVA. The mean and s.d. were calculated from the average of the percentages obtained in each repetition. In experiment 1, the cleavage rates were higher (P&amp;lt;0.05) in co-culture with fetal fibroblasts (46%±0.17), oviduct cells (50%±0.09), and ear fibroblasts (58%±0.17) than with granulosa cells (28%±0.12) and SOF IVC (30%±0.18). Also, the morula rates were higher (P&amp;lt;0.05) in co-culture with fetal fibroblasts (35%±0.16), oviduct cells (31%±0.01), and ear fibroblasts (35%±0.14) than with granulosa cells (11%±0.01) and SOF IVC (27%±0.17). In contrast, there were no differences in blastocyst rates between co-culture with granulosa cells (10%±0.04), SOF IVC (12%±0.09), and fetal fibroblasts (24%±0.14). However, there were differences between co-culture with oviduct cells (19%±0.06) and ear fibroblasts (32%±0.14). In Experiment 2, there were no differences in cleavage rates between TCM-199+FCS (28%±0.12), CR1aa+BSA (44%±0.24), KSOM+BSA (38%±0.19), and SOF-IVC+FCS (50%±0.20). However, there were differences in morula rates between CR1aa+BSA (7%±0.09) and SOF IVC+FCS (35%±0.21), TCM-199+FCS (23%±0.09), and KSOM+BSA (27%±0.15). We obtained a higher blastocyst rates in SOF IVC+BSA (24±0.12) and KSOM+BSA than with CR1aa+BSA (3±0.06) and TCM-199+FCS (9±0.03). In conclusion, KSOM and SOF-IVC were the best media for culture, and oviduct cells and ear and fetal fibroblasts were the best cells to produce alpaca embryos by IVF.

65 Co-culture of porcine epithelial oviductal cells and invitro-produced embryos: Effect on embryo development and quality

The oviduct is involved in many reproductive functions, including early embryo development. The epithelial cells that cover the oviduct produce oviducal fluid and could be used to recreate the invivo environment into which embryo development takes place. This study aimed to evaluate the co-culture of porcine embryos with a monolayer of porcine oviducal epithelial cells (POEC) and its effect on embryo development and quality. The POEC were obtained by pressing the isthmus (from diestrus sow oviducts) using slides and performing 3 cycles of vortexing and decanting in DMEM-F12 medium. Passage 1 cells were used for these experiments (POEC-1). Oocytes were obtained from follicular aspiration of slaughterhouse ovaries. Oocytes were invitro matured for 44h in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP during the first 22h. Invitro fertilization was performed with 17°C-refrigerated boar semen for 4h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and sodium pyruvate (20 denuded oocytes per drop, 1×106 spermatozoa mL−1). Presumptive zygotes were washed and randomly assigned to one of the following groups for invitro culture: control (50-µL drop of NCSU-23 with sodium pyruvate and lactate), POEC-1 (same as the control+POEC-1 50 000 cells mL−1), POEC-1+FBS (same as the control+POEC-1 50 000 cells mL−1 and 2.5% of fetal bovine serum). Culture conditions were 7% O2, 5% CO2, 39°C, and humidity. On Day 2, the cleavage rate was recorded, and embryos were transferred to NCSU-23 drops with glucose and without cells. The blastocyst rate was recorded on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoechst) and the apoptosis index (TUNEL-positive cells/total cells). Co-culture with POEC-1 significantly increased the blastocyst rate (control: 14%; POEC-1+FBS: 10%; POEC-1: 28%; P&amp;lt;0.05 Chi-squared test) and allowed embryo hatching (control: 0; POEC-1+FBS: 22.2%; POEC-1: 7; P&amp;lt;0.05 Chi-squared test). However, there was no significant difference in the number of cells per blastocyst (control: 58.6±6; POEC-1+FBS: 50.3±3.7; POEC-1: 50.6±4.8; nonparametric ANOVA) or in the apoptosis index (control: 8.1; POEC+FBS 8.3; POEC: 7.4; nonparametric ANOVA). The use of POEC-1 during the first 2 days of embryo culture enhanced embryo development and improved culture conditions, allowing embryo hatching. The effect on embryo development could be due to an effect of POEC itself or the effect of feeder cells. Other parameters of embryo quality should be evaluated in the future.

