Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle.

Prostaglandins (PGs) play important roles in regulation of the functions of the hen oviduct. However, little is known about the expression and localization of the rate-limiting cyclooxygenases (COX-1 and COX-2) in the oviduct. The aim of this study was to determine the COXs expression and localization in the different segments of the oviduct and to investigate changes in their expression levels during the ovulatory cycle of laying hens. White Leghorn laying hens were killed at 0, 4, 7, 16 and 24 h after oviposition, and samples from the infundibulum, magnum, isthmus, uterus, and vagina were collected. Gene and protein expressions were examined by real-time PCR and western blot, respectively, for both COX-1 and COX-2. Localization of COX-1 and COX-2 in the hen oviduct was determined by immunohistochemistry and PCR analysis of samples collected by laser capture microdissection (LCM). The expression level of COX-1 was highest in the infundibulum, while that of COX-2 was significantly higher in the uterus than in the other segments. The expression levels of COX-1 in the infundibulum and COX-2 in the uterus were higher at 0 and 24 h after oviposition, just prior to subsequent ovulation and oviposition. Western blot analysis confirmed the presence of COX-1 and COX-2 in all oviductal segments. The density of COX-2 was the highest in the uterus, and did not change during the ovulatory cycle. COX-1 and COX-2 were localized in the surface epithelium of all oviductal segments besides the uterine tubular glands. We conclude that both COXs are differentially expressed in the different oviductal segments with a temporal association to ovulation and oviposition. COX-1 and COX-2 may play an important role in the infundibulum and uterus, respectively, and COX-2 may be one of the factors regulating the induction of oviposition.

Spatial distribution of cannabinoid receptor 1 and fatty acid amide hydrolase in the cat ovary and oviduct.

Involvement of the endocannabinoid system in female reproduction has been extensively described in humans with the cognate receptors and ligands being found in the ovaries and genital tract. In human, an imbalance of the endocannabinoid system is linked with both ectopic pregnancy and infertility. In bovine species anandamide levels regulate aspects of sperm-oviduct interaction. Here we report the immunohistochemical distribution of cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) in cat ovary and oviduct, using paraffin-embedded tissue samples and commercially available antibodies. We found a differential expression of both CB1R and FAAH during different stages of ovarian function and in the oviduct. CB1R was detected only in tertiary follicle granulosa cells while more immature follicles were negative. FAAH was instead found in ovarian pre-antral follicles, the oocyte cytoplasm, and in granulosa cells of primary, secondary and tertiary follicles. Secondary and tertiary follicles were also FAAH immunoreactive. Luteal cells were immunopositive for both CB1R and FAAH. Because CBR1 in oviduct was found only in ciliated cells, it might represent a specific marker at least in cats. In contrast, FAAH immunoreactivity was observed in both ciliated and non-ciliated cells. The present study may thus serve as the starting point for further investigations on the role of the endocannabinoid system in cat reproduction. Additional work will be needed to assess whether the morphological distribution of CB1R and FAAH changes in different conditions such as pre-pubertal age, follicular phase of the sexual cycle and pregnancy.

Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during pause in laying induced by tamoxifen.

Induced pause in egg laying simulates natural molting events in which the hen's reproductive organs regress and rejuvenate. Such processes require extracellular matrix remodeling that is maintained, at least in part, by the action of proteolytic enzymes known as matrix metalloproteinases (MMPs). Nevertheless, information concerning the expression and hormonal regulation of MMP system members in chickens is scarce. Therefore, MMP-2, -7, and -9 and their tissue inhibitors (TIMP-2, -3) expression and localization were investigated in all segments of the domestic hen oviduct (infundibulum, magnum, isthmus, shell gland, vagina) during a pause in egg laying induced by tamoxifen (TMX)-an estrogen receptor modulator. Hy-Line Brown hens were treated daily with TMX (n = 6) at a dose of 6 mg/kg of body weight or a vehicle (n = 6) until complete cessation of egg laying (for 7 days). Chickens were decapitated on Day 7 of the experiment. Real-time polymerase chain reaction and Western blotting revealed section-dependent expression of MMP-2, -7, -9 and TIMP-2 and -3. Immunohistochemistry found tissue and cell-dependent localization of examined proteins in the wall of the oviduct. The MMP-2, TIMP-2, and TIMP-3 were localized mainly in the luminal epithelium, MMP-7 in the luminal and glandular epithelium, whereas MMP-9 was detected only in the connective tissue. Treatment of chickens with TMX markedly elevated the relative expression of MMP-7 and MMP-9 mRNA in the oviduct, but did not affect MMP-2, TIMP-2, and TIMP-3 mRNA levels. However, TMX increased the MMP-2 protein level in the infundibulum, shell gland, and vagina as well as activity of MMP-2 evaluated by gelatin zymography. The results obtained indicate that MMP-2, MMP-7, and MMP-9 are involved in chicken oviduct regression. Moreover, changes in the expression and activity of chosen MMPs after TMX treatment may indicate a contribution of estrogen in the regulation of transcription, translation, and/or the activity of selected elements of the MMP system.

