Bovine Oviduct Epithelial Cells Research Articles

The interactions between nerve growth factor and gonadotrophins in bovine oviduct

Nerve growth factor promotes the survival and differentiation of nervous cells and is thought to play an important role in the development of reproductive tissues. The aims of this work were to detect the presence of NGF and its receptor NTRK1 in bovine oviduct samples, and to investigate the regulatory interactions between NGF/NTRK1 and gonadotrophins in bovine oviduct epithelial cells. Both transcripts and proteins of NGF and NTRK1 were detected by RT-PCR and Western blotting, and the corresponding proteins were specifically immunolocalized in oviduct epithelial cells. In addition, real-time PCR experiments revealed that the levels of NGF and NTRK1 mRNA in oviduct epithelial cells treated with exogenous FSH or LH were greater than those in negative control cells (P<0.05). Similarly, treatment with NGF significantly increased the expression of FSHR and LHR in oviduct epithelial cells via its effects on NTRK1 (P<0.05). This process was suppressed by treatment with the NTRK1 inhibitor K252α. We conclude that NGF/NTRK1 may have a role in regulating the function of bovine oviducts via its interactions with gonadotrophins.

Bovine oviductal epithelial cells: Long term culture characterization and impact of insulin on cell morphology

In vitro models that resemble cell function in vivo are needed to understand oviduct physiology. This study aimed to assess cell functions and insulin effects on bovine oviductal epithelial cells (BOECs) cultured in an air–liquid interface. BOECs (n=6) were grown in conditioned Ham's F12, DMEM or Ham's F12/DMEM with 10% fetal calf serum (FCS) for 3 weeks. After selecting the most suitable medium (Ham's F12), increasing insulin concentrations (1ng/mL, 20ng/mL and 5μg/mL) were applied, and cell morphology and trans-epithelial electrical resistance (TEER; n=4) were evaluated after 3 and 6 weeks. Keratin immunohistochemistry and mRNA expression of oviductal glycoprotein 1 (OVGP1) and progesterone receptor (PGR) were conducted (n=4) to assess cell differentiation. BOECs grown without insulin supplementation or with 1ng/mL of insulin displayed polarization and secretory activity. However, cells exhibited only 50% of the height of their in vivo counterparts. Cultures supplemented with 20ng/mL insulin showed the highest quality, but the 5μg/mL concentration induced massive growth. TEER correlated negatively with insulin concentration (r=−0.459; p=0.009). OVGP1 and PGR transcripts were still detectable after 3 and 6 weeks. Cellular localization of keratins closely resembled that of BOECs in vivo. Cultures showed heterogeneous expression of PGR and OVGP1 in response to estradiol (10pg/mL). In summary, BOECs grown for long term in an air–liquid interface expressed markers of cell differentiation. Additionally, insulin supplementation (20ng/mL) improved the cell morphology in vitro.

Effect of zearalenone on estrogen receptor, IGF-I and IGF-II genes expression in bovine oviduct epithelial cell culture

Zearalenone (ZEA) is a nonsteroidal estrogenic component that we assumed ZEA can bind to estrogen receptors and causes estrogenic responses and affect Insulin like growth factors (IGF) secretion and cause embryo growth retardation and abortion in cattle. Therefore bovine oviduct epithelial cells cultured (BOEC) in DMEM-Ham F12 medium, 1% Penicillin-erythromycin and 5% fetal bovine serum. The cells passaged for 2 times. Culture flasks divided into 4 treatments and 3 replications. Amounts of 100, 160 and 250 µl of ZEA(10000 ng/ml) added to 3 treatment of experiment and one treatment maintained as control group. After 36 hours cells detach from flasks surface and total RNA extracted. Total RNA converted into cDNA and estrogen receptor, IGF-I and IGF-II genes expression assessed with quantitative Real Time PCR. Results analyzed with SPSS statistics 20. The results show that estrogen receptor gene expression in presence of different amount of ZEA had a significant difference (P<0.01) with control group, but IGF-I, II gene expressions had no significant difference (P<0.01) between treatments and control group. It means that ZEA is agonist to estrogen receptor and it can activate estrogen receptors transcription. Although ZEA is able to activate estrogen receptors and cause alteration in function of reproductive tract. IGFs genes expressed in both treatments and control group and there was no any difference between them. ZEA is group of endocrine disrupters, but it has no effect on IGFs system hormones.

Evaluation the bovine oviduct epithelial cells culture in monolayer system.

