Oviductal Cells Research Articles

Inflammatory Lesions in the Oviducts and Its Relationship With Endometrial Inflammation and Ovarian Activity in Criollo Mares

The equine oviduct is considered as a reproductive organ with essential functions for early reproductive events. There is extensive literature on the pathology of the equine endometrium relating to reduced fertility, but in only few studies, the influence of alterations of the equine oviduct has been examined. The aim of this study was to investigate the incidence of inflammatory lesions in the oviduct and its association with endometrial inflammation and ovarian activity in mares. One hundred fifty Criollo mares were selected from a population of horses sent to slaughter at an abattoir. Internal reproductive tracts were recovered within 10 minutes after slaughter, and uterine and oviducts samples were collected, processed, stained with hematoxylin-eosin, and analyzed. From the total mares examined, 69.3% were considered cyclic, 18.7% in anestrus, and 12% in the transitional phase. No significant (P > .05) correlations coefficients were observed among the degree of inflammatory cells in oviduct and in endometrium from cyclic and noncyclic mares. Most studied mares had some degree of lymphocytic infiltration in the oviduct mucosa (64%). The incidence of neutrophilic and eosinophilic infiltration in oviducts occurred in 8% and 4% of the mares studied, respectively. Cyclicity did not influence the neutrophilic and eosinophilic infiltration (P > .05). The incidence of oviduct inflammation in Criollo mares is more frequent than previously reported in other breeds; however, the neutrophilic and eosinophilic infiltration is rare. Endometrial infiltration is not an oviduct infiltration cause and is possible to observe oviduct inflammation without endometrial inflammation.

Spontaneous Transformation of Murine Oviductal Epithelial Cells: A Model System to Investigate the Onset of Fallopian-Derived Tumors.

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOELOW) was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH). The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperitoneal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers, such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOEHIGH and MOELOW were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53, and FOXM1 with reduced expression of p21. MOEHIGH were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.

Estradiol increases IP3 by a nongenomic mechanism in the smooth muscle cells from the rat oviduct.

Estradiol (E2) accelerates egg transport by a nongenomic action, requiring activation of estrogen receptor (ER) and successive cAMP and IP3 production in the rat oviduct. Furthermore, E2 increases IP3 production in primary cultures of oviductal smooth muscle cells. As smooth muscle cells are the mechanical effectors for the accelerated oocyte transport induced by E2 in the oviduct, herein we determined the mechanism by which E2 increases IP3 in these cells. Inhibition of protein synthesis by Actinomycin D did not affect the E2-induced IP3 increase, although this was blocked by the ER antagonist ICI182780 and the inhibitor of phospholipase C (PLC) ET-18-OCH3. Immunoelectron microscopy for ESR1 or ESR2 showed that these receptors were associated with the plasma membrane, indicating compatible localization with E2 nongenomic actions in the smooth muscle cells. Furthermore, ESR1 but not ESR2 agonist mimicked the effect of E2 on the IP3 level. Finally, E2 stimulated the activity of a protein associated with the contractile tone, calcium/calmodulin-dependent protein kinase II (CaMKII), in the smooth muscle cells. We conclude that E2 increases IP3 by a nongenomic action operated by ESR1 and that involves the activation of PLC in the smooth muscle cells of the rat oviduct. This E2 effect is associated with CaMKII activation in the smooth muscle cells, suggesting that IP3 and CaMKII are involved in the contractile activity necessary to accelerate oviductal egg transport.

Sperm storage and spermatozoa interaction with epithelial cells in oviduct of Chinese soft-shelled turtle, Pelodiscus sinensis.

Spermatozoa are known to be stored within the female genital tract after mating in various species to optimize timing of reproductive events such as copulation, fertilization, and ovulation. The mechanism supporting long-term sperm storage is still unclear in turtles. The aim of this study was to investigate the interaction between the spermatozoa and oviduct in Chinese soft-shelled turtle by light and electron microscopy to reveal the potential cytological mechanism of long-term sperm storage. Spermatozoa were stored in isthmus, uterine, and vagina of the oviduct throughout the year, indicating long-term sperm storage in vivo. Sperm heads were always embedded among the cilia and even intercalated into the apical hollowness of the ciliated cells in the oviduct mucosal epithelium. The stored spermatozoa could also gather in the gland conduit. There was no lysosome distribution around the hollowness of the ciliated cell, suggesting that the ciliated cells of the oviduct can support the spermatozoa instead of phagocytosing them in the oviduct. Immune cells were sparse in the epithelium and lamina propria of oviduct, although few were found inside the blood vessel of mucosa, which may be an indication of immune tolerance during sperm storage in the oviduct of the soft-shelled turtle. These characteristics developed in the turtle benefited spermatozoa survival for a long time as extraneous cells in the oviduct of this species. These findings would help to improve the understanding of reproductive regularity and develop strategies of species conservation in the turtle. The Chinese soft-shelled turtle may be a potential model for uncovering the mechanism behind the sperm storage phenomenon.

