Oviductal Cells Research Articles

Abstract 4039: PAX8 increases migration and metastasis of high grade serous ovarian cancer through upregulation of Rho GTPases

Abstract High grade serous ovarian cancer, the most lethal subtype of ovarian cancer, can originate in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). PAX8 is a lineage specific transcription factor that is ubiquitously expressed in HGSOC. We have shown that knockdown of PAX8 using shRNA in multiple ovarian tumor cells lines leads to apoptosis, suggesting that PAX8 plays an essential role in cancer survival. In this study, we used CRISPR technology to delete PAX8 from the OVCAR8 cell line. PAX8 deletion led to a decrease in migration and invasion in vitro and an increase in survival and a reduction in tumor volume in vivo. Previous work using RNA-sequencing and ChIP-sequencing identified cell adhesion as a top differentially expressed gene between malignant ovarian cancer and benign fallopian tube cell lines. We performed quantitative proteomic analysis of the OVCAR8-PAX8-/- cell line to define PAX8 altered proteins. We also performed quantitative proteomics and transcriptomic analyses on a previously generated murine OSE cell line with forced PAX8 expression (MOSE-PAX8). These analyses identified several genes that contribute to an increase in migration and EMT. Specifically, our data indicates PAX8 upregulates key drivers involved in altering cell morphology including the GTPases: RhoA, Cdc42, and Ras. Inhibition of RhoA led to a greater decrease in migration for both OVCAR8-PAX8-/- and MOSE-PAX8 when compared to control. Inhibition of Ras had a greater effect in OVCAR8-PAX8-/- while inhibition of Cdc42 had a greater effect in MOSE-PAX8. These data provide a mechanistic explanation for the role of PAX8 on increasing migration and metastasis in ovarian cancer. Citation Format: Laura Hardy, Melissa Pergande, Angel Hernandez, Stephanie Cologna, Joanna Burdette. PAX8 increases migration and metastasis of high grade serous ovarian cancer through upregulation of Rho GTPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4039.

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling.

The Wnt signaling pathway can be grouped into two classes, the β-catenin-dependent and β-catenin-independent pathways. Wnt5a signaling through a β-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.

Fallopian tube cells from high-risk women have altered adipokine signaling

Fallopian tube cells from high-risk women have altered adipokine signaling

Molecular signatures of epithelial oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica)

BackgroundIn this work we have determined molecular signatures of oviduct epithelial and progenitor cells. We have proposed a panel of selected marker genes, which correspond with the phenotype of oviduct cells of a laying hen (Gallus gallus domesticus) and quail (Coturnix japonica). We demonstrated differences in characteristics of those cells, in tissue and in vitro, with respect to different anatomical and functional parts of the oviduct (infundibulum (INF), distal magnum (DM, and proximal magnum (PM)). The following gene expression signatures were studied: (1) oviduct markers (estrogen receptor 1, ovalbumin, and SPINK7 - ovomucoid), (2) epithelial markers (keratin 5, keratin 14, and occludin) and (3) stem-like/progenitor markers (CD44 glycoprotein, LGR5, Musashi-1, and sex determining region Y-box 9, Nanog homebox, OCT4/cPOUV gene encoding transcription factor POU5F3).ResultsIn chicken, the expression of oviduct markers increased toward the proximal oviduct. Epithelial markers keratin14 and occludin were high in distal oviduct and decreased toward the proximal magnum. In quail oviduct tissue, the gene expression pattern of oviduct/epithelial markers was similar to chicken. The markers of progenitors/stemness in hen oviduct (Musashi-1 and CD44 glycoprotein) had the highest relative expression in the infundibulum and decreased toward the proximal magnum. In quail, we found significant expression of four progenitor markers (LGR5 gene, SRY sex determining region Y-box 9, OCT4/cPOUV gene, and CD44 glycoprotein) that were largely present in the distal oviduct. After in vitro culture of oviduct cells, the gene expression pattern has changed. High secretive potential of magnum-derived cells diminished by using decreased abundance of mRNA. On the other hand, chicken oviduct cells originating from the infundibulum gained ability to express OVM and OVAL. Epithelial character of the cells was maintained in vitro. Among progenitor markers, both hen and quail cells expressed high level of SOX9, LGR5 and Musashi-1.ConclusionAnalysis of tissue material revealed gradual increase/decrease pattern in majority of the oviduct markers in both species. This pattern changed after the oviductal cells have been cultured in vitro. The results can provide molecular tools to validate the phenotype of in vitro biological models from reproductive tissue.