DIO3, the thyroid hormone inactivating enzyme, promotes tumorigenesis and metabolic reprogramming in high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy with a need for better understanding the disease pathogenesis. The biologically active thyroid hormone, T3, is considered a tumor suppressor by promoting cell differentiation and mitochondrial respiration. Tumors evolved a strategy to avoid these anticancer actions by expressing the T3 catabolizing enzyme, Deiodinase type 3 (DIO3). This stimulates cancer proliferation and aerobic glycolysis (Warburg effect). We identified DIO3 expression in HGSOC cell lines, tumor tissues from mice and human patients, fallopian tube (FT) premalignant lesion and secretory cells of normal FT, considered the disease site-of-origin. Stable DIO3 knockdown (DIO3-KD) in HGSOC cells led to increased T3 bioavailability and demonstrated induced apoptosis and attenuated proliferation, migration, colony formation, oncogenic signaling, Warburg effect and tumor growth in mice. Proteomics analysis further indicated alterations in an array of cancer-relevant proteins, the majority of which are involved in tumor suppression and metabolism. Collectively this study establishes the functional role of DIO3 in facilitating tumorigenesis and metabolic reprogramming, and proposes this enzyme as a promising target for inhibition in HGSOC.

Oncogenes in high grade serous adenocarcinoma of the ovary.

High grade serous ovarian cancer is characterized by relatively few mutations occurring at low frequency, except in TP53. However other genetic aberrations such as copy number variation alter numerous oncogenes and tumor suppressor genes. Oncogenes are positive regulators of tumorigenesis and play a critical role in cancer cell growth, proliferation, and survival. Accumulating evidence suggests that they are crucial for the development and the progression of high grade serous ovarian carcinoma (HGSOC). Though many oncogenes have been identified, no successful inhibitors targeting these molecules and their associated pathways are available. This review discusses oncogenes that have been identified recently in HGSOC using different screening strategies. All the genes discussed in this review have been functionally characterized both in vitro and in vivo and some of them are able to transform immortalized ovarian surface epithelial and fallopian tube cells upon overexpression. However, it is necessary to delineate the molecular pathways affected by these oncogenes for the development of therapeutic strategies.

In vitro fertilization and embryo development in pigs

Considerable progress has been made in the in vitro production of pig embryos using improved methods for in vitro maturation (IVM) and fertilization (IVF). Despite the progress, polyspermic penetration remains a problem for in vitro-matured oocytes. Variation among boars, ejaculates and IVF protocols used in different laboratories appears to influence the incidence of polyspermy. Recent studies indicate that oviduct cells and their secretions play a role in reducing polyspermy. Very early attempts to culture in vivo-derived pig embryos met with little success and most were arrested at the four-cell stage. At present, many culture media are available that can overcome the four-cell block and support development to the blastocyst stage. In contrast, blastocyst development of in vitro-produced (IVP) embryos in these culture media varies significantly. Significant differences in morphology and numbers of cells have been observed in in vitro-produced blastocysts compared with in vivo-derived blastocysts. Surgical transfer of in vitro-produced embryos to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although several systems are available for the generation of in vitro-produced embryos, the problems of polyspermy and poor embryo survival prevent large-scale production of embryos. Further research should be directed to improve oocyte and embryo quality, and to develop methods to minimize polyspermy through development of better IVM, IVF and embryo culture techniques.