Regulation of Sperm Function by Oviduct Fluid and the Epithelium: Insight into the Role of Glycans.

In many vertebrates, females store sperm received at mating in specialized reservoirs until fertilization. In some species, sperm are routinely stored for up to a decade. But the structures used to store sperm vary considerably across taxa, suggesting the underlying mechanisms might be equally variable. In mammals, after mating, sperm pass through the utero-tubal junction and bind to epithelial cells of the oviduct isthmus to form a reservoir. This reservoir regulates sperm function, including viability and capacitation, ultimately affecting sperm lifespan. In addition, sperm binding to oviduct cells influences oviduct cell gene transcription and translation, perhaps to aid sperm storage and fertility. The sperm reservoir allows successful reproduction in species in which semen deposition and ovulation are not always synchronized. The focus of this review is on recent studies of the functions of oviduct fluid and of the adhesion molecules that allow sperm to adhere to the oviduct epithelium. The important of glycans on the oviduct epithelium is highlighted.

Fibronectin stimulates human sperm capacitation through the cyclic AMP/protein kinase A pathway.

Does fibronectin (Fn) stimulate the sperm capacitation process in humans? Fibronectin stimulates human sperm capacitation. Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α5β1) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.

Effect of stearic acid on in-vitro formation of sheep oocytes

The present study was undertaken to study the effect of stearic acid (SA), a non esterified fatty acid (NEFA) on oocyte development of ewes (Ovis aries). Sheep oocytes were matured in vitro in the presence of different concentration of stearic acid (0 μM, 10 μM, 20 μM, and 30 μM) for 24h. After in vitro maturation, oocytes were in vitro inseminated with cauda epidydimus sperm processed with BO medium with concentration of 2 million sperms/ml and cultured on oviductal cell culture for 8 days. Oocytes were evaluated for cleavage and fertilization rates and after 7–8 days post insemination zygotes were evaluated for moruale /blastocyst stages. The maturation, cleavage and morulae/ blastocyst production rates were significantly lowered in media containing 20 μM stearic acid. Increment of stearic acid to 30 μM in media further reduced the maturation, cleavage and morulae/ blastocyst production. In conclusion, the metabolic stressors NEFA (SA) impaired the maturation, viability, cleavage and embryo production rates at the level 20 μM in ewes.

Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein.

Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

Heat shock protein A8 stabilizes the bull sperm plasma membrane during cryopreservation: Effects of breed, protein concentration, and mode of use

Heat shock protein A8 (HSPA8) is a highly conserved member of the Hsp70 family, which is expressed in oviductal cells, translocated into oviductal fluid, and becomes attached to the sperm surface during sperm transport. Previous research has shown that HSPA8 supports mammalian sperm viability during in vitro incubation at both 5 °C and body temperature. The present series of experiments was designed to explore the possibility that bovine recombinant HSPA8 might therefore protect bull spermatozoa during cryopreservation through its beneficial effects on the sperm plasma membrane. Soy-based cryopreservation media were used in these experiments. The effects of HSPA8 addition before freezing were examined at concentrations ranging from 0.2 to 6.4 μg/mL, whereas the effects of postthaw HSPA8 addition were tested between 0.2 and 12.8 μg/mL. When bull spermatozoa (from beef and dairy breeds) were frozen in the presence of HSPA8, beneficial but complex effects on postthaw viability were observed. Low HSPA8 concentrations (0.2 and 0.4 μg/mL) resulted in significantly reduced postthaw sperm viability, but concentrations above 0.8 μg/mL improved plasma membrane integrity. If HSPA8 was added to spermatozoa after thawing, outcomes were also biphasic and beneficial effects on viability were only seen if the HSPA8 concentration exceeded 3.2 μg/mL. Beneficial effects were significantly more apparent with beef rather than dairy breeds. When HSPA8 was used in combination with cholesterol-loaded cyclodextrin, spermatozoa from the beef breeds showed significantly lower apoptotic effects. This was not observed with the dairy breeds.