STAT3/PIAS3 Levels Serve as "Early Signature" Genes in the Development of High-Grade Serous Carcinoma from the Fallopian Tube.

The initial molecular events that lead to malignant transformation of the fimbria of the fallopian tube (FT) through high-grade serous ovarian carcinoma (HGSC) remain poorly understood. In this study, we report that increased expression of signal transducer and activator of transcription 3 (pSTAT3 Tyr705) and suppression or loss of protein inhibitor of activated STAT3 (PIAS3) in FT likely drive HGSC. We evaluated human tissues-benign normal FT, tubal-peritoneal junction (TPJ), p53 signature FT tissue, tubal intraepithelial lesion in transition (TILT), serous tubal intraepithelial carcinoma (STIC) without ovarian cancer, and HGSC for expression of STAT3/PIAS3 (compared with their known TP53 signature) and their target proliferation genes. We observed constitutive activation of STAT3 and low levels or loss of PIAS3 in the TPJ, p53 signature, TILT, and STIC through advanced stage IV (HGSC) tissues. Elevated expression of pSTAT3 Tyr705 and decreased levels of PIAS3 appeared as early as TPJ and the trend continued until very advanced stage HGSC (compared with high PIAS3 and low pSTAT3 expression in normal benign FT). Exogenous expression of STAT3 in FT cells mediated translocation of pSTAT3 and c-Myc into the nucleus. In vivo experiments demonstrated that overexpression of STAT3 in FT secretory epithelial cells promoted tumor progression and metastasis, mimicking the clinical disease observed in patients with HGSC. Thus, we conclude that the STAT3 pathway plays a role in the development and progression of HGSC from its earliest premalignant states.Significance: Concomitant gain of pSTAT3 Tyr705 and loss of PIAS3 appear critical for initiation and development of high-grade serous carcinoma. Cancer Res; 78(7); 1739-50. ©2018 AACR.

Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells

A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.

Effect of Different Culture Conditions on In vitro Development and Quality of Buffalo Embryos

This work was carried out to evaluate the quality and the developmental competence of the in vitro buffalo embryos produced under different culture conditions. Oocytes were matured in TCM 199 for 24 h at 38.5°C and 5% CO2. After maturation, oocytes were in vitro fertilized by frozen thawed sperms capacitated in vitro by caffeine at final concentration 2 million sperm /ml. The sperm-oocytes were incubated in TALP medium for 24 hrs. Fertilized oocytes were cultured in Cell free media (CR1aa) or with cumulus or oviductal cell monolayers (Experiment 1), CR1aa with addition of (10, 20, 50 and 100 µg/mL) lactoferrin (Experiment 2) inside CO2 incubator with 5% CO2 at 38°C. The cleavage, morula, blastocyst rates were recorded and embryo cell number and mitochondrial function were evaluated. Addition of bovine oviductal cell monolayer (BOCM) to culture media significantly increased the morula and the blastocyst compared with cell free media and media with cumulus cell monolayer and also significantly increased the cell number and the mitochondrial function of in vitro produced buffalo embryos. Addition of lactoferrin by concentration 50µg/ml to culture media resulted in a significant increase in rates of cleavage; morula and blastocyst compared with control or other concentrations and also resulted in an increase in the cell number and the mitochondrial function of in vitro produced buffalo embryos. It could be concluded that, addition of oviductal cells (monolayer) or lactoferrin to culture medium significantly improved the quality of in vitro produced buffalo embryos.

PTEN loss in the fallopian tube induces hyperplasia and ovarian tumor formation.

The signaling events involved in the onset of ovarian cancer from the fallopian tube epithelium (FTE) are crucial for early detection and treatment of the disease, but they remain poorly defined. Conditional homozygous knockout of PTEN mediated by PAX8-cre recombinase was sufficient to drive endometrioid and serous borderline ovarian carcinoma, providing the first model of FTE-derived borderline tumors. In addition, heterozygous PTEN deletion in the FTE resulted in hyperplasia, providing a model to study early events of human ovarian pathogenesis. To uncover the mechanism underlying the invasion of cancerous oviductal cells to the ovary, PTEN-deficient murine oviductal cells were developed and tagged with green fluorescent protein. Loss of PTEN increased cell migration, invasion, and upregulated WNT4, a key regulator of Müllerian duct development during embryogenesis. Further investigation revealed that WNT4 was required for increased migration and colonization of the ovary by PTEN-deficient oviductal cells in a β-catenin independent manner. Human tumor microarrays and ovarian cancer cells lines confirmed WNT4 expression in cancer and its role in migration. Together, these findings provide a novel model to study the mechanism of fallopian tube tumor initiation and invasion to the ovary mediated by loss of PTEN, which may help to define early events of human ovarian carcinogenesis.