The oviduct is an important site in reproduction, such as fertilization and early embryonic development. In this study bovine oviduct epithelial cells (BOEC) isolated and cultured in monolayer system to evaluate the proliferation and viability of cells in quality manner before and after two time passages. The epithelial cells isolated mechanically from oviduct tissue in sterile condition. Isolated cells floated in culture medium, fetal bovine serum and antibiotics in a T-24 culture flask. Two days after seeding have been observed that cells adhered to flask surface and are proliferating. The rate of cell proliferation was well. After one week the cells covered the flask surface and were confluent. Then the cells isolated from flask by trypsin-EDTA enzyme and passage into 4 flasks. Cells cover the flasks in 10 days and passage into 12 flasks. In the second passage the cells was confluent in 12 days. We observed that in first culture there was a little dead cell during the culture and cells proliferation rate was high. After each passage the number of dead cells in flasks increased and cells proliferation rate in each passage decreased.

Development rate and gene expression of IVP bovine embryos cocultured with bovine oviduct epithelial cells at early or late stage of preimplantation development

The use of somatic cells for coculture with embryos has been amply investigated to study embryo maternal interactions. The use of bovine oviduct epithelial cells (BOEC) has been shown to improve the blastocyst rate and quality, affecting their gene expression profile. In this study, we evaluated different timings of BOEC coculture for the development of in-vitro–produced embryos and their effects on blastocysts rate and mRNA abundance of some genes that are important for embryo development. Our results confirmed the positive effects of BOEC on early development of bovine embryos. The presence of the cells during the first four days or during the last four days of development was enough to produce the full BOEC effect. When the presence of BOEC was restricted to the four first days, the kinetics of blastocyst development was accelerated, with significantly more blastocysts at Days 6 and 7 than when the cells were present all along the culture or only during the last four days. Older cells used at early stage were not active anymore. Using young cells at late stage did not improve the cell effect, compared with the older ones. Therefore, the lower effect of BOEC at late stage, compared with early period, may not be explained by cell aging. In addition, the presence of BOEC, at early or late stages, induced changes in the embryos expression profile of genes known to be related to embryo quality, suggesting reduced apoptosis and increased capacity to struggle against oxidative stress after coculture. In conclusion, we confirmed the effect of BOEC on the rate and quality of bovine IVP embryos development. We found for the first time that the presence of BOEC during the four first days of the 8-days development is enough to produce these effects. These first four days represent the period of the presence of the embryos in the oviduct in vivo, highlighting the physiological relevance of this in vitro model of coculture. In addition, we found that the presence of BOEC at early stages of development induced modification of transcription profile in the blastocyst, four days later, suggesting an epigenetic regulation induced by BOEC in growing embryos.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10μM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes, and is involved in fertilization-related events

Fertilization is a calcium-dependent process that involves sequential cell–cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein β-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and β-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine–induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. β-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell–cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm–OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.

Bone morphogenetic proteins in the bovine oviduct: Differential expression of BMP-5 in the isthmus during the estrous cycle

Bone morphogenetic proteins (BMPs) play a crucial role in mammalian reproduction, but little is known about their expression and function in the oviduct, where preimplantation events take place. In the present study, messenger RNA (mRNA) expression of BMPs was examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in bovine oviduct epithelial cells obtained from ampulla and isthmus at different stages of the estrous cycle. Expression of BMP-2, -3, -4, -7, -10 and -15 mRNA was detected in epithelial cells of both anatomic regions, whereas BMP-5 mRNA was specifically expressed in isthmus epithelial cells throughout the estrous cycle. High expression levels for BMP-5 and for BMP-2, -4, and -7 mRNA were observed during the preovulatory stage. Considering the region-specific gene expression of BMP-5, its protein localization in the oviduct and its presence in the oviductal fluid were evaluated by immunohistochemistry and Western blot analysis. BMP-5 protein staining was observed in isthmus sections with a more intense signal in the luminal epithelial cell layer. In addition, a 21 kDa protein corresponding to the BMP-5 mature monomeric form was detected in bovine oviductal fluid throughout the estrous cycle. In conclusion, these results demonstrate that different members of the BMP family are expressed in the bovine oviduct during the estrous cycle, and reveal that BMP-5 is differentially expressed in the isthmus. The expression of this factor in the oviduct epithelium and its presence in the luminal fluid suggest a possible action of BMP-5 as a new autocrine and/or paracrine regulator of the reproductive events that occur in the bovine oviductal environment.