Characteristics, tissue-specific expression, and hormonal regulation of expression of tyrosine aminotransferase in the avian female reproductive tract.

Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine to p-hydroxyphenylpyruvate. Accumulation of tyrosine in the body due to a genetic mutation in the TAT gene causes tyrosomia type II in humans. The TAT gene is regarded as a model for studying steroid-inducible factors regulating a variety of biological functions of TAT. However, little is known of the effects of estrogen on the expression of the TAT gene in chickens. Therefore, in the present study, we identified expression of the avian TAT gene in various organs. The results showed the TAT was detected predominantly in the liver and reproductive organs including testis, oviduct, and ovary. Specifically, TAT mRNA was expressed abundantly in the glandular and luminal epithelia of the oviducts in response to endogenous and exogenous estrogens which also induce dramatic morphological changes in the oviduct of chickens. In addition, target microRNAs of TAT (miR-1460, miR-1626-3p, miR-1690-5p, and miR-7442-3p) were found to modulate expression of the TAT gene. Especially, miR-1690-5p influenced TAT gene transcription by binding directly to its 3'-UTR region. Moreover, the expression of TAT was abundant in glandular epithelia of cancerous but not normal ovaries from laying hens. Taken together, our findings suggest that TAT plays an important role in the cytodifferentiation of oviducts in response to estrogen and in the progression of ovarian cancer in chickens.

Involvement of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct regression and recrudescence.

This study was undertaken to examine mRNA and protein expression, localization of selected MMPs and their tissue inhibitors (TIMPs), and the activity of MMPs in all segments of the chicken oviduct (infundibulum, magnum, isthmus, shell gland, vagina) during a pause in egg laying induced by food deprivation. Control chickens (n = 18) were fed ad libitum, while experimental birds (n = 18) were fasted for 5 days, followed by being fed every other day, and then fed daily from day 10 onwards. Chickens were decapitated on day 6 (when the oviduct was regressed), day 13 (during oviduct recrudescence), and days 17-20 (oviduct rejuvenation) of the experiment. A pause in laying occurred between days 6 and 13 and resumption of laying before days 17-20. Real-time polymerase chain reaction and western blot revealed different expression of MMP-2, -7, -9 and TIMP-2 and -3 on mRNA and protein levels, respectively, as well as activity of MMP-2 and -9 by activity assay in the oviduct parts. Immunohistochemistry showed cell- and tissue-dependent localization of the examined members of the MMP system. Regression of the oviduct was accompanied predominantly by an increase in the relative expression of MMP-2, -7 and -9, and TIMP-2 and -3 mRNAs. The protein expression levels and localization of MMPs and TIMPs did not show pronounced changes during the examined period. During oviduct regression and recrudescence, elevated activity of MMP-2 and -9 was found. In summary, the results showing the stage of reproductive cycle-dependent changes as well as cell- and tissue-dependent differences in the expression of selected MMPs and TIMPs, and activity of MMP-2 and MMP-9 point to the significance that these molecules might be the local regulators of remodeling and functions of the hen oviduct.

Histochemical structure and immunolocalisation of the hyaluronan system in the dromedary oviduct.

We investigated the local modulation of some histochemical properties of oviducts of the dromedary (Camelus dromedarius), focusing on the immnolocalisation of hyaluronic acid (HA) synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL1) and the HA receptor CD44 in the ampulla and isthmus. Abundant acidic mucopolysaccharides (glycosaminoglycans) were detected by Alcian blue staining along the luminal surface of both ciliated and non-ciliated epithelial cells (LE). Staining for HAS2 was higher in the primary epithelial folds of the ampulla compared with the isthmus, especially in secretory cells, adluminal epithelial surface and supranuclear cell domain. HAS3 staining was stronger in the LE of the isthmus than ampulla. HYAL2 was detected in the LE in the ampulla and isthmus and was more intense in the adluminal projections of secretory cells. HYAL1 was weakly detected in the LE with no difference between the ampulla and isthmus. Strong CD44 immunostaining was present in the LE of the ampulla and isthmus. CD44 staining was higher in secretory cells than in ciliated epithelial cells and was higher in the supranuclear region than the basal region of the cytoplasm. In conclusion, we provide evidence that HA synthesis and turnover occur in the camel oviduct. Differences in HAS2 and HAS3 expression suggest regional differences in the molecular size of HA secreted in oviductal fluid that may influence oviduct-gamete interaction in the camel.