Sperm binding to porcine oviductal cells is mediated by SRCR domains contained in DMBT1.

The oviduct is an organ in which a subpopulation of sperm is stored in a reservoir, preserving its fertilizing potential. In porcine, two oviductal proteins have been identified in relation to sperm binding, Annexin A2 and Deleted in Malignant Brain Tumor 1 (DMBT1). DMBT1 is a multifunctional, multidomain glycoprotein, and the characteristics of all of its domains, as well as its carbohydrates, make them candidates for sperm binding. In this work, we challenge sperm for binding to pig oviductal cells on primary culture, after treatment with antibodies specific for the different domains present in DMBT1. Only anti-SRCR antibodies produced inhibition of sperm binding to cells. Thus, SRCR is the main domain in DMBT1 promoted sperm binding to form the reservoir in the oviduct, and this function is probably elicited through the polypeptide itself.

Nitric oxide activation by progesterone suppresses ATP-induced ciliary activity in oviductal ciliated cells.

Ciliary beat frequency (CBF) regulates the oviductal transport of oocytes and embryos, which are important components of the reproductive process. Local release of ATP transiently increases CBF by increasing [Ca2+]i. Ovarian hormones also regulate ciliary activity and oviductal transport. Progesterone (P4) induces nitric oxide (NO) production and high P4 concentrations induce ciliary dysfunction. However, the mechanism by which P4 affects CBF has not been elucidated. To evaluate the role of P4 in NO production and its effect on ATP-induced increases in CBF, we measured CBF, NO concentrations and [Ca2+]i in cultures of oviductal ciliated cells treated with P4 or NO signalling-related molecules. ATP induced a [Ca2+]i peak, followed by an increase in NO concentrations that were temporally correlated with the decreased phase of the transiently increased CBF. Furthermore, P4 increased the expression of nitric oxide synthases (iNOS and nNOS) and reduced the ATP-induced increase in CBF via a mechanism that involves the NO signalling pathway. These results have improved our knowledge about intracellular messengers controlling CBF and showed that NO attenuates oviduct cell functions. Furthermore, we showed that P4 regulates neurotransmitter (ATP) actions on CBF via the NO pathway, which could explain pathologies where oviductal transport is altered and fertility decreased.

Elevated non-esterified fatty acids impair survival and promote lipid accumulation and pro-inflammatory cytokine production in bovine endometrial epithelial cells.

Elevated non-esterified fatty acids (NEFAs) are associated with negative effects on bovine theca, granulosa and oviductal cells but the effects of NEFAs on bovine endometrial epithelial cells (bEECs) are not as well documented. The objective of this study was to define the effects of NEFAs on bEECs. Postprimary bEECs were treated with 150, 300 or 500µM of either palmitic acid (PA), stearic acid (SA) or oleic acid (OA) or a mixture of NEFAs (150µM of each FA) or 0.5% final concentration of vehicle ethanol (control). Viability and proliferation of bEECs exposed to 150µM of each NEFA or a mixture of NEFAs were unaffected. Increased lipid accumulation was found in all treated groups (P<0.01). In cells exposed to 500µM of each NEFA and 300µM PA decreased cell viability (P<0.001), proliferation (P<0.05) and increased apoptosis (P<0.05) were observed. Treatment with 500µM OA, PA and SA had the strongest effects on cell viability, proliferation and apoptosis (P<0.05). Treatment with PA and OA increased interleukin-6 (IL-6) concentrations (P<0.05), whereas only the highest concentration of PA, OA and SA stimulated IL-8 production (P<0.05). These results suggest that high concentrations of NEFAs may impair endometrial function with more or less pronounced effects depending on the type of NEFA and time of exposure.

Natural and environmental oestrogens induce TGFB1 synthesis in oviduct cells.