TRANSFECTION OF EXOGENOUS DNA IN CHICKEN OVIDUCT CELLS IN VITRO

Effective production of pharmaceutical proteins using transgenic poultry may be achieved only from a construction of expression vector for chicken oviduct bioreactor. This study was focused on development of temporary expression system by using primary oviduct epithelial cells in which transfected gene expression can be studied. The present work was aimed to study three manners for preparation of primary oviduct cells, but only one was effective. Selective digestion of chicken oviduct tissue pieces in trypsin at 38° C and mechanical withdrawal of upper cell layer resulted in the isolation of purity primary culture. Experiments indicated that monolayer cultured for 2-3 d were fit to be passaged. Then after 1-2-d, the cells were transfected. Based on our result with primary culture, we did not used complicated enzyme treatment and extra equipment in order to increase the purity of epithelial cells. Liposomes «Lipofectine® 2000» were used for primary oviduct cells transfection of a plasmid designed based on the pIRES EGFP2 vector (Clontech, United States). The ratio of cells carrying GFP activity was 5-8% of the total number of cultured cells. This researches was focused on development in vitro of temporary expression system in which transfected gene expression can be tested for 4-5 d.

Loss of LKB1 and p53 synergizes to alter fallopian tube epithelial phenotype and high-grade serous tumorigenesis.

Liver kinase B1 (LKB1) is a tumor suppressor ubiquitously expressed serine/threonine protein kinase involved in energy metabolism and cellular polarity. In microarray experiments that compared normal tubal epithelium with high-grade serous carcinoma (HGSC), we observed a decrease in LKB1 mRNA expression in HGSC. In this study, we demonstrate that loss of cytoplasmic and nuclear LKB1 protein expression is frequently observed in tubal cancer precursor lesions as well as in both sporadic and hereditary HGSCs compared with other ovarian cancer histotypes. Bi-allelic genomic loss of LKB1 in HGSC did not account for the majority of cases with a decrease in protein expression. In vitro, shLKB1-fallopian tube epithelial (FTE) cells underwent premature cellular arrest and in ex vivo FTE culture, LKB1 loss and p53 mutant synergized to disrupt apical to basal polarity and decrease the number of ciliated cells. Overexpression of cyclin E1 allowed for bypass of LKB1-induced cellular arrest, and increased both proliferation and anchorage-independent growth of transformed FTE cells. These data suggest that LKB1 loss early in ovarian serous tumorigenesis has an integral role in tumor promotion by disrupting apical to basal polarity in the presence of mutated p53 in fallopian tube cells.

Expression pattern of inflammatory response genes and their regulatory micrornas in bovine oviductal cells in response to lipopolysaccharide: implication for early embryonic development.

In the present study, we used an in vitro model to investigate the response of the oviduct with respect to inflammatory mediators and their regulatory microRNAs in case of bacterial infection and subsequent association with embryo survival. For this, we conducted two experiments. In the first experiment, cultured primary bovine oviductal cells (BOEC) were challenged with lipopolysaccharide (LPS) for 24h and the temporal expression pattern of inflammatory mediators and their regulatory microRNAs were measured at 0, 3, 6, 12, 24 and 48h after LPS treatment. Intriguingly, the temporal patterns of all miRNAs except miR-21 were significantly up-regulated at 6h after LPS treatment. Whereas, we observed significant overexpression of pro-inflammatory mediators as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) after LPS challenge for 24h. On the other hand, the expression level of essential elements like oviductal glycoprotein 1 (OVGP1) and insulin-like growth factor 2 (IGF2) was significantly decreased in challenged groups compared with control. Moreover, miR-155, miR-146a, miR-223, miR-21, miR-16 and miR-215 have shown a clear suppression in challenged group after LPS treatment. In the 2nd experiment there were four groups of blastocysts produced, namely embryo+LPS free media, embryo+LPS, BOEC+embryo and BOEC+embryo+LPS. The suboptimal oviduct environment due to LPS challenge is found to have a significant influence on the expression of inflammatory response genes (TNFα and CSF1), stress response genes (SOD and CAT), mitochondrial activity, reactive oxygen species (ROS) accumulation and apoptotic level either in cultured or co-cultured blastocysts. Collectively, LPS challenge led to aberrant changes in oviductal transcriptome profile, which could lead to a suboptimal environment for embryo development.