Regulation of cyclooxygenase-2 expression in rat oviductal epithelial cells: Evidence for involvement of GPR30/Src kinase-mediated EGFR signaling.

The oviduct plays a crucial role in female reproduction by regulating gamete transport, providing a specific microenvironment for fertilization and early embryonic development. Cyclooxygenase (COX)-derived prostaglandins play essential role in carrying out these oviduct-specific functions. Estrogen upregulates COX-2 expression in rat oviduct; however, the mechanisms responsible for regulation of COX-2 expression in rat oviductal epithelial cells (OECs) remain unclear. In the present study, we proposed that estrogen induces COX-2 expression via G-protein coupled receptor i.e., GPR30 in OECs. To investigate this hypothesis, we examined the effects of E2-BSA, ICI 182,780, GPR30 agonist and GPR30 antagonist on COX-2 expression and explored potential signaling pathway leading to COX-2 expression. Co-localization experiments revealed GPR30 to be primarily located in the peri-nuclear space, which was also the site of E2-BSA-fluorescein isothiocyanate (E2-BSA-FITC) binding. The E2-BSA induced-COX-2 and prostaglandin release were subjected to regulation by both EGFR and PI3K signaling as inhibitors of c-Src kinase (PP2), EGFR (EGFR inhibitor) and PI-3 kinase (LY294002) attenuated E2-BSA mediated effect. These results suggest that EGFR transactivation leading to activation of PI-3K/Akt pathway participates in COX-2 expression in rat OECs. Interestingly, E2-BSA induced COX-2 expression and subsequent prostaglandin release were abolished by NF-κB inhibitor. In addition, E2-BSA induced the nuclear translocation of p65-NF-κB and up-regulated the NF-κB promoter activity in rat OECs. Taken together, results demonstrated that E2-BSA induced the COX-2 expression and consequent PGE2 and PGF2α release in rat OECs. These effects are mediated through GPR30-derived EGFR transactivation and PI-3K/Akt cascade leading to NF-κB activation.

Estrogen regulation of phosphoserine phosphatase during regression and recrudescence of female reproductive organs.

Phosphoserine phosphatase (PSPH) is a well-known mediator of l-serine biosynthesis in a variety of tissues and its dysregulation causes various diseases, specifically most cancers. However, little is known about the expression and hormonal regulation of PSPH gene in the female reproductive tract. Therefore, in the current study, we focused on relationships between PSPH expression and estrogen during growth, development, differentiation, remodeling and recrudescence of the chicken oviduct and in the progression of epithelial-derived ovarian carcinogenesis in laying hens. The results revealed that PSPH mRNA and protein levels increased in the glandular (GE) and luminal epithelial (LE) cells in the developing oviduct of chicks treated with exogenous estrogen. Additionally, PSPH mRNA and protein expression was up-regulated in GE and LE of the oviduct in response to endogenous estrogen during the recrudescence phase after induced molting. Furthermore, PSPH mRNA and protein were predominantly detected in GE of cancerous, but not normal ovaries. In conclusion, PSPH is a novel estrogen-responsive gene involved in development of the oviduct of chicks and recrudescence of the oviduct of laying hens after molting. PSPH is also a potential target molecule that may help elucidate mechanism responsible for the progression of epithelial cell-derived ovarian carcinogenesis and be of use in therapeutic applications as a biomarker for early diagnosis of epithelial cell-derived ovarian cancer in laying hen as well as women.

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-ß1,4-N-acetylglucosamine (Galß1,4GlcNAc) 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.

Avian prostatic acid phosphatase: estrogen regulation in the oviduct and epithelial cell-derived ovarian carcinomas.