An acute-phase protein as a regulator of sperm survival in the bovine oviduct: alpha 1-acid-glycoprotein impairs neutrophil phagocytosis of sperm in vitro.

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.

Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator

This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E2 (PGE2), which dose-dependently (10(-6), 10(-7), and 10(-8) M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP2 receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE2 or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE2 released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP2 receptors. Thus, the bovine oviduct provides a PGE2-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1.

Beneficial effects of silymarin against nitric oxide-induced oxidative stress on cell characteristics of bovine oviduct epithelial cell and developmental ability of bovine IVF embryos

The antioxidative effects of silymarin were examined against nitric oxide-induced oxidative stress on cell characteristics of bovine oviduct epithelial cell (BOEC) and developmental rates of bovine in vitro fertilisation (IVF) embryos. The cell viability and morphology in BOEC which were treated with sodium nitroprusside (SNP) declined, as concentrations of SNP increase. The additions of silymarin 50, 100 and 200 µM into SNP 1000 µM increased the cell viability inhibited by SNP 1000 µM alone, but addition of silymarin 500 µM into SNP 1000 µM group did not recover the inhibitory effect of SNP 1000 µM (p<0.05). The levels of malondialdehyde (MDA), as the indicator of lipid peroxidation (LPO) in the treatments of SNPs increased with dose-dependant manners (p<0.05), whereas additions of silymarin 50, 100 and 200 µM into SNP 1000 µM significantly decreased MDA levels by 1.6, 1.4 and 1.3 folds of control (p<0.05). Antioxidant and Bcl-2 genes were expressed in control or SNP 1000 µM plus silymarin 200 µM treatment group, whereas pro-apoptotic genes (caspase-3 and Bax) were expressed only in SNP treatment groups. When bovine IVF embryos were co-cultured with BOEC supplemented with silymarin in the presence or absence of SNP, the development ability to blastocyst in 200 µM of silymarin alone was the highest among all treatments. These results suggest that silymarin has positive effects on cell characteristics such as viability, morphology and LPO of BOEC, and the increase of bovine IVF embryo development rate might be through antioxidative and anti-apoptotic actions.

Altered expression of inflammation-associated genes in oviductal cells following follicular fluid exposure: Implications for ovarian carcinogenesis

Evidence indicates that high-grade serous ovarian carcinoma (HGSOC) may originate from lesions within the distal fallopian tube epithelium (FTE). Our previous studies indicate that fallopian tube epithelial cells from carriers of germline mutations in breast cancer susceptibility genes exhibit a pro-inflammatory gene expression signature during the luteal phase, suggesting that delayed resolution of postovulatory inflammatory signaling may contribute to predisposition to this ovarian cancer histotype. To determine whether exposure of tubal epithelial cells to periovulatory follicular fluid alters expression of inflammation-associated genes, we used an ex vivo culture system of bovine oviductal epithelial cells. Oviductal cells grown on collagen IV-coated transwell membranes assumed a cobblestone appearance and immunocytochemistry for FoxJ1 and Pax8 indicated that both ciliated and secretory epithelial cells were maintained in the cultures. Oviductal cells were exposed to human follicular fluid or culture medium for 24 h following which total cellular RNA was extracted at various time points. Expression of genes associated with inflammation was determined by quantitative real-time RT-PCR. Exposure to follicular fluid transiently increased the transcript levels of interleukin 8 (IL8) and cyclooxygenase 2 (PTGS2), and decreased the expression of mitochondrial superoxide dismutase (SOD2), glutathione peroxidase 3 (GPX3), disabled homolog 2 (DAB2), and glucocorticoid receptor (NR3C1). Tumor necrosis factor (TNF) and IL6 levels were also decreased while those of nicotinomide phosphoribosyltransferase (NAMPT) were unaffected. This study demonstrates that periovulatory follicular fluid can act directly upon oviductal epithelial cells to alter gene expression that might contribute to early carcinogenic events. Furthermore, these findings illustrate the potential use of bovine oviductal cells to study signaling events implicated in ovarian carcinogenesis.

Bovine oviduct epithelial cells down-regulate phagocytosis of sperm by neutrophils: PGE2 as a major physiological factor toward ovulation

Bovine oviduct epithelial cells down-regulate phagocytosis of sperm by neutrophils: PGE2 as a major physiological factor toward ovulation

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

Long-term culture of primary bovine oviduct epithelial cells: Impact of insulin and EGF supplementation

Long-term culture of primary bovine oviduct epithelial cells: Impact of insulin and EGF supplementation

Regulation of Innate Immune Function in Bovine Oviduct Epithelial Cells in Culture: The Homeostatic Role of Epithelial Cells in Balancing Th1/Th2 Response

This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.