Seminal fluid promotes in vitro sperm–oviduct binding in the domestic cat (Felis catus)

From many endangered or threatened species which are expected to profit from assisted reproduction techniques, mainly epididymal sperm of dead or freshly castrated males are available. These sperm had contact to epididymal secretion products but not to seminal fluid components. Notably, products of accessory sex glands have been shown in domestic animals to condition sperm for fertilization, in particular by mediating sperm–oviduct interaction. We report for the first time that motile epididymal sperm from domestic cats are able to bind to fresh oviduct epithelial cell explants from preovulatory females (median [min, max] of 10 [8, 16] and 10 [8, 17] sperm per 0.01 mm2 explant surface from both isthmic and ampullar regions, respectively). More sperm attach to the explants when epididymal sperm were preincubated for 30 minutes with seminal fluid separated from electroejaculates of mature tomcats (median [min, max] of 17 [13, 25] and 16 [12, 21] sperm per 0.01 mm2 explant surface from isthmus and ampulla, respectively). The proportion of bound sperm increased from a median of 54% to 62% by seminal fluid treatment. Sperm–oviduct binding could be facilitated by the decelerated sperm motion which was observed in seminal fluid–treated samples or supported by seminal fluid proteins newly attached to the sperm surface. Seminal fluid had no effect on the proportion of sperm with active mitochondria. Extent and pattern of sperm interaction in vitro were independent of explant origin from isthmus or ampulla. Sperm were attached to both cilia and microvilli of the main epithelial cell types present in all explants. In contrast to published sperm-binding studies with porcine and bovine oviduct explants where predominantly the anterior head region of sperm was attached to ciliated cells, the tails of some cat sperm were firmly stuck to the oviduct cell surfaces, whereas the heads were wobbling. Whether this response is a preliminary step toward phagocytosis or a precondition to capacitation and fertilization remains to be determined. In conclusion, treatment of epididymal sperm with seminal fluid or particular protein components should be considered in future investigations for its potential to improve the outcome of artificial insemination in felids.

Von Hippel-Lindau gene expression on the human fallopian tube epithelium during the menstrual cycle.

The Von Hippel-Lindau gene (VHL) is a tumor suppressor gene, which is widely expressed in kidney, lung, breast, ovary, and cervix. VHL gene mutations can induce VHL disease and tumorigenesis. However, whether this gene is expressed in the human fallopian tube has not been evaluated. The objectives of this study were to investigate whether the VHL gene is expressed in human fallopian tube, and to investigate its expression changes during the menstrual cycle. Twenty‑seven patients undergoing abdominal hysterectomy with adnexectomy for benign uterine disease were enrolled in the study. Human fallopian tubes were divided into proliferative stage(n=14) and secretory stage(n=13) according to the stage of the menstrual cycle they were isolated from. The expression of the VHL gene and protein was studied by reverse transcription-polymerase chain reaction(RT-PCR), western blotting and immunohistochemistry, respectively. The results revealed positive expression of the VHL protein in the cytoplasm of ciliated cells of the human fallopian tube. The mRNA and protein expression of VHL in the fallopian tubes was higher in the proliferative compared to the secretory phase of the menstrual cycle, but this difference was not significant (P>0.05). Overall, this study presents data on the VHL mRNA and protein expression in the human fallopian tube, which may be relevant to the process of differentiation of ciliated and secretory cells.

Evaluation of immunological interaction between spermatozoa and fallopian tube epithelial cells.

Toll-like receptors (TLR) are one of the major compartments of innate immune system. It was revealed that the TLR have relevance in ovulation, sperm capacitation and fertilisation. So, in this study, the expression of TLR, their adaptor molecules and cytokines in human fallopian tube cell line under the effect of human normal spermatozoa was evaluated. TLR mRNA and protein were evaluated in OE-E6/E7 cell line. Semen samples from 10 donors were collected and co-incubated with OE-E6/E7 cell line and used as sperm group, and cell line without spermatozoa was used as control group. Afterwards, the level of TLR, their adaptor molecule and cytokine mRNA expression was compared using qPCR in sperm and control groups, and supernatant was used for ELISA. To determine whether elevated cytokine reaction to spermatozoa in OE-E6/E7 cell line is mediated via TLR, TLR3 function-blocking antibody was used. OE-E6/E7 cell line expressed TLR1-6 genes and proteins. TLR expressions, especially TLR3 and TLR5, in OE-E6/E7 cell line under the effect of spermatozoa were significantly higher. Also, levels of adaptor molecules and cytokine production were increased in sperm group than in control group (P < 0.05). So, it may be hypothesised that TLR are essential for spermatozoa and fallopian tube immunological interaction and for preparing safe environment for important events in fallopian tube.