Prostatic acid phosphatase (ACPP) is a glycoprotein that is mainly synthesized and secreted by glandular epithelial cells (GE) of the prostate, and it is well known as a biomarker for prostate cancer. Although ACPP was used as prognostic/diagnostic indicator and studied to elucidate regulatory mechanism(s) during several decades in humans, its role is not clearly understood. Gene profiling data using a chicken DNA microarray revealed that ACPP increased significantly during remodeling and recrudescence of the oviduct in response to estrogen. Thus, in this study, we investigated the expression and hormonal regulation of ACPP gene in the reproductive tracts of chickens. ACPP was specifically detected in the luminal cells (LE) and GE of chicken oviduct, and diethylstilbestrol (a synthetic nonsteroidal estrogen) stimulated its expression during development of the oviduct. In addition, ACPP mRNA and protein were localized to LE and GE during the regeneration phase of the oviduct of laying hens during induced molting. Furthermore, ACPP mRNA and protein were abundant in GE of ovarian carcinoma, but not in normal ovaries. Moreover, strong expression of ACPP protein was detected in epithelial cells of cancerous ovaries from women. Collectively, results of the present study are the first to show that ACPP is a novel estrogen-stimulated gene in the oviductal epithelial cells of the chicken and that its expression increases significantly in epithelial cells of ovarian carcinoma, which indicates that it may be a candidate biomarker for diagnosis of epithelia-derived ovarian cancer in women.

Superoxide dismutase activity in the oviductal and uterine fluid of the bitch and the effects of the enzyme on viability, motility and hyperactivation of canine sperm in vitro.

Superoxide dismutase (SOD) activity in flushings from oviducts and uterine horns of 8 anestrous, 5 estrous and 7 diestrous bitches was measured. SOD activity in oviductal fluid in estrous bitches was significantly higher than that in anestrous and diestrous bitches (P<0.01). SOD activity in uterine fluid of diestrous bitches was, however, significantly higher than that in anestrous and estrous bitches (P<0.01). Additionally, sperm collected from normal dogs were incubated in MEM and in MEM containing SOD (SOD-MEM) for 24 hr. The percentages of sperm with viability, motility and hyperactivation in SOD-MEM were higher than those in MEM. SOD produced in oviduct and uterus may be able to maintain or improve sperm quality and fertility in the dog.

Summer heat stress affects prostaglandin synthesis in the bovine oviduct.

Summer heat stress (HS) negatively affects reproductive functions, including prostaglandin (PG) F2α secretion in the endometrium, and decreases fertility in cattle. In the present study, we examined the effects of elevated temperatures on PG synthesis in oviductal epithelial cells. The epithelial cells obtained from the ampulla and isthmus of the oviduct were incubated at various temperatures (38.5, 39.5, 40.0, and 40.5 °C) for 24 h. In the ampulla, PGE2 concentration was higher at 40.5 °C than at 38.5 °C, while PGF2α production was not affected by the temperatures in this range. The expressions of microsomal PGE synthase 1 (PTGES (mPGES1)), cytosolic PGES (PTGES3 (cPGES)), and heat shock protein 90 (HSP90AA1 (HSP90)) mRNAs and proteins were higher at 40.5 °C than at 38.5 °C in the ampullary epithelial cells. Seasonal changes in the expressions of PGES and HSP90AA1 mRNAs in oviductal tissues were also investigated. The expressions of PTGES3 and HSP90AA1 mRNAs were higher in the ampullary tissues in summer than in winter. In summary, elevated temperatures stimulated PGE2 production in the ampullary oviduct by increasing the expressions of PGESs and HSP90AA1, which can activate cPGES. The overall results suggest that HS upsets PG secretions and reduces oviductal smooth muscle motility, which in turn could decrease gamete/embryo transport through the oviduct.

In vivo and in vitro expression of the plasminogen activators and urokinase type plasminogen activator receptor (u-PAR) in the pig oviduct.

Plasminogen activator activities have previously been reported in oviductal fluid. At present the question was whether the source of these activities is molecules come from blood plasma or if these activators are synthesized by the oviduct. Gene expression and protein synthesis of urokinase type (u-PA) and tissue type (t-PA) occur in different regions of the pig oviduct. Their relative concentrations do not vary between the ampulla and isthmus regions and are similar throughout the estrous cycle. However, while relative amounts of t-PA mRNA were not different between the different stages of the estrous cycle, u-PA mRNA was greater after ovulation (P<0.05). Regarding the function of u-PA, its receptor (u-PAR) was distinguished by immunohistochemistry at the apical region of the epithelial cells and was more noticeable in the isthmus. Expression of u-PA, t-PA, u-PAR and PAI-1 genes in primary oviductal epithelial cell cultures was studied under 17-ß-estradiol (100 pg/ml) and progesterone (100 ng/ml). u-PA mRNA increased in the presence of progesterone (P<0.05), but not by action of 17-ß-estradiol. t-PA, PAI-1 and u-PAR were similar when cultured with the hormones. These results suggest that u-PA could be regulated by progesterone at a transcriptional level, by the balance of their activity for PAI-1 or at the epithelial surface through the binding of u-PAR. In conclusion, plasminogen activation system components might cooperate in the oviductal lumen to control plasmin generation.