96 EXPRESSION OF THE LONG FORM OF THE LEPTIN RECEPTOR IN BOVINE OVIDUCT EPITHELIAL, UTERINE EPITHELIAL, AND BLASTOCYST-STAGE EMBRYOS

Critical reproductive events of animals are affected via metabolic cues that directly modulate the hypothalamic-pituitary-gonadal axis. The adipose-derived 146-amino-acid protein-hormone leptin has been associated with puberty and reproduction in many mammals and may be of critical importance in the regulation of reproduction. Leptin receptors are expressed in the brain, pituitary, and gonads, and there is growing evidence that leptin may be required for normal embryonic implantation and development in some animals. To date 6 isoforms of the leptin receptor have been identified; the long form (Ob-Rb) is the only isoform to be associated with signal transduction. The objective of this experiment was to test the hypothesis that the Ob-Rb isoform is expressed in cultured bovine oviduct and uterine epithelial cells, in vivo-derived mid-luteal uterine biopsies, and expanded bovine blastocyst-stage embryos. For cells, cell lines from mid-luteal abattoir-derived reproductive tracts (n = 5) were used; for uterine biopsies (n = 20), biopsies were collected from both uterine horns at the greater curvature from mature beef cows (n = 4) in mid-luteal phase; expanded IVP blastocyst-stage embryos (n = 40) were produced from abattoir-derived ovaries. For fluorescence microscopy samples were incubated in rabbit anti-human leptin receptor (#4781-L, Linco Research, St. Charles, MO) followed by Alexa Fluor 488 anti-rabbit IgG (#A-11008, Molecular Probes, Carlsbad, CA). We confirmed that the Ob-Rb isoform is expressed in both oviductal and uterine in vitro-cultured epithelial cells and uterine biopsies. All oviduct but not all uterine-cultured epithelial cells were immunopositive for the Ob-Rb isoform. All uterine biopsies were positive for the Ob-Rb isoform along the luminal side of the uterine endometrium with an apparent lack of staining occurring in myometrial tissue. Moreover, we demonstrated that all bovine blastocysts were immunopositive with staining occurring within the trophoblast cell type but not the inner cell mass. These results show for the first time that bovine oviduct and uterine epithelial cells express the long form of the leptin receptor and support earlier findings that blastocyst-stage embryos contain Ob-Rb isoforms. This suggests that the Ob-Rb isoform is a component of the bovine maternal-embryonic complex; however, its function in pre-implantation embryo development remains to be clarified.

94 A POLARIZED CELL-CULTURE SYSTEM TO STUDY THE EFFECTS OF ELEVATED SERUM NONESTERIFIED FATTY ACID CONCENTRATIONS ON THE BOVINE OVIDUCTAL MICROENVIRONMENT IN VITRO

The detrimental consequences of elevated serum nonesterified fatty acid concentrations (NEFA) typical in negative energy balance, obesity, or type II diabetes have previously been demonstrated in ovarian follicles. However, regardless its key role in reproduction, the consequences of elevated NEFA on oviductal physiology are unclear. Therefore, the aim of this study was to 1) determine whether serum NEFAs can be found in the oviductal microenvironment using a polarized oviductal epithelial cell culture and more specifically to 2) study the effect of the BSA gradient and the direction of the NEFA gradient. Bovine oviductal epithelial cells from slaughterhouse oviducts in Day 3 to 5 of the luteal phase were mechanically isolated and cultured for 7 days in a flask. Cells were transferred in a polarized oviductal epithelial cell culture-system (Snapwell® polyester membrane, pore size 0.4 µm, 4 × 106 pores cm–2) with an apical (A) and basal (B) medium supporting cell growth until the transepithelial electric resistance reached 700 Ωcm2 (Day 6). Then, cells were exposed to a transmembranary BSA (0.75 or 3% BSA) or NEFA gradient (0 or 360 µM NEFA: 115 µM oleic acid, 140 µM stearic acid, 105 µM palmitic acid) for 48 h in 4 experimental setups: 1) A and B containing 0.75% BSA and B supplemented with NEFA, 2) same as in 1 but NEFAs were added in A, 3) A containing 0.75% BSA and B supplemented with 3% BSA and NEFA, 4) same as in 3 but with 3% BSA + NEFA in A and B contained 0.75% BSA. Samples were analyzed for total NEFA and specific fatty-acid concentrations by photometric and gas chromatographic assays. In total, 72 wells in 4 replicates were cultured and analyzed. Data were processed by paired sample t-tests and student t-tests. Exposure to NEFA did not alter the transepithelial electric resistance. In Exp. 1, the total NEFA concentration in A increased with 0.013 µM (21.11%) over 48 h of NEFA exposure in the basal compartment explained by a significant rise of stearic (8.52 µM, 20.68%) and oleic acid (12.86 µM, 45.17%) accompanied with only a 19.45% decrease of total NEFA in B (P &lt; 0.05). When the transport direction was reversed in Exp. 2, the total NEFA concentration decreased 53.4% in A, with no obvious NEFA rise in B. Implementing a BSA gradient (Exp. 3 and 4) was associated with a slight decrease (13 and 15.7%, respectively; P &lt; 0.05) of NEFA concentration in the supplemented compartment, but no obvious NEFA increase in the opposite compartment could be detected. Overall, these results suggest that NEFA transport in this culture system is tightly regulated and can only be influenced by the transport direction of the NEFA. Further research should focus on transport and metabolisation of the fatty acids added.