Autocrine/paracrine factors generated in response to 17β-oestradiol (E2), within the oviduct, facilitate early embryo development for implantation. Since transforming growth factor beta 1 (TGFB1) plays a key role in embryo implantation, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Here, we investigated whether oviduct cells synthesize TGFB1 and whether E2 and environmental oestrogens (EOEs; xenoestrogens and phytoestrogens) modulate its synthesis. Under basal conditions, bovine oviduct cells (OCs; oviduct epithelial cells and oviduct fibroblasts; 1:1 ratio) synthesized TGFB1. E2 concentration-dependent induced TGFB1 levels in OCs and these effects were mimicked by some, but not all EOEs (genistein, biochanin A and 4-hydroxy-2',4',6'-trichlorobiphenyl, 4-hydroxy-2',4',6'-dichlorobiphenyl); moreover, EOEs enhanced (P < 0.05) the stimulatory effects of E2 on TGFB1 synthesis. The OCs expressed oestrogen receptors alpha and beta and aryl hydrocarbon; moreover, co-treatment with ER antagonist ICI182780 blocked the stimulatory effects of E2 and EOEs on TGFB1 synthesis. Treatment with non-permeable E2-BSA failed to induce TGFB1, thereby ruling out the involvement of membrane ERs. Cycloheximide (protein synthesis inhibitor) blocked E2-induced TGFB1 synthesis providing evidence for de novo synthesis. The stimulatory effects of E2 and EOEs, were inhibited (P < 0.05) by MAPK inhibitor (PD98059), whereas intracellular-Ca2+ chelator (BAPTA-AM) and adenylyl cyclase inhibitor (SQ22536) abrogated the effects of E2, but not EOEs, suggesting that post-ER effects of E2 and EOEs involve different pathways. Our results provide the first evidence that in OCs, E2 and EOEs stimulate TGFB1 synthesis via an ER-dependent pathway. Exposure of the oviduct to EOEs may result in continuous/sustained induction of TGFB1 levels in a non-cyclic fashion and may induce deleterious effects on reproduction.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P &lt; 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

69 Can Bovine Sperm Interaction with the Oviduct Cells After Artificial Insemination Affect the Transcriptome Profile of the Oviduct?

During their transit through the female genital tract, spermatozoa bind to oviduct cells, where they are maintained alive for a long period. Although some studies have been carried out to evaluate the effects of the oviduct on spermatozoa, less is known about the effect of spermatozoa on the oviduct. The aim of this study was to evaluate the effect of sperm on the transcriptome profile of the bovine oviduct after fixed-time AI (FTAI). Nellore cows submitted to FTAI were randomly inseminated using 8 × 106 frozen–thawed spermatozoa (sperm group, n = 9) or saline solution (control group, n = 9). Eighteen hours after FTAI, the cows were slaughtered, and the cells from oviducal isthmus were collected and stored in liquid nitrogen until transcriptome analysis. For RNA extraction, 3 pools of 3 oviducts/cows from each group were formed. Total RNA was extracted and the RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The transcription profiling from each group was performed by HiSEqn 2500 (Illumina Inc., San Diego, CA, USA). After sequencing, the data were analysed by edgeR software, and differential gene expression analysis between sperm and control group was performed using the generalized linear model (P = 0.05). The P-value was adjusted by false discovery rate (FDR) Benjamini-Hochberg method, to avoid false positives. Moreover, using mRNA expression levels (log fold change and P-values) as coefficients, functional enrichment analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. A total of 9,374 genes were found to be expressed in bovine oviducal isthmus, of which 219 genes were differentially expressed between sperm and control group. Differentially expressed genes genes were classified according to 3 major GO classifications, with 10 gene categories in molecular function, 3 gene categories in cellular component, and zero categories in biological process. In the molecular function category, genes related to calcium ion binding (n = 113), molecular transducer activity (n = 111), and receptor activity (n = 111), were predominant. For cellular component, the highest number of genes (n = 176) was related to extracellular space. Moreover, KEGG assignments were used to classify the functional annotations of the pathways genes to further understand the biological functions of the expressed genes. It was shown that steroid hormone biosynthesis, arachidonic acid metabolism, calcium signalling, cytokine-cytokine receptor interaction, neuroactive ligant-receptor interaction, extracellular matrix-receptor interaction, cell adhesion molecules, coagulation and complement cascades, ovarian steroidogenesis, protein digestion and absorption and mineral absorption pathways had genes with higher or lower expression than the median of gene expression out of the tested category. The results suggested that bovine sperm affect the transcriptome profile of the bovine oviduct after FTAI. Moreover, based on the functional enrichment analysis, an effect of the sperm-oviduct interaction on some pathways analysed by GO and KEGG was identified. This research was financially supported by FAP-DF, FAPESP, CNPq.

Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a control. Sperm binding to oviduct cells was reduced by more than half in the three sorted samples when compared to the control sperm that were not sorted. The percentage of sperm that were motile 24 h after sorting was also decreased significantly in each of the sorted sample groups when compared to the unsorted control. In contrast, sorting did not decrease the percentage of sperm that bound purified soluble glycans or the location on sperm to which they bound. There was also no difference in sperm acrosome status among the four groups. In summary, sorting reduced sperm binding to the complex matrix around oviductal cell aggregates but sperm binding to purified soluble oviduct glycans was not affected. The requirement for higher affinity and motility to bind glycans immobilized on oviduct cells may explain this difference. The reduction in sperm fertility observed following sex-sorting may be explained partially by a reduced or altered ability to bind to the oviduct epithelium.

Flow cytometry sex sorting affects bull sperm longevity and compromises their capacity to bind to oviductal cells

This study assessed the effect of flow cytometry sexing on sperm longevity and the capacity of sperm to bind to oviductal cells. Each ejaculate from four bulls was divided into two fractions: the first was immediately frozen as non sexed sperm (NS) and the second was sexed originating X- and Y-bearing sperm. The fourth treatment had sex-sorted X and Y sperm (XY) combined. Sperm from each group was assessed for sperm characteristics after thawing, after washing and at 2, 4, 8 and 12 h of incubation at 39 °C in 5% CO2 in air. For the binding test, sperm were incubated in sp-TALP medium for 30 min or 24 h with oviductal explants. Percentages of motility (58.1 ± 4.3 and 35.2 ± 4.4), progressive motility (46.1 ± 6.1 and 25.7 ± 4.8), mitochondrial membrane potential (79.2 ± 8.3 and 69.0 ± 6.3), plasma membrane stability (77.4 ± 4.6 and 19.4 ± 4.2), and live sperm with intact acrosome (57.2 ± 8.5 and 31.3 ± 7.9) were higher in NS than in XY, respectively (P < 0.05). Those differences were maintained for up to 8 h. The sexing process did not affect the sperm binding to the explants after 30 min. However, after 24 h, XY had less (6.7 ± 2.0) sperm bound to explants than NS (23.6 ± 7.2). In conclusion, even though XY was of lower quality than NS, the decreases in quality in both NS and XY groups were similar between groups during incubation. Moreover, the sex-sorting process affected the ability of sperm to remain bound to oviductal explants.

Different inflammatory responses of bovine oviductal epithelial cells in vitro to bacterial species with distinct pathogenicity characteristics and passage number

The bovine oviduct provides the site for fertilization and early embryonic development. Modifications to this physiological environment, for instance the presence of pathogenic bacterial species, could diminish reproductive success at early stages of pregnancy. The aim of this study was to elucidate the inflammatory responses of bovine oviductal epithelial cells (BOEC) to a pathogenic bacterial species (Trueperella pyogenes) and a potentially pathogenic bacterium (Bacillus pumilus). BOEC from four healthy animals were isolated, cultured in passage 0 (P0) and passaged until P3. Trypan blue staining determined BOEC viability during 24 h co-culture with different multiplicities of infection (MOI) of T. pyogenes (MOI 0.01, 0.05, 0.1 and 1) or B. pumilus (MOI 1 and 10). BOEC remained viable when co-cultured with T. pyogenes at MOI 0.01 and with B. pumilus at MOI 1 and 10. Extracted total RNA from control and bacteria co-cultured samples was subjected to reverse transcription-quantitative polymerase chain reaction (RTq-PCR) to determine mRNA expression of various studied genes. The rate of release of interleukin 8 (IL8) and prostaglandin E2 (PGE2) from BOEC was measured by ELISA after 24 h co-culture with bacteria. RT-qPCR of various selected pro-inflammatory factors revealed similar mRNA expression of pro-inflammatory factors in BOEC co-cultured with T. pyogenes and in the controls. Higher mRNA expression of IL 1A, -1B, tumor necrosis factor alpha and CXC ligand (CXCL) 1/2, −3, −5 and IL8 and PG synthesis enzymes in BOEC co-cultured with B. pumilus was observed. In the presence of B. pumilus a higher amount of IL8 and PGE2 was released from BOEC than from controls. The viability and pro-inflammatory response of P3 BOEC incubated with bacteria was lower than in P0 BOEC. These findings illustrate the pathogenicity of T. pyogenes towards BOEC in detail and the potential role of B. pumilus in generating inflammation in oviductal cells. Culturing conditions influenced the pro-inflammatory responses of BOEC towards bacteria. Therefore, researchers conducting epithelial-bacterial in vitro co-culture should not underestimate the effects of these parameters.