Are glucocorticoids auto- and/or paracrine factors in early bovine embryo development and implantation?

We determined the transcript content of three genes involved in the metabolism of glucocorticoids (GC) in bovine in vitro fertilized embryos (2-blastomere stage until hatched blastocyst), trophoblast as well as the oviduct (Day 2-4 of the estrous cycle) and endometrium (Day 16 of the cycle and pregnancy). Since mRNA expression of the glucocorticoid receptor and two enzymes responsible for GC production (11ß-HSD1 and 2) was demonstrated in the embryos in all pre-implantation stages as well as in the endometrium and oviduct, it is suggested that GC may serve as auto-/paracrine factors in the development of bovine pre-implantation embryos.

Phosphodiesterase inhibitor 3-isobutyl-methyl-xanthine affects rabbit ovaries and oviduct.

The aim of our study was to examine the influence of 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterases on rabbit reproductive system. The ovarian cycle and ovulation of control rabbits were induced by gonadotropin releasing hormone (GnRH) or equine chorionic gonadotropin (eCG) followed by the human chorionic gonadotropin (hCG) administration. Experimental animals received GnRH together with IBMX (at 50 or 500 microg/animal) or eCG and hCG together with IBMX (at 5 or 257 microg/animal). After mating and insemination, blood samples were collected and analyzed for concentrations of progesterone and estradiol by RIA; some animals from the control and IBMX-treated groups were killed. The presence of follicles at different stages of development was evaluated by a light microscopy. Isolated ovarian fragments were cultured for 2 days. The secretion of progesterone and estradiol was assessed by RIA. The expression of markers of proliferation (PCNA and cyclin B1) and apoptosis (bax) in ovarian fragments was evaluated by Western-blotting. Epithelial cells were isolated from oviducts and cultured for 2 days. The expression of markers of proliferation (PCNA, ERK1,2-related MAP kinase) and apoptosis (bax, bcl-2) in the oviductal cells was evaluated by immunocytochemistry. It was observed, that the ovaries of rabbits treated with IBMX contained more secondary follicles, than control rabbits. Administration of IBMX reduced blood level of progesterone, but did not affect blood estradiol. Fragments of ovaries isolated from rabbits treated with IBMX released more estradiol, but not progesterone, than ovarian cells isolated from the control animals. IBMX injections substantially decreased the expression of the upper (23 kD), but not the bottom (24 kD) fraction of bax. IBMX administration did not affect PCNA, but it caused a decrease in the upper fraction (54 kD) and an increase in the bottom fraction (55 kD) of cyclin B1. Oviductal cells isolated from the IBMX-treated animals, contained less marker of apoptosis - bax (but not bcl-2) and proliferation - ERK1,2-related MAP kinase (but not PCNA) than control animals. These observations demonstrate the involvement of cyclic nucleotide-dependent intracellular mechanisms in control of rabbit reproductive functions: ovarian folliculogenesis, apoptosis, proliferation and steroid hormone release, as well as proliferation and apoptosis of the oviductal cells.

Expression of prostaglandin E synthases in the bovine oviduct.

The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E(2) is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH(2.) The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH(2) to PGE(2), the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE(2) production were present in the Bos taurus oviduct.

Localization and expression of peroxiredoxin II in the mouse ovary, oviduct, uterus, and preimplantation embryo.

Peroxiredoxin (Prx) II belongs to a recently discovered family of peroxidases that play important roles in antioxidation and signal transduction. In this study, we aimed to study the localization and expression of Prx II in the mouse ovary, oviduct, and uterus, and preimplantation embryos. Immunohistochemical staining analysis showed that, in the ovary, Prx II was expressed in the oocyte cytoplasm of the primary follicle, the secondary follicle, and the premature follicle; Prx II was expressed in germinal vesicle-intact oocytes (GV oocytes) and metaphase II eggs (MII eggs), as well as at various stages in early embryos. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that the Prx II mRNA was expressed at a high level in GV eggs, slightly lower levels in MII eggs, and had no detectable expression in four-cell embryos and early blastocysts. In the oviduct, Prx II was expressed in the epithelia, while in the uterus Prx II was mainly distributed in the endometrial stroma. Taken together, our results suggest that Prx II plays a key antioxidation role in the maturation of oocytes and development of early embryos, thus providing crucial experimental evidence for further exploring the function of Prx II in the development of oocytes and preimplantation embryos.