The Effect of Cysteamine and Oviductal Cells in Different Culture Media on the Development of Sheep Embryos

The Effect of Cysteamine and Oviductal Cells in Different Culture Media on the Development of Sheep Embryos

105 EFFECT OF LYSOPHOSPHATIDIC ACID ON PROSTAGLANDIN PRODUCTION IN THE BOVINE OVIDUCT

The oviduct is an essential organ for successful pregnancy in mammals. The transport of gametes and early embryos is mainly induced by contraction and relaxation of smooth muscle. The contraction and relaxation of bovine oviductal smooth muscle are induced by prostaglandin (PG) F2α and PGE2, respectively. Lysophosphatidic acid (LPA), a type of phospholipid, is involved in various physiological actions such as promoting inflammation and cellular proliferation in various organs. LPA acts through at least 6 G protein-coupled receptors. Both LPA and LPA receptors are expressed in endometrium and, moreover, LPA affects PG production by the endometrium in cow. Based on the above findings, we hypothesised that LPA is locally involved in PG production by oviductal cells to promote motility of oviductal smooth muscle in cow. Oviductal samples ipsilateral to a corpus luteum or a dominant follicle at peri-ovulation (0–6 and 19–21 days after ovulation) were collected in abattoir. Messenger RNA expression of LPA receptors (LPAR1–6) and LPA-producing enzymes (ATX, PLA1α, PLA1β) was examined in ampullary and isthmic tissues. Expression in cultured epithelial and stromal cells isolated from the bovine oviduct were also examined to determine the cells possessing LPA receptors and LPA-producing enzymes. In addition, the effect of LPA (0.1, 1, 10 μM) on the expression of cyclooxygenase (COX)-1 and COX-2 (PG-synthesising enzymes) and on PGE2 and PGF2α production by cultured epithelial and stromal cells was investigated. The significant differences (P &lt; 0.05) were determined by Student's t-test for 2 groups, and by one-way ANOVA followed by Tukey's multiple comparison test for more than 3 groups. LPAR1–6, ATX, PLA1α, and PLA1β were expressed in both ampullary and isthmic tissues as well as in both cultured epithelial and stromal cells. The expression of LPAR1–3 was significantly lower in the isthmic tissues than in the ampullary tissues, whereas the expression of LPAR4–6 was significantly higher in the isthmic tissues than in the ampullary tissues. The expression of COX-2 was significantly stimulated by 10 μM LPA in cultured isthmic stromal cells. In addition, LPA significantly stimulated both PGE2 and PGF2α production by cultured isthmic stromal cells. In the isthmus of the oviduct, LPA produced by epithelial and stromal cells may stimulate the expression of COX-2 in the stromal cells, followed by increased PG production. Because the mRNA expression of LPAR4–6 is higher in the isthmus than in the ampulla, those effects of LPA might be mediated by activation of LPAR4–6. The overall findings suggest that LPA is one of the regulating factors for transport of gametes and early embryos by controlling the motility of smooth muscle in the bovine oviduct.

Analyses of chicken sialyltransferases related to N-glycosylation

Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-β1,4-N-acetylglucosamine (Galβ1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.

211 TRANSCRIPTS EXPRESSION RELATED TO MAPK/SMAD2 PATHWAY IN OVIDUCT CELL, CUMULUS CELL, AND OOCYTE DERIVED FROM DIESTRUS BITCHES