123 GENISTEIN TRANSPORT ACROSS THE BOVINE OVIDUCT EPITHELIUM

The oviduct plays a vital role in regulating the environment surrounding the gametes and early mammalian embryo. However, the permeability of the oviduct to circulating dietary-derived compounds remains relatively unknown. The present study has investigated the barrier properties of the oviduct epithelium in vitro to the movement of genistein, a soya isoflavone and analogue of 17β-estradiol that naturally occurs in the diet and has been reported to exert teratogenic effects. Bovine oviduct epithelial cells (BOECs) were isolated from abattoir-derived reproductive tracts at the mid-luetal phase. The purity of the cell isolate was confirmed using flow cytometry to determine the proportion of cells that expressed CK18 (epithelia), and Vimentin (fibroblasts). Cells were seeded at a density of 1 × 106 mL–1 on polyethylene terephthalate transwell porous supports (Corning) and maintained between two media-filled chambers for 10 to 12 days, until they formed a polarised confluent monolayer, as confirmed by transepithelial resistance (TEER) greater than 700 Ωcm2. To assess the rate of transport, genistein was added to the basal compartment at physiologically relevant levels (100 µM) and the apical compartment was sampled at regular time points for 120 mins. The concentration of genistein in the apical and basal media was measured by HPLC. Furthermore, we compared the rate of genistein transport at physiological (39°C) and room temperature to indicate whether transport was temperature dependent. Rates of transport are expressed as mean apparent permeability coefficient and were compared between groups by a Student’s t-test. Genistein crossed the bovine oviduct epithelium at a linear rate that was higher than spontaneous diffusion across a blank membrane support (12.7 × 10–3 cm–2 µM–1 v. 7.32 × 10–3 cm–2 µM–1, n = 4; P = 0.0075). The rate of genistein transport by epithelial cells was unchanged when cells were assayed at room temperature (12.7 × 10–3 cm–2 µM–1 v. 13.11 × 10–3 cm–2 µM–1, n = 3; P = 0.76), respectively. No significant difference in the directionality of transport was found. Furthermore, TEER was maintained at approximately 700 Ωcm2, indicating that the cells remained confluent for the duration of the experiment. These data suggest that the bovine oviduct epithelial cell monolayer facilitates genistein movement from the basal to the apical compartment in vitro. Furthermore, the observation that the rate of transport is unchanged by temperature suggests a passive, trans-cellular, physicochemical mechanism, rather than an active biological process. Regardless of the mechanism, the oviduct epithelium is permeable to genistein, and may even facilitate its transport into the lumen, suggesting that gametes and early embryos could be exposed to this compound in vivo. This is relevant given the previously reported finding indicating that this naturally occurring dietary isoflavone has detrimental effects on early development (Newbold et al. 2001 Cancer Res. 61, 4325–4328). Furthermore, the results demonstrate the potential use of this epithelial model in characterising the transport or barrier properties of the oviduct epithelium towards a range of circulating xenobiotics.

Local immune tolerance via Toll-like receptor 2 and 4 in the bovine oviduct epithelial cells

Local immune tolerance via Toll-like receptor 2 and 4 in the bovine oviduct epithelial cells