Mechanisms mediating nutritional effects on embryonic survival in pigs

The inconsistency of data from experiments designed to show nutritional effects on embryonic survival is perplexing. However, a number of experimental models have provided some insight into the mechanisms that potentially mediate interactions between nutrition, metabolic state and embryonic survival. The developing ovarian follicle provides the maturational environment for the oocyte, and differences in follicular maturation are associated with differences in the ability of these follicles to support oocyte maturation. In turn, the rate of oocyte maturation and the maturational state of the oocyte immediately before ovulation may contribute to differences in embryonic survival. Therefore, evidence that nutritionally induced differences in metabolic state affect follicular development and the maturational state of the oocyte in the late follicular phase may constitute a mechanism by which nutrition affects the very early stages of conceptus development. Once ovulation occurs, the process of fertilization and early cleavage occurs in the environment of the oviduct. Nutritional state might affect the secretory and motile activity of the oviduct both directly, by influencing the physiology of the oviductal cells, or indirectly by affecting the secretion of key regulatory hormones. Thus evidence for nutritionally dependent effects on plasma progesterone concentrations in early pregnancy and associations with differences in embryonic survival may be partly mediated at the oviductal stage of development. Nutritional effects on circulating progesterone concentrations may also affect the uterine environment. However, the metabolic state of the gilt or sow, or specific nutrients in the diet, may directly affect the integrity of the endometrium and thus affect embryonic survival at this stage of development.

Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting

To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.

RETRACTED: Two Cu(II)-based coordination polymers: Enhancement on acupuncture treatment activity during salpingitis

• Two coordination polymers (CPs) were successfully synthesized. • The coordination polymers were characterized through single crystal diffraction of X-ray. • Compounds 1 and 2 were studied in our lab for the treatment of salpingitis combined with acupuncture. Via utilizing the mixed-ligand method, two coordination polymers (CPs) of { [Cu 2 (SDB) 2 (L 1 )]·(H 2 O) 4 ·(DMF)} n ( 1 ) and {[Cu(L 2 )(SDB)]} n ( 2 ) [L 2 = 4,4′-(2,5-bis(methylthio)-1,4-phenylene)dipyridine and the L 1 = 1,4-bis(4-pyridyl)-2,3-diaza-1,3-butadiene] were synthesized hydrothermally and then the coordination polymers were characterized through X-ray powder diffraction, IR, single crystal X-ray vdiffraction, and the measurements of thermogravimetry, as well as elemental analysis. Compounds 1 and 2 were studied in our lab for the treatment of salpingitis combined with acupuncture, the biological functional was evaluated and the mechanism was discussed. The inflammatory levels in the fallopian tube epithelial cells were measured through the ELISA test. Afterward, the JAK-STAT signaling pathway relative expression in the epithelial cells of fallopian tube was determined through the real time RT-PCR.

The effect of polychlorinated biphenyls (PCBs) on bovine oviductal contractions and LIF synthesis during estrous cycle, in vitro studies

Polychlorinated biphenyls (PCBs) are a group of synthetic xenobiotics that have been used in many industrial applications. Currently, PCBs are among the most prominent environmental contaminants. Previously we showed that PCBs impair secretion of prostaglandins (PGs) at the oviduct. PGs are involved in the regulation of oviductal contractions and the synthesis of leukemia inhibitory factors LIF. Since oviductal contractions are crucial for gamete and embryo transport, and LIF is essential for embryo implantation, the direct effect of PCBs on oviductal motor activity and LIF mRNA expression were investigated. Oviductal strips and cells were taken from cows during the estrous cycle and were treated with PCBs at concentrations close to their environmental ranges. All the studied PCBs decreased the force of the contractions of the longitudinal and circular muscles of the isthmus. Additionally, these PCBs decreased the amplitude of the longitudinal muscle of the oviduct. Moreover, PCB-30-OH and PCB-153 increased the mRNA expression of LIF. Since PCBs inhibit the motor function of the oviduct and stimulate the synthesis of LIF, it is possible that PCBs can slow gamete or embryo transport and increase the potential for pathological embryo implantation in the oviduct.