Effect of co-culture canine cumulus and oviduct cells with porcine oocytes during maturation and subsequent embryo development of parthenotes in vitro

In the estrus stage, canine oocytes are surrounded by cumulus cells and undergo maturation in the oviduct for 2–3 days after ovulation. We hypothesized that canine oviduct cells (cOC) and canine cumulus cells (cCC) during this stage might affect the maturation of oocytes and thereby improve subsequent embryo development. Therefore, the objective of this study was to compare the effects of a cOC and cCC co-culture on oocyte in vitro maturation (IVM) and subsequent embryo development, and to analyze the gene expressions in a molecular fashion what co-culture actually gives the specific pathways in which the co-culture cells act to improve maturation and embryo development.The effect of co-culture using cOC and cCC on porcine oocyte IVM was investigated. Thereafter, oocytes were activated using electrical stimulation and embryo developmental competence was estimated. The expression of the genes related to oocyte maturation, embryo development and apoptosis were analyzed. Also, reactive oxygen species (ROS) levels after IVM was analyzed.The IVM rate and embryo development including cleavage, blastocyst formation rates, and total blastocyst cell numbers from cOC group were significantly higher than other groups (P < 0.05). The expression of SMAD2/3 and growth differentiation factor 9 (GDF9) was significantly increased in cOC and oocytes from the cOC group compared with other groups. Moreover, the levels of GDF9, prostaglandin-endoperoxide synthase 2 (PTGS2), WNT3A and matrix metalloproteinase 2 (MMP2) were significantly up-regulated in blastocysts from the cOC group. The concentration of ROS was significantly lower in the supernatant of cOC groups compared with other groups. Also, the expression of BCL2 was significantly increased in porcine cumulus cells and oocytes from cOC group.The present study demonstrated that co-culture with cOC improved in vitro porcine oocyte maturation and subsequent embryo development competence. Also, co-culture with cOC during IVM induces a suitable environment for oocyte maturation by enhancing the mRNA level of SMAD2/3 and GDF9, and for embryo development by elevating the expression level of PTGS2, WNT3A and MMP2. In addition, the decreased ROS level in cOC co-culture could have a beneficial influence on oocyte maturation.

In vivo genetic cell lineage tracing reveals that oviductal secretory cells self-renew and give rise to ciliated cells

The epithelial lining of the fallopian tube is vital for fertility, providing nutrition to gametes and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions primarily consist of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing fallopian tube epithelial homoeostasis are unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and various Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple-transgenic mouse models, we showed that Wnt/β-catenin signaling is essential for self-renewal as well as the differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

Analysis of fructose and mannose – regulatory peptides signaling pathway in porcine epithelial oviductal cells (OECs) primary cultured long-term in vitro

AbstractThe morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the compound process responsible for proper oocytes transport and successful fertilization. However, the main mechanisms which regulated this process are still not entirely known. Moreover, the OECs metabolism, which may be identified as the “cellular activity” marker, is poorly recognized. In this study we investigated the fructose and mannose metabolic pathway in porcine OECs primary long-term cultured in vitro.In our study, we employ a primary long term in vitro culture (IVC) and microarray approach (the Affymetrix microarray were used for analysis of transcriptomic profile of OECs) for expression levels analysis.We found that from the whole analyzed transcriptome, 1537 genes were upregulated and 995 were down regulated after 7 days of culture, 1471 genes were upregulated and 1061 were downregulated after 15 days of culture and 1329 genes were upregulated and 1203 were downregulated after 30 days of culture. Moreover, the differential expression of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 at 24 hours, 7 day, 15 day and 30 day, was also observed.We suggested that fructose and mannose metabolism may be important molecular bio-marker of porcine OECs capability in in vitro model. The metabolic profile is significantly accompanied by cells proliferation in vitro. The transcriptomic profile of SORD, FPGT, PFKFB4, TPI1, MPI, ALDOC, HK2 and PFKFB3 expression may be identified as “fingerprint” of fructose and mannose metabolism in OECs as well as involved in cellular in vitro developmental capacity in pigs.