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are known to play an essential role in regulating ovarian follicular development and ovulation rate via SMAD pathway in several species and have different functions depending on the species. However, their expression and function have not yet been reported in dogs. Therefore, the aim of present study is to analyse expressions of transcripts related to the MAPK/SMAD2 pathway in oviduct cell, cumulus cell, and oocyte derived from diestrus bitches. Ovariohysterectomy was performed in 5 large, mixed breed dogs 3 days after serum progesterone reached 5–10 ng mL–1. Oviduct cells were obtained by scraping the superficial oviduct tissue and oocytes were obtained by mincing the ovary. Both cumulus cells derived from non-ovulated oocytes (IM-cumulus) and ovulated oocytes (M-cumulus) were obtained. Oviduct, IM-cumulus, and M-cumulus cells were cultured using RCMEP media and cryopreserved using FBS containing 10% DMSO. Total RNA was extracted from Oviduct, IM-cumulus, and M-cumulus cells and oocytes. After cDNA synthesis, transcript expression of β-actin, GDF9, MAPK1, BMP6, and SMAD2 genes were analysed. The data was analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). The threshold for statistical significance was set at P &lt; 0.05. In results, mRNA expression of MAPK1 was not significantly different among the cells. However, expressions of BMP6, GDF9, and SMAD2 were significantly higher in oocytes compared to oviduct, IM-cumulus, and M-cumulus cells. Although oviduct cells showed increased GDF9 and SMAD2 transcripts similar to that of oocytes, there was no significant difference between them. In conclusion, MAPK/SMAD2 pathway may have important roles in growth of immature oocytes in dogs and oviduct cells may help the maturation of ovulated immature oocytes. Further studies will be conducted to analyse MAPK/SMAD2 pathway in anestrus, proestrus, and oestrus periods.This research was supported by RDA (#PJ008975022014), IPET (#311062-04-3SB010), Research Institute for Veterinary Science, the BK21 plus program, and global PhD Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014H1A2A1021187).

104 EXPRESSION OF ACTIVIN A AS A LOCAL REGULATOR IN THE BOVINE OVIDUCT

Activin (ACV) is known as a local regulator of several reproductive functions including follicular development and implantation in mammals. ACVA is a glycopeptide belonging to the transforming growth factor β superfamily, and is a homodimer of inhibin ßA (INHBA) subunits. Follistatin (FST), an ACV-specific binding protein, inhibits ligand-receptor binding. ACV receptor (ACVR) is a hetero-tetramer consisting of 2 kinds of protein, ACVR1 or ACVR1B and ACVR2A or ACVR2B. The oviduct provides an optimal environment for sperm capacitation, fertilization, and early embryonic development. Previous reports have demonstrated that ACVRs were expressed in bovine oocytes and embryos, and that early embryonic development is facilitated by ACVA in vitro. ACVA produced by the bovine oviduct may affect gametes and embryos as well as oviductal cells as a local regulator in cow. Bovine oviductal samples were classified into 6 stages of the oestrous cycle (day of ovulation; Days 2–3 after ovulation; Days 5–6; Days 8–12; Days 15–17; Days 19–21). We examined (1) protein expression of ACVA and FST in oviductal fluid collected from the ampulla and isthmus, (2) mRNA expression of INHBA and FST in the ampullary and isthmic oviductal tissues during the oestrous cycle, (3) the effects of oestradiol-17β (E2: 0.1, 1, 10 nM) and progesterone (P4: 1, 10, 100 nM) on mRNA expressions of INHBA and FST in cultured oviductal epithelial cells isolated from the ampulla and isthmus, and (4) mRNA expression of ACVRs in tissues and in cultured epithelial and stromal cells. The main findings were as follows: (1) Both ACVA and FST were detected throughout the oestrous cycle in the oviductal fluid of the ampulla and isthmus. (2) INHBA expression was higher in the isthmus than in the ampulla. FST expression in the ampulla was lowest at peri-ovulation, INHBA expression in the isthmus was highest on the day of ovulation and FST in the isthmus during Days 2–6 was highest. Because an increase of ACVA production and a decrease of FST production raise ACVA bioactivity, ACVA seems to be most active at peri-ovulation in both the ampulla and isthmus. (3) In the cultured isthmic oviductal epithelial cells, 10 nM E2 significantly stimulated INHBA expression, but tended to suppress FST expression. Therefore, the bioactivity of ACVA seems to be controlled by E2 during the oestrous cycle in the isthmus. (4) The expression of ACVR1B and ACVR2A was clearly detected in the tissues as well as in cultured epithelial and stromal cells. The overall findings suggest that ACVA secreted into oviductal fluid plays an important role in oviductal functions, including fertilization in the ampulla and sperm motility and viability in the isthmus. It is also suggested that ACVA acts on both epithelial and stromal cells as a local regulator of cellular functions, such as cellular proliferation and secretion in the cow.