REGULATION OF OVIDUCT HOMEOSTASIS AND FERTILITY BY Pax2 AND Pax8 GENES

In mouse and humans, the development regulatory genes Pax2 and Pax8 are expressed in ovarian duct surface epithelia, yet the function of these genes and proteins in adult females remains unclear. By Generating double & single KO mouse modules of Pax2 and Pax8 we investigate their roles in maintaining the integrity of the oviductal cells. Our studies directly address aspects of oviduct epithelial homeostasis and the effects on fertility. To create Pax2, Pax8, and Pax2/8 double mutants specifically in oviduct epithelia and characterize changes in gene expression, epithelial cell integrity, and fertility. We will implement detailed analyses of oviducts isolated at various times post tamoxifen administration from mice with conditional Pax2, Pax8, or both alleles, using the Ovigp1-CreER driver. Mice will also carry reporter allele (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo) to mark all active Cre expressing cells with cell surface EGFP. In addition to defining novel functions for Pax proteins in the oviduct, we will utilize state-of-the-art methods for single cell analyses, transcriptomics, and chromatin remodeling. Mutation of Pax2 and Pax8 in adult female oviducts results in significant changes in gene expression and morphology. Loss of both Pax2 and Pax8 resulted in infertility (Table 1). To date, most of the preliminary data was obtained from Pax2/8 double mutants since we were concerned with redundancy and expected the most significant phenotypes in the double mutants. Analyses of histology, immunohistochemistry and changes in gene expression of total RNA from whole oviducts isolated after tamoxifen administration. For a rapid first pass screen, we compared controls (Pax2f/f;Pax8f/f) to single Pax mutants and Pax2/8 double mutants (Pax2f/f;Pax8f/f; Ovigp1-CreER) by Affymetrix microarrays. These data show hundreds of changes in gene expression levels upon Pax2 or Pax8 deletion, with significant overlap between the Pax2 and Pax8 mutants but also expression changes unique to each single mutant.Tabled 1TABLE 1. Fertility of Oviduct Specific Pax MutantsGenotype # females # plugs Litters Litter sizePax2f/f, Pax8f/f, Ovi-Cre 5 15 0 0Pax2f/f, Pax8f/f, 4 12 12 6-9Pax8f/f, Ovi-Cre 3 6 2 6-7 Open table in a new tab Little is known regarding the mechanisms underlying epithelial homoeostasis, the proteins that determine cell fates, and epithelial integrity in the adult oviduct. Pax8 expressing secretory cells are thought to give rise to ciliated cells, which help move the oocyte down the duct and Pax2 is associated with cilia motility and physiology. Using a conditional KO model of Pax2 & Pax8 will allow us to understand those mechanisms and identify future novel therapeutic targets for diagnostics and treatments

Abstract 1378: Targeting the metabolic-epigenetic axis to sensitize HR-proficient ovarian cancer to PARP inhibitors

Abstract Overexpression of the oncogene cyclin E1 is considered one of the initiating factors in fallopian tube transformation and ovarian tumorigenesis. Patients with high cyclin E1 expression have worse overall survival than patients with low expression. Cyclin E1 overexpression increases expression of DNA damage response (DDR) genes in order to tolerate DNA damage and bypass senescence, a state of cell cycle arrest. However, the mechanism by which cyclin E1-high cells transcriptionally increase DDR gene expression to bypass senescence remains unclear. We found that cyclin E1 overexpression alters the metabolic-epigenetic axis through wildtype isocitrate dehydrogenase I (wtIDH1), a TCA cycle enzyme. Upregulation of wtIDH1 in cyclin E1-high cells increased the transcription of multiple DDR genes related to homologous recombination (HR), including BRCA1, BRCA2, and RAD51. Published data from our lab demonstrates that wtIDH1 primarily converts isocitrate to alpha-ketoglutarate (αKG) in cyclin E1-high ovarian cancer cells, and suppression of wtIDH1 and αKG pools induces senescence through increased repressive histone marks. Our current data suggest that wtIDH1 is both necessary and sufficient for HR gene expression in cyclin E1-high cells in part through modulation of histone methylation. Functionally, inhibition of wtIDH1 in cyclin E1-high fallopian tube cells induced senescence via decreased HR and marked accumulation of DNA damage. Cyclin E1-high ovarian cancer tumors are HR-proficient and resistant to emerging poly(ADP-ribose) polymerase (PARP) inhibitors. Interestingly, inhibition of both wtIDH1 and PARP in combination increased apoptosis of cyclin E1-high ovarian cancer cells in vitro, suggesting this may be a novel therapy for HR-proficient ovarian cancers. Together, our data suggest that wtIDH1-mediated metabolism affects the epigenome in cyclin E1-high cells, which contributes to both fallopian tube transformation and HR-proficiency. Targeting wtIDH1 with current FDA-approved inhibitors may therefore be a rational therapeutic strategy for cyclin E1-high ovarian cancer patients. Citation Format: Erika S. Dahl, Kelly E. Leon, Chi-Wei Chen, Qingyuan Jia, Raquel Buj, Nathaniel W. Snyder, Katherine M. Aird. Targeting the metabolic-epigenetic axis to sensitize HR-proficient ovarian cancer to PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1378.