Glycosidase determination in bovine oviducal fluid at the follicular and luteal phases of the oestrous cycle.

Gamete recognition and binding of spermatozoa to the oviduct are carbohydrate-mediated processes in which several glycosidases are thought to have a role, although this has not been demonstrated unequivocally. Oviducal fluid is the biological milieu in which fertilisation and early embryo development take place, but the enzyme composition of oviducal fluid is largely unknown. The aim of the present study was to determine glycosidase activity and protein content in bovine oviducal fluid (bOF) and the volume of fluid collected per oviduct. Oviducts obtained from a slaughterhouse were classified as either in the follicular or luteal phase on the basis of ovarian luteal morphology. Oviducal fluid was aspirated, centrifuged and the volume determined. Samples were then frozen until assay. Substrates conjugated to 4-methylumbelliferyl were used to screen for the activity of seven glycosidases at pH 7.2. The results indicate that bOF has alpha-l-fucosidase, beta-N-acetyl-glucosaminidase, beta-d-galactosidase, alpha-D-mannosidase and beta-N-acetyl-galactosaminidase activity during both phases of the cycle, with the specific activity of the latter two enzymes being higher during the follicular phase. There was no N-acetyl-neuraminidase or alpha-d-galactosidase activity detected in bOF at either phase of the oestrous cycle at pH 7.2, but activity for both glycosidases was detected at pH 4.4. There were no differences in protein concentration or the volume of bOF collected between the two phases of the cycle. These findings indicate that oviducal fluid exhibits glycosidase activity, with specific variations throughout the oestrous cycle, suggesting that these enzymes play a role in carbohydrate-mediated events.

Transgenic cyclooxygenase-2 expression and high salt enhanced susceptibility to chemical-induced gastric cancer development in mice.

Cyclooxoygenase (COX)-2 overexpression is involved in gastric carcinogenesis. While high-salt intake is a known risk factor for gastric cancer development, we determined the effects of high salt on gastric chemical carcinogenesis in COX-2 transgenic (TG) mice. COX-2 TG mice were developed in C57/BL6 strain using the full-length human cox-2 complementary DNA construct. Six-week-old COX-2 TG and wild-type (WT) littermates were randomly allocated to receive alternate week of N-methyl-N-nitrosourea (MNU, 240 p.p.m.) in drinking water or control for 10 weeks. Two groups of mice were further treated with 10% NaCl during the initial 10 weeks. All mice were killed at the end of week 50. Both forced COX-2 overexpression and high-salt intake significantly increased the frequency of gastric cancer development in mice as compared with WT littermates treated with MNU alone. However, no additive effect was observed on the combination of high salt and COX-2 expression. We further showed that MNU and high-salt treatment increased chronic inflammatory infiltrates and induced prostaglandin E(2) (PGE(2)) production in the non-cancerous stomach. Whereas high-salt treatment markedly increased the expression of inflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1 beta and IL-6) in the gastric mucosa, COX-2 overexpression significantly altered the cell kinetics in the MNU-induced gastric cancer model. In conclusion, both high salt and COX-2 overexpression promote chemical-induced gastric carcinogenesis, possibly related to chronic inflammation, induction of PGE(2), disruption of cell kinetics and induction of inflammatory cytokines.

Identification and characterization of the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) in hen egg.

Using zymography and mass spectrometry, we identified for the first time the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) as a complex with TIMP-2 (tissue inhibitor of metalloproteinases) in egg white and yolk. Real-time polymerase chain reaction confirmed that MMP-2 and its inhibitors TIMP-2 and TIMP-3 were expressed all along the oviduct and in the liver of laying hens. We also demonstrated that the processing of pro-MMP-2 into mature MMP-2 by serine proteases does not occur in vivo, although purified pro-MMP-2 undergoes proteolytic maturation by these proteases in vitro. Moreover, the relative pro-MMP-2 activity assessed by gelatin zymography was shown to decrease in egg white during the storage of unfertilized or fertilized eggs. However, the mature form of 62 kDa MMP-2 could not be detected. The fact that MMP-2 is found as a proform in fresh eggs suggests that the activity of this metalloprotease is regulated under specific conditions during embryonic development.