Cytologic studies of in vivo fallopian tube specimens in patients undergoing salpingo-oophorectomy.

Objectives:Recent research shows that most high grade ovarian cancer (OC) originates from the fallopian tube (FT). Cytologic evaluation of FT cells may enable early detection of OC.Material and Methods:This was a prospective study with patients enrolled from 3 centers (October 2016– August 2017). Forty-two women undergoing salpingo-oophorectomy for a pelvic mass suspicious for malignancy or undergoing risk-reducing surgery for BRCA mutations were included in the study. At the time of scheduled surgery, a novel catheter was used to collect FT cells through hysteroscopy. A pathologist blinded to surgical or pathologic findings evaluated FT cytology, and results were compared to pathology.Results:Of the 61 samples collected, 72% (44/61) met the adequacy criteria (≥5 clusters of cells with 20 cells in each cluster). Cytology classification criteria were established and applied to adequate samples. Forty-four samples were benign with mixed population of cells with round, oval, and spindled nuclei; 2-dimensional clusters; columnar cell configuration; flat sheets; cilia presence; no/mild nuclear pleomorphism; no nuclear membrane irregularities; and no nucleoli. Five samples had benign features with reactive nuclear and cytoplasmic changes and/or background inflammation, which were categorized as “reactive atypia.” Two malignant samples had features of 3-dimensional (3D) clusters, loss of mixed population of cells; increased nuclear/cytoplasmic ratio; nuclear membrane irregularity and nucleoli presence. Three samples with some but not all of malignant features were categorized as “neoplastic” (anisonucleosis; small nucleoli and features suggestive of 3D clusters). Malignant/ neoplastic samples were labeled as “Positive” (n = 5) while benign/reactive samples were labeled as “Negative” (n = 39). A high concordance rate (95%, 42/44) was observed between cytology results and histology.Conclusions:We characterized cytologic features for pathologically distinct FT samples collected in vivo using a novel catheter and demonstrated its value in detecting OC.

Bovine Follicular Fluid and Extracellular Vesicles Derived from Follicular Fluid Alter the Bovine Oviductal Epithelial Cells Transcriptome.

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

The role of GtxA during Gallibacterium anatis infection of primary chicken oviduct epithelial cells

Gallibacterium anatis (G. anatis), one of the major pathogens causing reproductive tract disorders in laying hens, leads to a reduction in egg production and increased mortality, caused by either single or mixed infections with other pathogens. As a specific virulence factor of G. anatis, the role of GtxA in layers’ salpingitis remains unclear. In this study, we explored the effect of GtxA on G. anatis infection by comparing wild strain Yu-PDS-RZ-1-SLG (RZ) and its GtxA deleted counterpart RZΔgtxA in primary chicken oviduct epithelial cells (COEC). Their adherence, invasion, cytoxicity, and ability to induce apoptosis and and cytokine secretion were evaluated and the cytotoxicity and cytokine secretion of the recombinant GtxA protein and its N-terminal adenylate cyclase and C-terminal RTX hemolysin domain were also analyzed. We found that the adhesion ability of RZΔgtxA was significantly lower than that of parental strain RZ, and its toxicity to COEC was weakened; Meanwhile, apoptosis was inhibited and the expression of IL-6, IL-2, TNF-α and IFN-γ were dramatically reduced in COEC infected by RZΔgtxA. In contrast, the recombinant protein GtxA inhibited the proliferation of oviduct cells and induced obvious cytotoxicity, and the expression of IL-6, TNF-α and IFN-γ were up-regulated in COEC interacted with recombinant proteins. Our study indicates that GtxA promotes G. anatis adherence to cells, changes cells permeability and expression of inflammatory factors, resulting in cell damage and apoptosis.

Abstract A35: Targeting the IDH1-mediated metabolic-epigenetic axis in cyclin E-high ovarian cancer

Abstract Cyclin E is an oncogene that is overexpressed in approximately 20% of high-grade serous ovarian carcinomas (HGSOC) and is associated with worse survival and resistance to DNA-damaging agents due to homologous recombination (HR) proficiency. Additionally, cyclin E is considered an initiating factor in the transformation of fallopian tube cells, the cell of origin of HGSOC. In normal cells, increased cyclin E induces replication stress and subsequent DNA damage that leads to cellular senescence, a state of cell cycle arrest. Previous studies demonstrated that cyclin E overexpression in fallopian tube increases expression of DNA damage response (DDR) genes. This presumably allows for tolerance of replication stress, repair of DNA damage, and bypass of senescence. However, the mechanism by which cyclin E-high cells bypass senescence and increase DDR gene expression remains unclear. We previously published that the TCA cycle enzyme isocitrate dehydrogenase I (IDH1) is high in HGSOC cells compared to fallopian tube. Importantly, IDH1 is critical for HGSOC cell proliferation as knockdown or inhibition of IDH1-induced senescence through increased repressive histone H3K9me2 at multiple proliferation-promoting gene loci. Excitingly, we observed a positive correlation between high cyclin E expression and high IDH1 in both fallopian tube and HGSOC cells. Indeed, overexpression of cyclin E increased IDH1 expression in both fallopian tube and HGSOC cells. Therefore, we hypothesized that upregulation of IDH1 in cyclin E high cells increases DDR gene expression by altering the epigenetic landscape and thereby suppressing senescence induction. Our data demonstrate that IDH1 overexpression in fallopian tube phenocopies cyclin E overexpression by increasing expression of several DDR genes, including BRCA2. Indeed, knockdown of IDH1 in cyclin E-high cells decreased DDR gene expression, demonstrating that IDH1 is necessary for DDR gene expression in cyclin E-high cells. Mechanistically, we determined that this is through a metabolic-epigenetic axis, which regulates occupancy of repressive H3K9 methylation at DDR gene loci. Finally, to determine whether the alteration in DDR gene expression is functionally relevant, we treated cyclin E-high PARPi-resistant cells with an IDH1 inhibitor. While neither inhibitor affected proliferation of cyclin E-high HGSOC cells alone, the combination was highly synergistic, suggesting that the IDH1 inhibitor converted HR-proficient cyclin E-high cells to an HR-deficient phenotype. Together, our data suggest that IDH1-mediated metabolism affects the epigenome in cyclin E-high cells, which contributes to both fallopian tube transformation and HR proficiency. Targeting IDH1 with currently FDA-approved inhibitors may therefore be a rational therapeutic strategy for cyclin E-high HGSOC patients. Citation Format: Erika S. Dahl, Raquel Buj, Qingyuan Jia, Kelly E. Leon, Katherine M. Aird. Targeting the IDH1-mediated metabolic-epigenetic axis in cyclin E-high ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A35.